CIRCULAR RNA ENCODING VEGF POLYPEPTIDES, FORMULATIONS, AND METHODS OF USES1. Related applications
The application claims priority to, and the benefit of PCT Application No. PCT/CN2023/115512, filed on August 29, 2023, the contents of which are incorporated herein by reference in their entirety.
2. Incorporation by reference of sequence listing
The contents of the electronic sequence listing (TFH01061PCT-Sequence listing. xml; Size: 170, 721 bytes; and Date of Creation: August 28, 2024) are herein incorporated by reference in its entirety.
3. FieldThe present invention relates to the field of molecular biology and immunology, in particular to constructs and methods for the manufacture and therapeutic use of circular RNAs encoding VEGF polypeptides and formulations comprising the circular RNAs in treating subjects in need of VEGF therapy.
4. BackgroundCircular RNAs (circRNAs) are a category of RNA molecules formed by head-to-tail ligation, which were demonstrated to have multiple biological functions in recent years. (Yang et al., Cell Research, 27 (5) : 626-641 (2017) ; Abe et al., Scientific Reports, 5: 16435 (2015) ; Gao et al., Nature Cell Biology, 23 (3) : 278-291 (2021) ; Pamudurti et al., Molecular Cell, 66 (1) : 9-21 (2017) ) . Compared with linear RNAs, circRNAs have better stability and therefore provide a promising new platform for RNA drugs.
Vascular endothelial growth factor A (VEGF-A) is a dimeric glycoprotein that plays a essential role in neurons, and is considered to be the main, dominant inducer to the growth blood vessels. VEGF-A is essential for adults during organ remodeling and diseases that involve blood vessels, for example, in wound healing, tumor angiogenesis, diabetic retinopathy, and age-related macular degeneration.
The compositions provided herein, which comprise circular RNAs encoding VEGF-A polypeptides, as well as related methods and systems address the VEGF-A related therapeutic need and provide related advantages.
5. SummaryProvided herein are compositions comprising a circular ribonucleotide acid (circRNA) , wherein the circRNA comprises, a translation initiation (TI) sequence and a protein-coding (Z) sequence, and wherein the Z sequence comprises a VEGF sequence encoding a VEGF polypeptide sequence.
Provided herein are compositions comprising a circular ribonucleotide acid (circRNA) , wherein the circRNA comprises, a translation initiation (TI) sequence and a protein-coding (Z) sequence, and wherein the Z sequence comprises a VEGF-A sequence encoding a VEGF-A polypeptide sequence (VF) .
In some embodiments, the VF is selected from an amino acid sequence listed in Table 1.
In some embodiments, the VF is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to an amino acid sequence listed in Table 1.
In some embodiments, the VEGF-A sequence has a nucleotide sequence listed in Table 2.
In some embodiments, the VEGF-A sequence has a nucleotide sequence which is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence listed in Table 2.
In some embodiments, the circRNA comprises a linker sequence, wherein the linker sequence is a 2A self-cleaving peptide.
In some embodiments, the TI sequence comprises: an IRES, an IRES-like nucleotide sequence, or a combination thereof.
In some embodiments, the TI sequence is at least 95%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence listed in Table 4.
In some embodiments, the TI sequence is 100%identical to a nucleotide sequence listed in Table 4.
In some embodiments, the circRNA comprises a nucleotide sequence that is at least 95%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence listed in Table 3.
In some embodiments, the circRNA comprises a modified RNA nucleotide and/or modified nucleoside.
In some embodiments, the circRNA comprises at least 10%modified RNA nucleotides and/or modified nucleosides.
In some embodiments, at least one of the modified RNA nucleotide and/or modified nucleoside is m5C (5-methylcytidine) , m5U (5-methyluridine) , m6A (N6-methyladenosine) , Y (pseudouridine) , or m1A (1-methyladenosine) .
In some embodiments, at least one of the modified RNA nucleotide and/or modified nucleoside is introduced at in vitro transcription (IVT) .
In some embodiments, the composition further comprises a buffer, preferably a citrate saline buffer, a phosphate-buffered saline (PBS) buffer, or a tromethamine (THAM) buffer.
In some embodiments, the buffer is substantially free of divalent cations.
In some embodiments, said buffer substantially free of divalent cations is a citrate saline buffer.
In some embodiments, the citrate saline buffer is substantially free of calcium and magnesium.
In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.
In some embodiments, the pharmaceutically acceptable excipient is chosen from a solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, core-shell nanoparticles, polymer, peptide, protein, cell, hyaluronidase, and mixtures thereof.
Provided herein also methods for treating a subject suffering from a disease responsive to VEGF-A therapy, in a subject comprising administering to the subject a therapeutically effective amount of the composition disclosed herein.
In some embodiments, the disease is chosen from heart failure with reduced or preserved ejection fraction, kidney disease, a disease involving skin grafting and tissue grafting, post-MI cardiac dysfunction, ischemic heart disease, a vascular injury from trauma or surgery, a skin ulcer including a diabetic ulcer, diabetic foot, critical limb ischemia, lower limb ischemia, pulmonary hypertension, stable severe angina pectoris, refractory angina, congenital and acquired maxillofacial defects, alveolar bone atrophy, ligament or tendon lesions, ocular disease (e.g. proliferative diabetic retinopathy and macular edema) , and peripheral arterial disease.
In some embodiments, the disease is heart failure with reduced or preserved ejection fraction. In some embodiments, the disease is kidney disease. In some embodiments, the disease is a disease involving skin grafting and tissue grafting. In some embodiments, the disease is post-MI cardiac dysfunction. In some embodiments, the disease is ischemic heart disease. In some embodiments, the disease is a vascular injury from trauma or surgery. In some embodiments, the disease is a skin ulcer including a diabetic ulcer. In some embodiments, the disease is diabetic foot. In some embodiments, the disease is critical limb ischemia. In some embodiments, the disease is lower limb ischemia. In some embodiments, the disease is pulmonary hypertension. In some embodiments, the disease is stable severe angina pectoris. In some embodiments, the disease is refractory angina. In some embodiments, the disease is maxillofacial defects. In some embodiments, the disease is congenital or acquired maxillofacial defects. In some embodiments, the disease is alveolar bone atrophy. In some embodiments, the disease is ligament or tendon lesions. In some embodiments, the disease is ocular disease. In some embodiments, the disease is peripheral arterial disease.
In some embodiments, the composition is administered to the subject via intramuscular, intradermal, subcutaneous, intracranial, intrathecal, vitreous, and subretinal, muscular, cardiac, intracardiac, or epicardiac route, through a portal vein catheter, through a coronary sinus catheter, and/or by direct administration into the area to be treated.
In some embodiments, the composition is delivered to the subject via microneedles, hydrogels, or sprays. In some embodiments, the composition comprises a buffer for delivering to the subject via microneedles, hydrogels, or sprays.
In some embodiments, the composition is administered to the subject intracardially or epicardially, preferably at a fixed-dosage in multiple administrations. In some embodiments, the composition is administered to the subject through a portal vein catheter, preferably at a fixed-dosage in multiple administrations. In some embodiments, the composition is administered to the subject through a coronary sinus catheter, preferably at a fixed-dosage in multiple administrations. In some embodiments, the composition is administered to the subject by direct administration into the area to be treated, preferably at a fixed-dosage in multiple administrations.
In some embodiments, the composition is suitable for a naked formulation. In some embodiments, the naked formulation is administered to the subject via intradermal, subcutaneous, intramuscular, intracranial, intrathecal, vitreous, muscular, cardiac, and subretinal.
Provided herein also methods for modulating a physiological process in a mammalian cell, tissue, or subject comprising contacting said mammalian cell, tissue, or subject with the composition disclosed herein.
In some embodiments, the modulating is chosen from inducing angiogenesis, stimulating vascular cell proliferation, increasing proliferation and/or altering the fate of epicardial derived progenitor cells, upregulating endothelialization, inducing cardiac regeneration, increasing revascularization of tissue grafts for wound healing, improving vascular function, increasing tissue perfusion and new vessel formation, reducing scar tissue, promoting stent biofunctionality, inducing lymphangiogenesis, inducing bone repair, and improving cardiac function.
Provided herein also methods for expressing VEGF-A in a mammalian cell or tissue, comprising contacting said mammalian cell or tissue with the composition disclosed herein.
Provided herein also methods for producing VEGF-A in a subject, comprising administering to said subject the composition disclosed herein. In some embodiments, the subject is suffering from a disease responsive to VEGF-A therapy. In some embodiments, the disease is chosen from heart failure with reduced or preserved ejection fraction, kidney disease, a disease involving skin grafting and tissue grafting post-MI cardiac dysfunction, ischemic heart disease, a vascular injury from trauma or surgery, a skin ulcer including a diabetic ulcer, diabetic foot, critical limb ischemia, lower limb ischemia, pulmonary hypertension, stable severe angina pectoris, refractory angina, congenital and acquired maxillofacial defects, alveolar bone atrophy and peripheral arterial disease.
Provided herein also methods for increasing wound healing in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition disclosed herein.
Provided herein also methods for inducing neovascularization in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition disclosed herein.
Provided herein also methods for inducing angiogenesis in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition disclosed herein.
Provided herein also methods for inducing vasodilation in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition disclosed herein.
Provided herein also methods for inducing blood flow upregulation in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition disclosed herein.
Provided herein also methods for increasing capillary and/or arteriole density in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition disclosed herein.
Provided herein also methods for attenuating fibrosis in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition disclosed herein.
The subjects treated in accordance with the methods described herein can be any mammals such as rodents, domestic animals such as dogs or cats, or primates, e.g. non-human primates. In a preferred embodiment, the subject is a human. In some embodiments, the subject treated in accordance with the methods described herein is female. In some embodiments, the subject treated in accordance with the methods described herein is male. In some embodiments, the subject treated in accordance with the methods described herein can be of any age. In some embodiments, the subject treated in accordance with the methods described herein is less than 18 years old. In a specific embodiment, the subject treated in accordance with the methods described herein is less than 13 years old. In another specific embodiment, the subject treated in accordance with the methods described herein is less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, or less than 5 years old. In another specific embodiment, the subject treated in accordance with the methods described herein is 1-3 years old, 3-5 years old, 5-7 years old, 7-9 years old, 9-11 years old, 11-13 years old, 13-15 years old, 15-20 years old, 20-25 years old, 25-30 years old, or greater than 30 years old. In another specific embodiment, the subject treated in accordance with the methods described herein is 30-35 years old, 35-40 years old, 40-45 years old, 45-50 years old, 50-55 years old, 55-60 years old, or greater than 60 years old. In another specific embodiment, the subject treated in accordance with the methods described herein is 18-64 years old, 65-74 years old, or greater than 75 years old.
6. Illustrative Embodiments
Set 1
The composition comprising a circular ribonucleotide acid (circRNA) , wherein the circRNA comprises, a translation initiation (TI) sequence and a protein-coding (Z) sequence, and wherein the Z sequence comprises a VEGF sequence encoding a VEGF polypeptide sequence.
The composition comprising a circular ribonucleotide acid (circRNA) , wherein the circRNA comprises, a translation initiation (TI) sequence and a protein-coding (Z) sequence, and wherein the Z sequence comprises a VEGF-A sequence encoding a VEGF-A polypeptide sequence (VF) .
The composition of paragraph [0049] , wherein the VF is selected from an amino acid sequence listed in Table 1.
The composition of paragraph [0049] , wherein the VF is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to an amino acid sequence listed in Table 1.
The composition of paragraph [0049] , wherein the VEGF-A sequence has a nucleotide sequence listed in Table 2.
The composition of paragraph [0049] , wherein the VEGF-A sequence has a nucleotide sequence which is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence listed in Table 2.
The composition of any one of paragraphs [0049] to [0053] , wherein the circRNA comprises a linker sequence, wherein the linker sequence is a 2A self-cleaving peptide.
The composition any one of paragraphs [0049] to [0054] , wherein the TI sequence comprises: an IRES, an IRES-like nucleotide sequence, or a combination thereof.
The composition of paragraph [0055] , wherein the TI sequence is at least 95%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence listed in Table 4.
The composition of paragraph [0055] , wherein the TI sequence is 100%identical to a nucleotide sequence listed in Table 4.
The composition of paragraph [0049] , wherein the circRNA comprises a nucleotide sequence that is at least 95%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence listed in Table 3.
The composition of any one of paragraphs [0049] to [0058] , wherein the circRNA comprises a modified RNA nucleotide and/or modified nucleoside.
The composition of paragraph [0059] , wherein the circRNA comprises at least 10%modified RNA nucleotides and/or modified nucleosides.
The composition of paragraph [0060] , wherein at least one of the modified RNA nucleotide and/or modified nucleoside is m5C (5-methylcytidine) , m5U (5-methyluridine) , m6A (N6-methyladenosine) , Y (pseudouridine) , or m1A (1-methyladenosine) .
The composition of any one of paragraphs [0059] to [0061] , wherein at least one of the modified RNA nucleotide and/or modified nucleoside is introduced at in vitro transcription (IVT) .
The composition of any one of paragraphs [0049] to [0062] , the composition further comprise a buffer, preferably a citrate saline buffer, a phosphate-buffered saline (PBS) buffer, or a tromethamine (THAM) buffer.
The composition of paragraph [0063] , wherein the buffer is substantially free of divalent cations.
The composition of paragraph [0064] , wherein said buffer substantially free of divalent cations is a citrate saline buffer.
The composition of paragraph [0065] , wherein the citrate saline buffer is substantially free of calcium and magnesium.
The composition of paragraph [0065] , wherein the citrate saline buffer contains no calcium or magnesium.
The composition of paragraph [0064] , further comprising a pharmaceutically acceptable excipient.
The composition of paragraph [0068] , wherein the pharmaceutically acceptable excipient is chosen from a solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, core-shell nanoparticles, polymer, peptide, protein, cell, hyaluronidase, and mixtures thereof.
A method for treating a subject suffering from a disease responsive to VEGF-A therapy, in a subject comprising administering to the subject a therapeutically effective amount of the composition of any one of paragraphs [0049] to [0063] .
The method of paragraph [0070] , wherein the buffer that is substantially free of divalent cations in said composition or formulation is a citrate saline buffer.
The method of paragraph [0071] , wherein the citrate saline buffer is substantially free of calcium and magnesium.
The method of paragraph [0071] , wherein the citrate saline buffer contains no calcium or magnesium.
The method of paragraph [0070] , wherein the composition further comprises a pharmaceutically acceptable excipient.
The method of paragraph [0074] , wherein the pharmaceutically acceptable excipient is chosen from a solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, core-shell nanoparticles, polymer, peptide, protein, cell, hyaluronidase, and mixtures thereof.
The method of paragraph [0070] , wherein the disease is chosen from heart failure with reduced or preserved ejection fraction, kidney disease, a disease involving skin grafting and tissue grafting, post-MI cardiac dysfunction, ischemic heart disease, a vascular injury from trauma or surgery, a skin ulcer including a diabetic ulcer, diabetic foot, critical limb ischemia, lower limb ischemia, pulmonary hypertension, stable severe angina pectoris, refractory angina, congenital and acquired maxillofacial defects, alveolar bone atrophy, ligament or tendon lesions, ocular disease, and peripheral arterial disease.
The method of paragraph [0070] , wherein the disease is heart failure with reduced or preserved ejection fraction.
The method of paragraph [0070] , wherein the disease is post-MI cardiac dysfunction.
The method of paragraph [0070] , wherein the disease is ischemic heart disease.
The method of paragraph [0070] , wherein the disease is a vascular injury from trauma or surgery.
The method of paragraph [0070] , wherein the disease is a skin ulcer including a diabetic ulcer.
The method of paragraph [0070] , wherein the disease is diabetic foot.
The method of paragraph [0070] , wherein the disease is critical limb ischemia.
The method of paragraph [0070] , wherein the disease is lower limb ischemia.
The method of paragraph [0070] , wherein the disease is pulmonary hypertension.
The method of paragraph [0070] , wherein the disease is stable severe angina pectoris.
The method of paragraph [0070] , wherein the disease is refractory angina.
The method of paragraph [0070] , wherein the disease is maxillofacial defects.
The method of paragraph [0088] , wherein the disease is congenital or acquired maxillofacial defects.
The method of paragraph [0070] , wherein the disease is alveolar bone atrophy.
The method of paragraph [0070] , wherein the disease is ligament or tendon lesions.
The method of paragraph [0070] , wherein the disease is ocular disease.
The method of paragraph [0070] , wherein the disease is peripheral arterial disease.
The method of paragraph [0070] , wherein the composition is administered to the subject via intramuscular, intradermal, subcutaneous, intracranial, intrathecal, vitreous, and subretinal, muscular, cardiac, intracardiac, or epicardiac route, through a portal vein catheter, through a coronary sinus catheter, and/or by direct administration into the area to be treated.
The method of paragraph [0070] , wherein the composition is delivered to the subject via microneedles, hydrogels, or sprays.
The method of paragraph [0070] , wherein the composition is administered to the subject intramuscularly.
The method of paragraph [0070] , wherein the composition is administered to the subject intradermally.
The method of paragraph [0070] , wherein the composition is administered to the subject subcutaneously.
The method of paragraph [0070] , wherein the composition is administered to the subject intracranially.
The method of paragraph [0070] , wherein the composition is administered to the subject intrathecally.
The method of paragraph [0070] , wherein the composition is administered to the subject vitreously.
The method of paragraph [0070] , wherein the composition is administered to the subject subretinally.
The method of paragraph [0070] , wherein the composition is administered to the subject muscularly.
The method of paragraph [0070] , wherein the composition is administered to the subject cardiacly.
The method of paragraph [0070] , wherein the composition is administered to the subject intracardially or epicardially, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [0070] , wherein the composition is administered to the subject through a portal vein catheter, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [0070] , wherein the composition is administered to the subject through a coronary sinus catheter, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [0070] , wherein the composition is administered to the subject by direct administration into the area to be treated, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [0070] , wherein the composition is suitable for naked formulation.
The method of paragraph [00109] , wherein the naked formulation is administered to the subject via intradermal, subcutaneous, intramuscular, intracranial, intrathecal, vitreous, muscular, cardiac, and subretinal.
A method for modulating a physiological process in a mammalian cell, tissue, or subject comprising contacting said mammalian cell, tissue, or subject with the composition of any one of paragraphs [0049] to [0063] .
The method of paragraph [00111] , wherein the modulating is chosen from inducing angiogenesis, stimulating vascular cell proliferation, increasing proliferation and/or altering the fate of epicardial derived progenitor cells, upregulating endothelialization, inducing cardiac regeneration, increasing revascularization of tissue grafts for wound healing, improving vascular function, increasing tissue perfusion and new vessel formation, reducing scar tissue, promoting stent biofunctionality, inducing lymphangiogenesis, inducing bone repair, and improving cardiac function.
The method of paragraph [00111] , wherein the modulating comprises inducing angiogenesis.
The method of paragraph [00111] , wherein the modulating comprises stimulating vascular cell proliferation.
The method of paragraph [00111] , wherein the modulating comprises increasing proliferation and/or altering the fate of epicardial derived progenitor cells.
The method of paragraph [00111] , wherein the modulating comprises upregulating endothelialization.
The method of paragraph [00111] , wherein the modulating comprises inducing cardiac regeneration.
The method of paragraph [00111] , wherein the modulating comprises increasing revascularization of tissue grafts for wound healing.
The method of paragraph [00111] , wherein the modulating comprises improving vascular function.
The method of paragraph [00111] , wherein the modulating comprises increasing tissue perfusion and new vessel formation.
The method of paragraph [00111] , wherein the modulating comprises reducing scar tissue.
The method of paragraph [00111] , wherein the modulating comprises promoting stent biofunctionality.
The method of paragraph [00111] , wherein the modulating comprises inducing lymphangiogenesis.
The method of paragraph [00111] , wherein the modulating comprises inducing bone repair.
The method of paragraph [00111] , wherein the modulating comprises improving cardiac function.
The method of paragraph [00111] , wherein the buffer that is substantially free of divalent cations in said composition is a citrate saline buffer.
The method of paragraph [00126] , wherein the citrate saline buffer is substantially free of calcium and magnesium.
The method of paragraph [00126] , wherein the citrate saline buffer contains no calcium or magnesium.
A method for expressing VEGF-A in a mammalian cell or tissue, comprising contacting said mammalian cell or tissue with the composition of any one of paragraphs [0049] to [0063] .
The method of paragraph [00129] , wherein the buffer that is substantially free of divalent cations in said composition or formulation is a citrate saline buffer.
The method of paragraph [00130] , wherein the citrate saline buffer is substantially free of calcium and magnesium.
The method of paragraph [00130] , wherein the citrate saline buffer contains no calcium or magnesium.
A method of producing VEGF-A in a subject, comprising administering to said subject the composition of any one of paragraphs [0049] to [0063] .
The method of paragraph [00133] , wherein the buffer that is substantially free of divalent cations in said composition is a citrate saline buffer.
The method of paragraph [00134] , wherein the citrate saline buffer is substantially free of calcium and magnesium.
The method of paragraph [00134] , wherein the citrate saline buffer contains no calcium or magnesium.
The method of paragraph [00133] , wherein the buffer is suitable for naked formulation
The method of paragraph [00137] , wherein the buffer is a sodium chloride solution or Ringer's solution contains NaCl, KCl, CaCl2, NaHCO3, and/or MgCl2.
The method of paragraph [00138] , wherein the concentration of Ca2+ is in a range of 0.75 mM-1.5 mM.
The method of paragraph [00133] , wherein the buffer is a 10 mM citric acid buffer solution and 130 M sodium chloride solution, with a pH range of 6-7.5.
The method of paragraph [00133] , wherein the buffer is phosphate buffer and/or acetate buffer.
The method of paragraph [00133] , wherein the buffer contains glucose, sucrose, and/or trehalose.
The method of paragraphs [00138] to [00142] , wherein a naked RNA formulation comprises buffer and one or more circRNA (s) .
The method of paragraph [00143] , wherein the concentration of circRNA (s) is in a range of 500 ng/μL-2000 ng/μL.
The method of paragraph [00133] , wherein the composition further comprises a pharmaceutically acceptable excipient.
The method of paragraph [00145] , wherein the pharmaceutically acceptable excipient is chosen from a solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, core-shell nanoparticles, polymer, peptide, protein, cell, hyaluronidase, and mixtures thereof.
The method of paragraph [00133] , wherein the subject is suffering from a disease responsive to VEGF-A therapy.
The method of paragraph [00147] , wherein the disease is chosen from heart failure with reduced or preserved ejection fraction, kidney disease, a disease involving skin grafting and tissue grafting post-MI cardiac dysfunction, ischemic heart disease, a vascular injury from trauma or surgery, a skin ulcer including a diabetic ulcer, a diabetic foot, critical limb ischemia, lower limb ischemia, pulmonary hypertension, stable severe angina pectoris, refractory angina, congenital and acquired maxillofacial defects, alveolar bone atrophy, ligament or tendon lesions, ocular disease, and peripheral arterial disease.
The method of paragraph [00147] , wherein the disease is heart failure with reduced or preserved ejection fraction.
The method of paragraph [00147] , wherein the disease is post-MI cardiac dysfunction.
The method of paragraph [00147] , wherein the disease is ischemic heart disease.
The method of paragraph [00147] , wherein the disease is a vascular injury from trauma or surgery.
The method of paragraph [00147] , wherein the disease is a skin ulcer including a diabetic ulcer.
The method of paragraph [00147] , wherein the disease is diabetic foot.
The method of paragraph [00147] , wherein the disease is critical limb ischemia.
The method of paragraph [00147] , wherein the disease is lower limb ischemia.
The method of paragraph [00147] , wherein the disease is pulmonary hypertension.
The method of paragraph [00147] , wherein the disease is stable severe angina pectoris.
The method of paragraph [00147] , wherein the disease is refractory angina.
The method of paragraph [00147] , wherein the disease is maxillofacial defects.
The method of paragraph [00160] , wherein the disease is congenital or acquired maxillofacial defects.
The method of paragraph [00147] , wherein the disease is alveolar bone atrophy.
The method of paragraph [00147] , wherein the disease is ligament or tendon lesions.
The method of paragraph [00147] , wherein the disease is ocular disease.
The method of paragraph [00147] , wherein the disease is peripheral arterial disease.
The method of paragraph [00133] , wherein the composition is administered to the subject via intramuscular, intradermal, subcutaneous, intracranial, intrathecal, vitreous, and subretinal, muscular, cardiac, intracardiac, or epicardiac route, through a portal vein catheter, through a coronary sinus catheter, and/or by direct administration into the area to be treated.
The method of paragraph [00133] , wherein the composition is delivered to the subject via microneedles, hydrogels, or sprays.
The method of paragraph [00133] , wherein the composition is administered to the subject intramuscularly.
The method of paragraph [00133] , wherein the composition is administered to the subject intradermally.
The method of paragraph [00133] , wherein the composition is administered to the subject subcutaneously.
The method of paragraph [00133] , wherein the composition is administered to the subject intracranially.
The method of paragraph [00133] , wherein the composition is administered to the subject intrathecally.
The method of paragraph [00133] , wherein the composition is administered to the subject vitreously.
The method of paragraph [00133] , wherein the composition is administered to the subject subretinally.
The method of paragraph [00133] , wherein the composition is administered to the subject muscularly.
The method of paragraph [00133] , wherein the composition is administered to the subject cardiacly.
The method of paragraph [00133] , wherein the composition is administered to the subject intracardially or epicardially, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [00133] , wherein the composition is administered to the subject through a portal vein catheter, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [00133] , wherein the composition is administered to the subject through a coronary sinus catheter, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [00133] , wherein the composition is administered to the subject by direct administration into the area to be treated, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [00133] , wherein the composition is suitable for a naked formulation.
The method of paragraph [00181] , wherein the naked formulation is administered to the subject via intradermal, subcutaneous, intramuscular, intracranial, intrathecal, vitreous, muscular, cardiac, and subretinal.
A method for increasing wound healing in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition of any one of paragraphs [0049] to [0063] .
A method for inducing neovascularization in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition of any one of paragraphs [0049] to [0063] .
A method for inducing angiogenesis in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition of any one of paragraphs [0049] to [0063] .
A method for inducing vasodilation in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition of any one of paragraphs [0049] to [0063] .
A method for inducing blood flow upregulation in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition of any one of paragraphs [0049] to [0063] .
A method for increasing capillary and/or arteriole density in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition of any one of paragraphs [0049] to [0063] .
A method for attenuating fibrosis in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition of any one of paragraphs [0049] to [0063] .
The method of paragraph [0070] wherein the subject is a human.
Set 2
A composition comprising a circular ribonucleotide acid (circRNA) , wherein the circRNA comprises, a translation initiation (TI) sequence and a protein-coding (Z) sequence, and wherein the Z sequence comprises a VEGF-A sequence encoding a VEGF-A polypeptide sequence (VF) .
The composition paragraph [00192] , wherein the VF is selected from the group of amino acid sequences listed in Table 1.
The composition of paragraph [00192] , wherein the VF is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to an amino acid sequence listed in Table 1.
The composition of paragraph [00192] , wherein the VEGF-A sequence has a nucleotide sequence listed in Table 2.
The composition of paragraph [00192] , wherein the VEGF-A sequence has a nucleotide sequence which is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence listed in Table 2.
The composition of any one of paragraphs [00192] to [00196] , wherein the circRNA comprises a linker sequence, wherein the linker sequence encodes a 2A self-cleaving peptide. 6
The composition of any one of paragraphs [00192] to [00197] , wherein the TI sequence comprises: an IRES, an IRES-like nucleotide sequence, or a combination thereof.
The composition of paragraph [00198] , wherein the IRES is at least 95%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence listed in Table 4.
The composition of paragraph [00198] , wherein the IRES-like sequence is at least 95%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence listed in Table 4.
The composition of paragraph [00192] , wherein the circRNA comprises a nucleotide sequence that is at least 95%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence listed in Table 3.
The composition of any one of paragraphs [00192] to [00201] , wherein the circRNA comprises a modified RNA nucleotide and/or modified nucleoside.
The composition of paragraph [00202] , wherein the circRNA comprises at least 10%modified RNA nucleotides and/or modified nucleosides.
The composition of paragraph [00203] , wherein at least one of the modified RNA nucleotide and/or modified nucleoside is m5C (5-methylcytidine) , m5U (5-methyluridine) , m6A (N6-methyladenosine) , Y (pseudouridine) , or m1A (1-methyladenosine) .
The composition of any one of paragraphs [00202] to [00204] , wherein at least one of the modified RNA nucleotide and/or modified nucleoside is introduced at in vitro transcription (IVT) .
The composition of any one of paragraphs [00192] to [00205] , wherein the circRNA is encapsulated in a lipid nanoparticle (LNP) .
The composition of paragraph [00206] , wherein the LNP comprises MC3, ALC-0315 or SM-102.
The composition of paragraph [00206] , wherein the LNP comprises MC3, DSPC, Cholesterol, and PEG-2000 at molar ratio of 50: 10: 38.5: 1.5.
The composition of paragraph [00206] , wherein the LNP comprises SM-102, DSPC, Cholesterol, and PEG-2000 at molar ratio of 50: 10: 38.5: 1.5.
The composition of paragraph [00206] , wherein the LNP comprises ALC-0315, DSPC, Cholesterol, and ALC-0159 .
The composition of any one of paragraphs [00192] to [00205] wherein the composition further comprise a buffer, preferably a citrate saline buffer, a phosphate-buffered saline (PBS) buffer, or a tromethamine (THAM) buffer.
The composition of paragraph [00211] , wherein the buffer is substantially free of divalent cations.
The composition of paragraph [00212] , wherein said buffer substantially free of divalent cations is a citrate saline buffer.
The composition of paragraph [00213] wherein the citrate saline buffer is substantially free of calcium and magnesium.
The composition of paragraph [00213] , wherein the citrate saline buffer contains no calcium or magnesium.
The composition of paragraph [00212] , further comprising a pharmaceutically acceptable excipient.
The composition of paragraph [00216] , wherein the pharmaceutically acceptable excipient is selected from the group consisting of a solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, core-shell nanoparticles, polymer, peptide, protein, cell, hyaluronidase, and mixtures thereof.
A method for treating a subject suffering from a disease responsive to VEGF-A therapy, in a subject comprising administering to the subject a therapeutically effective amount of the composition of any one of paragraphs [00192] to [00211] .
The method of paragraph [00218] , wherein the buffer that is substantially free of divalent cations in said composition or formulation is a citrate saline buffer.
The method of paragraph [00219] , wherein the citrate saline buffer is substantially free of calcium and magnesium.
The method of paragraph [00219] , wherein the citrate saline buffer contains no calcium or magnesium.
The method of paragraph [00218] , wherein the composition further comprises a pharmaceutically acceptable excipient.
The method of paragraph [00222] , wherein the pharmaceutically acceptable excipient is selected from the group consisting of a solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, core-shell nanoparticles, polymer, peptide, protein, cell, hyaluronidase, and mixtures thereof.
The method of paragraph [00218] , wherein the disease is selected from the group consisting of heart failure with reduced or preserved ejection fraction, kidney disease, a disease involving skin grafting and tissue grafting, post-MI cardiac dysfunction, ischemic heart disease, a vascular injury from trauma or surgery, a skin ulcer including a diabetic ulcer, diabetic foot, critical limb ischemia, lower limb ischemia, pulmonary hypertension, stable severe angina pectoris, refractory angina, congenital and acquired maxillofacial defects, alveolar bone atrophy, ligament or tendon lesions, ocular disease, and peripheral arterial disease.
The method of paragraph [00218] , wherein the disease is heart failure with reduced or preserved ejection fraction.
The method of paragraph [00218] , wherein the disease is post-MI cardiac dysfunction.
The method of paragraph [00218] , wherein the disease is ischemic heart disease.
The method of paragraph [00218] wherein the disease is a vascular injury from trauma or surgery.
The method of paragraph [00218] , wherein the disease is a skin ulcer including a diabetic ulcer.
The method of paragraph [00218] , wherein the disease is diabetic foot.
The method of paragraph [00218] , wherein the disease is critical limb ischemia.
The method of paragraph [00218] , wherein the disease is lower limb ischemia.
The method of paragraph [00218] , wherein the disease is pulmonary hypertension.
The method of paragraph [00218] , wherein the disease is stable severe angina pectoris.
The method of paragraph [00218] , wherein the disease is refractory angina.
The method of paragraph [00218] , wherein the disease is maxillofacial defects.
The method of paragraph [00236] , wherein the disease is congenital or acquired maxillofacial defects.
The method of paragraph [00218] , wherein the disease is alveolar bone atrophy.
The method of paragraph [00218] , wherein the disease is ligament or tendon lesions.
The method of paragraph [00218] , wherein the disease is ocular disease.
The method of paragraph [00218] , wherein the disease is peripheral arterial disease.
The method of paragraph [00218] , wherein the composition is administered to the subject via intramuscular, intradermal, subcutaneous, intracranial, intrathecal, vitreous, and subretinal, muscular, cardiac, intracardiac, or epicardiac route, through a portal vein catheter, through a coronary sinus catheter, and/or by direct administration into the area to be treated.
The method of paragraph [00218] , wherein the composition is delivered to the subject via microneedles, hydrogels, or sprays.
The method of paragraph [00218] , wherein the composition is administered to the subject intramuscularly.
The method of paragraph [00218] , wherein the composition is administered to the subject intradermally.
The method of paragraph [00218] , wherein the composition is administered to the subject subcutaneously.
The method of paragraph [00218] , wherein the composition is administered to the subject intracranially.
The method of paragraph [00218] , wherein the composition is administered to the subject intrathecally.
The method of paragraph [00218] , wherein the composition is administered to the subject vitreously.
The method of paragraph [00218] , wherein the composition is administered to the subject subretinally.
The method of paragraph [00218] , wherein the composition is administered to the subject muscularly.
The method of paragraph [00218] , wherein the composition is administered to the subject cardiacly.
The method of paragraph [00218] , wherein the composition is administered to the subject intracardially or epicardially, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [00218] , wherein the composition is administered to the subject through a portal vein catheter, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [00218] , wherein the composition is administered to the subject through a coronary sinus catheter, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [00218] , wherein the composition is administered to the subject by direct administration into the area to be treated, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [00218] , wherein the composition is suitable for a naked formulation .
The method of paragraph [00257] , wherein the naked formulation is administered to the subject via intradermal, subcutaneous, intramuscular, intracranial, intrathecal, vitreous, muscular, cardiac, and subretinal.
A method for modulating a physiological process in a mammalian cell, tissue, or subject comprising contacting said mammalian cell, tissue, or subject with the composition of any one of paragraphs [00192] to [00211] .
The method of paragraph [00259] , wherein the modulating comprises inducing angiogenesis, stimulating vascular cell proliferation, inducing the proliferation and migration of vascular endothelial cells, increasing proliferation and/or altering the fate of epicardial derived progenitor cells, upregulating endothelialization, inducing cardiac regeneration, increasing revascularization of tissue grafts for wound healing, improving vascular function, increasing tissue perfusion and new vessel formation, reducing scar tissue, promoting stent biofunctionality, inducing lymphangiogenesis, inducing bone repair, or improving cardiac function, or any combination thereof.
The method of paragraph [00259] , wherein the modulating comprises inducing angiogenesis.
The method of paragraph [00259] , wherein the modulating comprises stimulating vascular cell proliferation.
The method of paragraph [00259] , wherein the modulating comprises increasing proliferation and/or altering the fate of epicardial derived progenitor cells.
The method of paragraph [00259] , wherein the modulating comprises upregulating endothelialization.
The method of paragraph [00259] , wherein the modulating comprises inducing cardiac regeneration.
The method of paragraph [00259] , wherein the modulating comprises increasing revascularization of tissue grafts for wound healing.
The method of paragraph [00259] , wherein the modulating comprises improving vascular function.
The method of paragraph [00259] , wherein the modulating comprises increasing tissue perfusion and new vessel formation.
The method of paragraph [00259] , wherein the modulating comprises reducing scar tissue.
The method of paragraph [00259] , wherein the modulating comprises promoting stent biofunctionality.
The method of paragraph [00259] , wherein the modulating comprises inducing lymphangiogenesis.
The method of paragraph [00259] , wherein the modulating comprises inducing bone repair.
The method of paragraph [00259] , wherein the modulating comprises improving cardiac function.
The method of paragraph [00259] , wherein the buffer that is substantially free of divalent cations in said composition is a citrate saline buffer.
The method of paragraph [00274] , wherein the citrate saline buffer is substantially free of calcium and magnesium.
The method of paragraph [00274] , wherein the citrate saline buffer contains no calcium or magnesium.
A method for expressing VEGF-A in a mammalian cell or tissue, comprising contacting said mammalian cell or tissue with the composition of any one of paragraphs [00192] to [00211] .
The method of paragraph [00277] , wherein the buffer that is substantially free of divalent cations in said composition or formulation is a citrate saline buffer.
The method of paragraph [00278] , wherein the citrate saline buffer is substantially free of calcium and magnesium.
The method of paragraph [00278] , wherein the citrate saline buffer contains no calcium or magnesium.
The method of paragraph [00278] , wherein the citrate saline buffer has 10 mM citric acid and 130 M sodium chloride, with a pH range 6-7.5.
The method of any one of paragraphs [00277] to [00281] , wherein the method expresses VEGF-A in heart cell or heart.
A method of producing VEGF-A in a subject, comprising administering to said subject the composition of any one of paragraphs [00192] to [00211] .
The method of paragraph [00283] , wherein the buffer that is substantially free of divalent cations in said composition is a citrate saline buffer.
The method of paragraph [00284] , wherein the citrate saline buffer is substantially free of calcium and magnesium.
The method of paragraph [00284] , wherein the citrate saline buffer contains no calcium or magnesium.
The method of paragraph [00283] , wherein the buffer is suitable for naked formulation.
The method of paragraph [00287] , wherein the buffer is a sodium chloride solution or Ringer's solution containing NaCl, KCl, CaCl2, NaHCO3, and/or MgCl2.
The method of paragraph [00288] , wherein the concentration of Ca2+ is in a range of 0.75 mM-1.5 mM.
The method of paragraph [00287] , wherein the buffer is a 10 mM citric acid buffer solution and 130 M sodium chloride solution, with a pH range of 6-7.5.
The method of paragraph [00287] , wherein the buffer is phosphate buffer and/or acetate buffer.
The method of paragraph [00287] wherein the buffer further comprises glucose, sucrose, and/or trehalose.
The method of paragraphs [00288] to [00292] , wherein a naked RNA formulation comprises buffer and one or more circRNA (s) .
The method of paragraph [00293] , wherein the concentration of circRNA (s) is in a range of 500 ng/μL-2000 ng/μL.
The method of paragraph [00283] , wherein the composition further comprises a pharmaceutically acceptable excipient.
The method of paragraph [00295] , wherein the pharmaceutically acceptable excipient is selected from the group consisting of a solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, core-shell nanoparticles, polymer, peptide, protein, cell, hyaluronidase, and mixtures thereof.
The method of paragraph [00283] , wherein the subject is suffering from a disease responsive to VEGF-A therapy.
The method of paragraph [00297] , wherein the disease is selected from the group consisting of heart failure with reduced or preserved ejection fraction, kidney disease, a disease involving skin grafting and tissue grafting post-MI cardiac dysfunction, ischemic heart disease, a vascular injury from trauma or surgery, a skin ulcer including a diabetic ulcer, diabetic foot, critical limb ischemia, lower limb ischemia, pulmonary hypertension, stable severe angina pectoris, refractory angina, congenital and acquired maxillofacial defects, alveolar bone atrophy, ligament or tendon lesions, ocular disease, and peripheral arterial disease.
The method of paragraph [00297] , wherein the disease is heart failure with reduced or preserved ejection fraction.
The method of paragraph [00297] , wherein the disease is post-MI cardiac dysfunction.
The method of paragraph [00297] , wherein the disease is ischemic heart disease.
The method of paragraph [00297] , wherein the disease is a vascular injury from trauma or surgery.
The method of paragraph [00297] , wherein the disease is a skin ulcer including a diabetic ulcer.
The method of paragraph [00297] , wherein the disease is diabetic foot.
The method of paragraph [00297] , wherein the disease is critical limb ischemia.
The method of paragraph [00297] , wherein the disease is lower limb ischemia.
The method of paragraph [00297] , wherein the disease is pulmonary hypertension.
The method of paragraph [00297] , wherein the disease is stable severe angina pectoris.
The method of paragraph [00297] , wherein the disease is refractory angina.
The method of paragraph [00297] , wherein the disease is maxillofacial defects.
The method of paragraph [00310] , wherein the disease is congenital or acquired maxillofacial defects.
The method of paragraph [00297] , wherein the disease is alveolar bone atrophy.
The method of paragraph [00297] , wherein the disease is ligament or tendon lesions.
The method of paragraph [00297] , wherein the disease is ocular disease.
The method of paragraph [00297] , wherein the disease is peripheral arterial disease.
The method of paragraph [00283] , wherein the composition is administered to the subject via intramuscular, intradermal, subcutaneous, intracranial, intrathecal, vitreous, and subretinal, muscular, cardiac, intracardiac, or epicardiac route, through a portal vein catheter, through a coronary sinus catheter, and/or by direct administration into the area to be treated.
The method of paragraph [00283] , wherein the composition is delivered to the subject via microneedles, hydrogels, or sprays.
The method of paragraph [00283] , wherein the composition is administered to the subject intramuscularly.
The method of paragraph [00283] , wherein the composition is administered to the subject intradermally.
The method of paragraph [00283] , wherein the composition is administered to the subject subcutaneously.
The method of paragraph [00283] , wherein the composition is administered to the subject intracranially.
The method of paragraph [00283] , wherein the composition is administered to the subject intrathecally.
The method of paragraph [00283] , wherein the composition is administered to the subject vitreously.
The method of paragraph [00283] , wherein the composition is administered to the subject subretinally.
The method of paragraph [00283] , wherein the composition is administered to the subject muscularly.
The method of paragraph [00283] , wherein the composition is administered to the subject cardiacly.
The method of paragraph [00283] , wherein the composition is administered to the subject intracardially or epicardially, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [00283] , wherein the composition is administered to the subject through a portal vein catheter, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [00283] , wherein the composition is administered to the subject through a coronary sinus catheter, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [00283] , wherein the composition is administered to the subject by direct administration into the area to be treated, preferably at a fixed-dosage in multiple administrations.
The method of paragraph [00283] , wherein the composition is suitable for a naked formulation.
The method of paragraph [00331] , wherein the naked formulation is administered to the subject via intradermal, subcutaneous, intramuscular, intracranial, intrathecal, vitreous, muscular, cardiac, and subretinal.
A method for increasing wound healing in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition of any one of paragraphs
to [00211] .
A method for inducing neovascularization in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition of any one of paragraphs [00192] to [00211] .
A method for inducing angiogenesis in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition of any one of paragraphs
to [00211] .
A method for inducing vasodilation in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition of any one of paragraphs
to [00211] .
A method for inducing blood flow upregulation in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition of any one of paragraphs [00192] to [00211] .
A method for increasing capillary and/or arteriole density in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition of any one of paragraphs [00192] to [00211] .
A method for attenuating fibrosis in a mammalian tissue or a subject comprising contacting said mammalian tissue or subject with the composition of any one of paragraphs
to [00211] .
The method of paragraph [00218] wherein the subject is a human.
The present disclosure also encompasses the delivery of naked one or more circRNA molecules or one or more circRNA complexes, and/or pharmaceutical, prophylactic, or diagnostic formulations thereof by any appropriate route taking into consideration likely advances in the sciences of drug delivery.
As non-limiting examples, in some embodiments, formulations in accordance with the present disclosure are administered to the subject via intramuscular, intradermal, subcutaneous, intracardiac or epicardiac route, through a portal vein catheter, through a coronary sinus catheter, and/or by direct administration into the area to be treated. In some embodiments, the formulation is administered to the subject intramuscularly. In some embodiments, the formulation is administered to the subject intradermally. In some embodiments, the formulation is administered to the subject subcutaneously.
In some embodiments, the composition is administered to the subject intracardially, preferably at a fixed-dosage in multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) administrations. In some embodiments, the composition is administered to the subject through a portal vein catheter, preferably at a fixed-dosage in multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) administrations. In some embodiments, the composition is administered to the subject through a coronary sinus catheter, preferably at a fixed-dosage in multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) administrations, In some embodiments, the composition is administered to the subject by direct administration into the area to be treated, preferably at a fixed-dosage in multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) administrations. For example, the composition is administered to the subject by direct injection to the damaged area during open heart surgery, preferably at a fixed-dosage in multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) administrations, In some embodiments, the composition is administered to the subject epicardially, preferably at a fixed-dosage in multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) administrations. For example, in patients undergoing coronary artery by-pass grafting (CABG) , the composition is administered to the patient from the external side of heart, preferably at a fixed-dosage in multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) administrations. In each of the embodiments in this paragraph, the "multiple administrations" can be separated from each other by short (l-5 mins) , medium (6-30 minutes) , or long (more than 30 minutes, hours, or even days) intervals of time.
In certain embodiments, compositions in accordance with the present disclosure are administered to a subject, wherein the formulations comprise a lipid-based complex (such as liposomes, lipoplexes, and lipid nanoparticles) . In other embodiments, naked one or more circRNA is administered in phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer. In other embodiments, naked one or more circRNA is administered in the absence of divalent cations, including calcium. In preferred embodiments, compositions for intracardiac or intradermal administration comprising the one or more circRNA are formulated in citrate saline buffer containing no calcium or magnesium. In some embodiments, the composition comprises a concentration of the one or more circRNA formulated in citrate saline buffer of between 0.1 and 1 μg/μL. In some embodiments, the composition comprises a concentration of the one or more circRNA formulated in citrate saline buffer of between l and 10 μg/μL. In some embodiments, the composition comprises a concentration of the one or more circ RNA formulated in citrate saline buffer of between 10 and 50 μg/μL.
In certain embodiments, compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about (0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0. l mg/kg to about 10 mg/kg, or from about l mg/kg to about 25 mg/kg, of one or more circRNA per subject body weight per day, one or more times a day, to obtain the desired therapeutic or prophylactic effect. The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, every four weeks or once as a single dose, either in a bolus dose or in multiple administrations over a period of second, minutes or hours in a 24 hour period. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more administrations) .
In some embodiments, compositions in accordance with the present disclosure may be administered at fixed-dosage levels. For example, compositions in accordance with the present disclosure may be administered at fixed-dosage levels from about 0.1 mg to about 1 mg, either per administration or per total dose. In some embodiments, compositions in accordance with the present disclosure may be administered at fixed-dosage levels from about 1 mg to about 10 mg, either per administration or per total dose. In some embodiments, compositions in accordance with the present disclosure may be administered at fixed-dosage levels from about 10 mg to about 25 mg, either per administration or per total dose. In some embodiments, compositions in accordance with the present disclosure may be administered at fixed-dosage levels from about 25 mg to about 50 mg, either per administration or per total dose. In some embodiments, compositions in accordance with the present disclosure may be administered at fixed-dosage levels from about 50 mg to about 100 mg, either per administration or per total dose. In some embodiments, compositions in accordance with the present disclosure may be administered at fixed-dosage levels from about 0.1 to about 25 mg, either per administration or per total dose. In some embodiments, compositions in accordance with the present disclosure may be administered at a fixed-dosage, preferably in multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) administrations. For example, in some embodiments, compositions in accordance with the present disclosure may be administered at 0.1 mg fixed-dosage, either per administration or per total dose, preferably in multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) administrations. In some embodiments, compositions in accordance with the present disclosure may be administered at 1 mg fixed-dosage, either per administration or per total dose, preferably in multiple {e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) administrations. In some embodiments, compositions in accordance with the present disclosure may be administered at 10 mg fixed-dosage, either per administration or per total dose, preferably in multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) administrations. In some embodiments, compositions in accordance with the present disclosure may be administered at 25 mg fixed-dosage, either per administration or per total dose, preferably in multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) administrations, in some embodiments, compositions in accordance with the present disclosure may be administered at 50 mg fixed-dosage, either per administration or per total dose, preferably in multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) administrations. In some embodiments, compositions in accordance with the present disclosure may be administered at 100 mg fixed-dosage, either per administration or per total dose, preferably in multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) administrations.
7. Brief Description of DrawingsFIG. 1 provides a circRNA which comprises, in the following order: 3’ intron fragment, TI, VEGF-A, 5’ intron fragment.
FIG. 2 provides a circRNA which comprises, in the following order: 3’ intron fragment, VEGF-A, TI, 5’ intron fragment .
FIG. 3 provides a circRNA which comprises, in the following order: 3’ intron fragment, L2, L1, TI, VEGF-A, 5’ intron fragment.
FIG. 4 provides a circRNA which comprises, in the following order: 3’ intron fragment, VEGF-A, L1, L2, TI, 5’ intron fragment.
FIG. 5 provides the dose-dependent expression of VEGF-A in the tested concentration range (25-400 ng) , which was observed in cardiomyocytes and cardiovascular cells across different species in human, rodent, and pig.
FIG. 6 provides the time-course expression of VEGF-A, which was studied in cardiomyocytes and cardiovascular cells across different species in human, rodent, and pig, and the expression of VEGF-A that can be sustained for more than 5 days across all cardiomyocytes.
FIG. 7 provides the test article and control solutions were then incubated for 6 hours in human VEGFR2 (Luc) HEK293 reporter cells, in which huVEGF-A binds to huVEGFR2 on the membrane of reporter cells and activates the downstream NFAT-Luciferase pathway.
FIG. 8 provides the activating Effect of A00A Expression Products in Cells of Different Species on HEK293-huVEGFR2-Luci Reporter Cells.
FIG. 9 provides the activation of VEGFR2 via A00A Expression Products in Cardiac Cells and fibroblast.
FIG. 10A provides the in vivo levels of VEGF-A circRNA, which is dose dependent.
FIG. 10B provides the in vivo levels of VEGF-A circRNA protein expression, which is dose dependent.
FIG. 11 provides the expression of VEGF-A circRNA and VEGF-A mRNA in mouse myocardial tissue is at the same level and dose-dependent.
FIG. 12 provides the comparison between the protein expression of VEGF-A circRNA and VEGF-A mRNA.
FIG. 13 provides a 7-day acclimation period when animals were entered the facility. The animals were randomly divided into 4 groups according to the EF and body weight during the adaptation period.
FIG. 14 provides the values of body weight in each group.
FIG. 15 provides the changes of body weight in each group.
FIG. 16 provides the EF%values of each group during the 28 days.
FIG. 17 shows the changes of EF values of each group during the 28 days.
FIG. 18 provides the FS%values of each group during the 28 days.
FIG. 19 shows the changes of FS%values of each group during the 28 days.
FIG. 20 provides the LVEDV%values of each group during the 28 days.
FIG. 21 shows the changes of LVEDV%values of each group during the 28 days.
FIG. 22 provides the LVESV%values of each group during the 28 days.
FIG. 23 shows the changes of LVESV%values of each group during the 28 days.
FIG. 24 provides the results of MI weight and ratio of MI in each group.
FIG. 25 shows the myocardial infarction rate of each group to illustrate the comparison.
FIG. 26 provides the TTC staining of myocardial infarction area.
FIG. 27 provides the values of myocardial fibrosis (%) of each group.
FIG. 28 shows the values of myocardial fibrosis (%) of each group to demonstrate the comparison.
FIG. 29 provides the evaluation of myocardial fibrosis by Masson staining.
FIG. 30 provides the relative changes (vs Day0) of EF (left) and FS (right) at different time points
FIG. 31 provides the VEGF-A circRNA expression when delivered by DLin-MC3-DMA, having of the original nitrogen-to-phosphorus ratio (N/P ratio) of DLin-MC3-DMA (referred to as MC3) : MC3_6/1 and the optimized N/P ratio: MC3_3/1 in 293T cells and immortalized human cardiomyocytes (IHC-SV40) , at gradient doses of 25 ng, 50 ng, 100 ng, and 200 ng per well in 1E4 cells.
FIG. 32 provides the dose-dependent trends of the VEGF-A circRNA expression, having of the original nitrogen-to-phosphorus ratio (N/P ratio) of DLin-MC3-DMA (referred to as MC3) : MC3_6/1 and the optimized N/P ratio: MC3_3/1 in 293T cells and immortalized human cardiomyocytes (IHC-SV40)
As provided in detail below, in the description of drawings above, TI refers to a translation initiation sequence. VEGF-A refers to a sequence encoding a VEGF-A protein. L refers to a linker sequence; L1 and L2 are each an independent linker sequence.
8. Detailed DescriptionProvided herein are immunogenic compositions comprising a circular ribonucleotide acid (circRNA) encoding VEGF polypeptides. In some embodiments, the immunogenic compositions provided herein comprise a circRNA comprising a translation initiation (TI) sequence and a protein-coding (Z) sequence, and wherein the Z sequence comprises a VEGF-Asequence encoding a VEGF-A polypeptide sequence (VF) . Methods of manufacture and methods of uses thereof are also provided herein.
8.1 Definitions
Unless otherwise defined herein, scientific and technical terms used in the present disclosures shall have meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.
As used herein in the specification, “a” or “an” may mean one or more. As used herein in the claim (s) , when used in conjunction with the word “comprising, ” the words “a” or “an” may mean one or more than one.
As used herein, the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or. ” As used herein “another” or “additional” may mean at least a second or more.
As used herein, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. The term “about” encompasses the exact number recited. In some embodiments, “about” means within plus or minus 10%of a given value or range. In certain embodiments, “about” means that the variation is ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1%of the value to which “about” refers. In some embodiments, “about” means that the variation is ±1%, ±0.5%, ±0.2%, or ±0.1%of the value to which “about” refers.
As used herein, “essentially free, ” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.1%, preferably below 0.05%, and more preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
The terms “peptide, ” “polypeptide” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids comprising at least two or more contiguous amino acids chemically or biochemically modified or derivatized amino acids. The term “peptide” as used herein refers to a class of short polypeptides. The term peptide may refer to a polymer of amino acids (natural or non-naturally occurring) having a length of up to about 100 amino acids. For example, peptides may be about 1 to about 10, about 10 to about 25, about 25 to about 50, about 50 to about 75, about 75 to about 100 amino acid residues in length. In some embodiments, the peptides may be about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1250, about 1500, about 1750, about 2000, about 2250, about 2500, about 2750, about 3000, about 3250, about 3500, about 3750, about 4000, about 4250, about 4500, about 4750, are about 5000 amino acid residues in length.
The terms “nucleic acid, ” “polynucleotide, ” and “oligonucleotide” are used interchangeably herein and refer to a polymer or oligomer of nucleotides of any length. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases (such as methylated, hydroxymethylated, or glycosylated) , non-natural nucleotides, non-nucleotide building blocks that exhibit similar structure and/or function as natural nucleotides (i.e., “nucleotide analogs” ) , and/or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. The nucleic acids or polynucleotides can be heterogenous or homogenous in composition, can be isolated from naturally occurring sources, or can be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and can exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. Nucleic acid structures also include, for instance, a DNA/RNA helix, peptide nucleic acid (PNA) , morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 4 (14) : 4503-4510 (2002) and U.S. Patent 5,034,506) , locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000) ) , cyclohexenyl nucleic acids (see Wang, Am. Chem. Soc., 122: 8595-8602 (2000) ) , and/or a ribozyme.
As is understood in the art, a nucleic acid strand is inherently directional, as the carbon atoms in the sugar ring are numbered from 1’ to 5’ and the “5’-end” has a free hydroxyl (or phosphate) on a 5’ carbon and the “3’ prime end” has a free hydroxyl (or phosphate) on a 3’ carbon. As used herein and understood in the art, a nucleic acid having certain sequence elements “from 5’ to 3’ ” means that these sequence elements are arranged linearly from the 5’ end to the 3’ end of the nucleic acid.
When referring to a nucleotide sequence or protein sequence, the term “identity” is used to denote similarity between two sequences. Sequence similarity or identity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981) , by the sequence identity alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48, 443 (1970) , by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988) , by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI) , the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12, 387-395 (1984) , or by inspection. Another algorithm is the BLAST algorithm, described in Altschul et al., J Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993) . A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Methods in Enzymology, 266, 460-480 (1996) ; blast. wustl/edu/blast/README. html. WU-BLAST-2 uses several search parameters, which are optionally set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. Further, an additional useful algorithm is gapped BLAST as reported by Altschul et al., (1997) Nucleic Acids Res. 25, 3389-3402. Unless otherwise indicated, percent identity is determined herein using the algorithm available at the internet address: blast. ncbi. nlm. nih. gov/Blast. cgi.
As used herein, terms “complementary” and “complementarity” refers to the relationship between two nucleic acid molecules having the capacity to form hydrogen bond (s) with one another by either traditional Watson-Crick base-paring or other non-traditional types of pairing. The two DNA/RNA strands with complementary sequences bind to form a duplex that follows the Watson–Crick base-pairing rules: A binds to T (U) with two hydrogen bonds; G binds to C with three hydrogen bonds. The degree of complementarity between two nucleotide sequences can be indicated by the percentage of nucleotides in a nucleotide sequence which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleotide sequence (e.g., about 50%, about 60%, about 70%, about 80%, about 90%, and 100%complementary) . Two nucleotide sequences are “perfectly complementary” or “100%complementary” if all the contiguous nucleotides of a nucleotide sequence will hydrogen bond with the same number of contiguous nucleotides in a second nucleotide sequence. Two nucleotide sequences are “substantially complementary” if the degree of complementarity between the two nucleotide sequences is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) over a region of at least 8 nucleotides (e.g., at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more nucleotides) , or if the two nucleotide sequences hybridize under at least moderate, or, in some embodiments high, stringency conditions. Exemplary moderate stringency conditions include overnight incubation at 37℃ in a solution comprising 20%formamide, 5%SSC (150 mM NaCl, 15 mM trisodium citrate) , 50 mM sodium phosphate (pH 7.6) , 5x Denhardt’s solution, 10%dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1*SSC at about 37-50℃, or substantially similar conditions, e.g., the moderately stringent conditions described in Sambrook, J., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; 4th edition (June 15, 2012) . High stringency conditions are conditions that use, for example (1) low ionic strength and high temperature for washing, such as 0.015 M sodium chloride/0.0015 M sodium citrate/0.1%sodium dodecyl sulfate (SDS) at 50℃, (2) employ a denaturing agent during hybridization, such as formamide, for example, 50% (v/v) formamide with 0.1%bovine serum albumin (BSA) /0.1%Ficoll/0.1%polyvinylpyrrolidone (PVP) /50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride and 75 mM sodium citrate at 42℃, or (3) employ 50%formamide, 5xSSC (0.75 M NaCl, 0.075 M sodium citrate) , 50 mM sodium phosphate (pH 6.8) , 0.1%sodium pyrophosphate, 5x Denhardt’s solution, sonicated salmon sperm DNA (50 pg/ml) , 0.1%SDS, and 10%dextran sulfate at 42℃, with washes at (i) 42℃ in 0.2*SSC, (ii) 55℃ in 50%formamide, and (iii) 55℃ in 0.1*SSC (optionally in combination with EDTA) . Additional details and an explanation of stringency of hybridization reactions are provided in, e.g., Sambrook, supra, and Ausubel et al., eds., SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 5th ed., John Wiley & Sons, Inc., Hoboken, N.J. (2002) .
The term “operably linked” as used herein and understood in the art with reference to sequence elements in nucleic acid molecules means that these sequence elements (e.g., an intron fragment, a target sequence, a promoter, and a coding sequence) are functionally related to each other. For example, a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation.
The term “hybridization” or “hybridized” when referring to nucleotide sequences is the association formed between and/or among sequences having complementarity.
The term “in vitro transcription, ” or “IVT, ” refers to versatile method to produce RNA in vitro that uses an RNA polymerase, ribonucleotides, and appropriate buffer conditions to synthesize RNA from a DNA template.
The term “expression construct” or “expression cassette, ” as used herein, means a nucleotide sequence that directs translation.
The terms “coding sequence, ” “coding sequence region, ” “coding region, ” and “CDS, ” as used interchangeably here refer to the portion of a nucleic acid (e.g., a DNA or an RNA) that is or can be translated to protein.
The terms “reading frame, ” “open reading frame, ” and “ORF” as used interchangeably herein refer to a nucleotide sequence that begins with an initiation codon (e.g., ATG) and, in some embodiments, ends with a termination codon (e.g., TAA, TAG, or TGA) .
The term “control elements” as used herein refers collectively to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRES) , enhancers, splice junctions, and the like, which collectively provide for the replication, transcription, post-transcriptional processing, and translation of a coding sequence in a recipient cell.
The term “promoter” as used herein refers to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene that is capable of binding to an RNA polymerase and allowing for the initiation of transcription of a downstream (3' direction) coding sequence. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors, to initiate the specific transcription of a nucleic acid sequence. A promoter that is “operatively positioned, ” “operatively linked” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence, which is “under control” and “under transcriptional control” of the promoter.
The term “enhancer” as used herein means a nucleic acid sequence that, when positioned proximate to a promoter, confers increased transcription activity relative to the transcription activity resulting from the promoter in the absence of the enhancer domain.
The terms “internal ribosome entry site” , “internal ribosome entry site sequence” , “IRES” and “IRES sequence region” as used interchangeably herein refer to cis elements of viral or human cellular RNAs (e.g., messenger RNA (mRNA) and/or circRNAs) that bypass the steps of canonical eukaryotic cap-dependent translation initiation. Previous methods for screening IRES and IRES-like sequence are described in PCT Appl. Nos. PCT/CN2020/077026, filed February 27, 2020; PCT/CN2022/095749, filed May 27, 2022; JP issued patent JP7297331B2.
The term “IRES-like sequence” and “Internal Ribosome Entry Site-like sequence, ” as used interchangeably herein refer to non-naturally occurring nucleotide sequences that display a function of a naturally occurring IRES.
The term “5’ intron fragment” and “3’ intron fragment” each refers to a fragment of a group II intron, wherein the 5’ intron fragment is located on the 5’ side of the 3’ intron fragment in the group II intron. The detailed information about group II intron is described in PCT Appl. Nos. PCT/CN2022/095749.
The term “vector” or “construct” (sometimes referred to as a gene delivery system or gene transfer “vehicle” ) refers to a vehicle that is used to carry genetic material (e.g., a nucleotide sequence) , which can be introduced into a host cell, where it can be replicated and/or expressed.
The term “treat” as used herein refers to executing a protocol or plan, which can include administering one or more drugs or active agents to a patient, in an effort to alleviate signs or symptoms of the disease or the recurrence of the disease. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission, increased survival, improved quality of life or improved prognosis. Alleviation or prevention can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. As used herein, a “treatment” does not require complete alleviation of signs or symptoms, and does not require a cure.
As used herein, the term “disease responsive to VEGF-A therapy” refers to a disease or disorder that shows improvement of one or more symptoms or clinical markers after administration of a pharmaceutical composition or a pharmaceutical formulation comprising VEGF-A protein or an agent capable of producing VEGF-A protein, such as the circRNA disclosed herein. Alternatively, a disease is "responsive" to VEGF-A therapy if the progression of the disease is reduced or halted with the administration of a pharmaceutical composition or a pharmaceutical formulation comprising VEGF-A protein or an agent capable of producing VEGF-A protein.
As used herein, the term “therapeutic beneficial” or “therapeutically effective” when used in connection with a therapeutic refers to the property of the therapeutic that promotes or enhances the well-being of the subject. This includes, but is not limited to, a reduction in the frequency, severity, or rate of progression of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or a reduction in the rate of metastasis or recurrence. Treatment of cancer can also refer to prolonging survival of a subject with cancer.
The term "effective amount" or "therapeutically effective amount" as used herein refers to the amount of therapeutic agent (for example, a circRNA) , pharmaceutical composition, or pharmaceutical formulation, sufficient to reduce at least one or more symptom (s) of the disease or disorder, or to provide the desired effect.
As used herein, the term “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. For animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required, e.g., by the FDA Office of Biological Standards.
As used herein, the term “pharmaceutically acceptable carrier” includes any and all aqueous biocompatible solvents (e.g., saline solutions, phosphate buffered saline, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc. ) , antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases) , isotonic agents, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.
The terms “transfection, ” “transformation, ” and “transduction” are used interchangeably herein and refer to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods.
As used herein, the term “subject” as used herein refers to any animal (e.g., a mammal) , including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. A subject can be a human. A subject can have a particular disease or condition.
As used herein, the term “Ringer's solution” refers to a solution of several salts dissolved in water for the purpose of creating an isotonic solution relative to the body fluids of an animal. In some embodiments, the Ringer's solution typically contains NaCl, KCl, CaCl2 and NaHCO3, sometimes with other minerals such as MgCl2, dissolved in distilled water.
As used herein, the term “naked formulation” refers to a pharmaceutical composition in which the active ingredient, such as a circRNA, is delivered in a simple aqueous buffer solution without the inclusion of additional complex delivery systems or encapsulating or protective agents such as lipid-based complexes (e.g., LNPs) ..
As used herein, the term “VEGF-A” refers to vascular endothelial growth factor A.
As used herein, the term “G-CSF” refers to granulocyte colony-stimulating factor, a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream.
As used herein, the term “administer” and its grammatical equivalents refer to the placement of a pharmaceutical composition or formulation comprising, e.g., a circRNA disclosed herein, into a subject by a method or route that results in at least partial localization of the pharmaceutical composition or the pharmaceutical formulation, at a desired site or tissue location. The pharmaceutical composition or the pharmaceutical formulation can be administered by any appropriate route that results in effective treatment in the subject, i.e., administration results in delivery to a desired location or tissue in the subject where at least a portion of the protein expressed by the circRNA is located at a desired target tissue or target cell location.
Nomenclature for nucleotides, nucleic acids, nucleosides, and amino acids used herein is consistent with International Union of Pure and Applied Chemistry (IUPAC) standards (see, e.g., bioinformatics. org/smsylupac. html) . Exemplary genes and polypeptides are described herein with reference to GenBank numbers, GI numbers and/or SEQ ID NOs. It is understood that one skilled in the art can readily identify homologous sequences by reference to sequence sources, including but not limited to Uniprot (https: //www. uniprot. org/) , GenBank (ncbi. nlm. nih. gov/genbank/) and EMBL (embl. org/) .
Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
Before the present disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments set forth herein, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments, and is not intended to be limiting.
8.2 Circular RNAs (circRNAs)
In some embodiments, provided herein are compositions comprising a circRNA, wherein the circRNA comprises, a translation initiation (TI) sequence and a protein-coding (Z) sequence, and wherein the Z sequence comprises a VEGF-A sequence encoding a VEGF-A polypeptide sequence (VF) . Additionally, in some embodiments, the circRNA can have one TI sequence. In some embodiments, the circRNA can have two or more TI sequences. See FIG. 1 to FIG. 4.
8.2.1 Structures with one TI sequence
In some embodiments, circRNAs provided herein comprise a translation initiation (TI) sequence and a protein-coding (Z) sequence, wherein the Z sequence comprises a VEGF-Asequence encoding a VEGF-A polypeptide sequence (VF) .
8.2.2 Structures with two TI sequences
In some embodiments, the circRNAs disclosed herein comprise, in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, and a second protein-coding (Z2) sequence, wherein the Z1 and the Z2 sequences each independently comprises a VEGF sequence, or a G-CSF sequence.
In some embodiments, the circRNAs disclosed herein comprise, in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, and a second protein-coding (Z2) sequence, wherein the Z1 and the Z2 sequences each independently comprises a VEGF111 sequence, VEGF121 sequence, VEGF145 sequence, VEGF165a sequence, VEGF165b sequence, VEGF189 sequence, VEGF206 sequence, or a VEGF-Ax sequence.
In some embodiments, the circRNAs disclosed herein comprise, in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, and a second protein-coding (Z2) sequence, wherein the Z1 and the Z2 sequences each independently comprises a VEGF-A sequence, or a platelet-derived growth factor (PDGF) sequence.
In some embodiments, the circRNAs disclosed herein comprise, in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, and a second protein-coding (Z2) sequence, wherein the Z1 and the Z2 sequences each independently comprises a VEGF-A sequence, or a basic fibroblast growth factor (bFGF) sequence.
In some embodiments, the circRNAs disclosed herein comprise, in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, and a second protein-coding (Z2) sequence, wherein the Z1 and the Z2 sequences each independently comprises a VEGF-A sequence, or an insulin-like growth factor 1 (IGF-1) sequence.
In some embodiments, the circRNAs disclosed herein comprise, in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, and a second protein-coding (Z2) sequence, wherein the Z1 and the Z2 sequences each independently comprises a VEGF-A sequence, or a transforming growth factor β1 (TGF-β1) sequence.
In some embodiments, the circRNAs disclosed herein comprise, in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, and a second protein-coding (Z2) sequence, wherein the Z1 and the Z2 sequences each independently comprises a VEGF-A sequence, or an epidermal growth factor (EGF) sequence.
In some embodiments, the circRNAs disclosed herein comprise, in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, and a second protein-coding (Z2) sequence, wherein the Z1 and the Z2 sequences each independently comprises a VEGF-A sequence, or a growth and differentiation factor (GDF) sequence.
In some embodiments, the circRNAs disclosed herein comprise, in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, and a second protein-coding (Z2) sequence, wherein the Z1 and the Z2 sequences each independently comprises a VEGF-A sequence, or a bone morphogenetic protein (BMP) sequence.
8.2.3 Fusion Proteins
CircRNAs provided herein comprise a protein-coding (Z) sequence. In other words, the encoded proteins can include two or more proteins joined together.
In some embodiments, bacterial protein platforms can be used. Non-limiting examples of these self-assembling proteins include ferritin, lumazine and encapsulin.
Ferritin is a protein whose main function is intracellular iron storage. Ferritin is made of 24 subunits, each composed of a four-alpha-helix bundle, that self-assemble in a quaternary structure with octahedral symmetry (Cho K.J. et al. J Mol Biol. 2009; 390: 83-98) . Several high-resolution structures of ferritin have been determined, confirming that Helicobacter pylori ferritin is made of 24 identical protomers, whereas in animals, there are ferritin light and heavy chains that can assemble alone or combine with different ratios into particles of 24 subunits (Granier T. et al. J Biol Inorg Chem. 2003; 8: 105-111; Fawson D.M. et al. Nature. 1991; 349: 541-544) . Ferritin self-assembles into nanoparticles with robust thermal and chemical stability.
Fumazine synthase (FS) is also well-suited as a nanoparticle platform. FS, which is responsible for the penultimate catalytic step in the biosynthesis of riboflavin, is an enzyme present in a broad variety of organisms, including archaea, bacteria, fungi, plants, and eubacteria (Weber S.E. Flavins and Flavoproteins. Methods and Protocols, Series: Methods in Molecular Biology. 2014) . The FS monomer is 150 amino acids long and consists of beta-sheets along with tandem alpha-helices flanking its sides. A number of different quaternary structures have been reported for FS, illustrating its morphological versatility: from homopentamers up to symmetrical assemblies of 12 pentamers forming capsids of 150 A diameter. Even FS cages of more than 100 subunits have been described (Zhang X. et al. J Mol Biol. 2006; 362: 753-770) .
Encapsulin, a novel protein cage nanoparticle isolated from thermophile Thermotoga maritima. Encapsulin is assembled from 60 copies of identical 31 kDa monomers having a thin and icosahedral T = 1 symmetric cage structure with interior and exterior diameters of 20 and 24 nm, respectively (Sutter M. et al. Nat Struct Mol Biol. 2008, 15: 939-947) . Although the exact function of encapsulin in T. maritima is not clearly understood yet, its crystal structure has been recently solved and its function was postulated as a cellular compartment that encapsulates proteins such as DyP (Dye decolorizing peroxidase) and Flp (Ferritin like protein) , which are involved in oxidative stress responses (Rahmanpour R. et al. FEBS J. 2013, 280: 2097-2104) .
CircRNAs provided herein comprise a protein-coding (Z) sequence. In some embodiments, the Z sequence also encodes a signal peptide. Signal peptides, comprising the N-terminal 15-60 amino acids of proteins, are typically needed for the translocation across the membrane on the secretory pathway and, thus, universally control the entry of most proteins both in eukaryotes and prokaryotes to the secretory pathway. In eukaryotes, the signal peptide of a nascent precursor protein (pre-protein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it for processing. ER processing produces mature proteins, wherein the signal peptide is cleaved from precursor proteins, typically by an ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor. A signal peptide can also facilitate the targeting of the protein to the cell membrane.
A signal peptide can have a length of 15-60 amino acids. For example, a signal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. In some embodiments, a signal peptide has a length of 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15-20 amino acids.
Signal peptides from heterologous genes (which regulate expression of genes other than VEGF in nature) are known in the art and can be tested for desired properties and then incorporated into a nucleic acid of the disclosure.
8.2.4 linkers
In some embodiments, the circRNAs provided herein encode more than one functional domain or peptide linked together as a fusion protein. In some embodiments, the circRNAs provided herein further comprise a linker (L) sequence that encodes a linker (L) located between at least two or each domain of the fusion protein. In some embodiments, circRNAs provided herein comprise more than one VEGF-encoding sequences and/or G-CSF-encoding sequences. In some embodiments, adjacent VEGF-encoding sequences and/or G-CSF-encoding sequences are linked by a linker sequence (L) .
The linker can be, for example, a cleavable linker or protease-sensitive linker. In some embodiments, the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, E2A linker, and combinations thereof. This family of self-cleaving peptide linkers, referred to as 2A peptides, has been described in the art (see for example, Kim, J.H. et al. (2011) PLoS ONE 6: el8556) . In some embodiments, the linker is an F2A linker. In some embodiments, the linker is a GGGS linker. In some embodiments, the fusion protein contains three domains with intervening linkers, having the structure: domain-linker-domain-linker-domain.
Cleavable linkers known in the art can be used in connection with the disclosure. Exemplary linkers include: F2A linkers, T2A linkers, P2A linkers, E2A linkers (See, e.g., WO2017127750) . The skilled artisan will appreciate that other art-recognized linkers may be suitable for use in the constructs of the disclosure (e.g., encoded by the nucleic acids of the disclosure) . The skilled artisan can likewise appreciate that other polycistronic constructs can be suitable for use as provided herein.
In some embodiments, the linker sequence comprises a 5’ UTR, 3’ UTR, poly-A sequence, polyA-C sequence, poly-C sequence, poly-U sequence, poly-G sequence, ribosome binding site, aptamer, riboswitch, ribozyme, small RNA binding site, translation regulation elements (e.g., a Kozak sequence) , a protein binding site (e.g., PTBP1 or HUR) a non-natural nucleotide, or a non-nucleotide chemical-linker sequence.
In some embodiments, the linker sequence comprises a 3’ UTR. In some embodiments, the 3’ UTR can be the 3’ UTR from human beta globin, human alpha globin xenopus beta globin, xenopus alpha globin, human prolactin, human GAP-43, human eEFlal, human Tau, human TNFa, dengue virus, hantavirus small mRNA, bunyavirus small mRNA, turnip yellow mosaic virus, hepatitis C virus, rubella virus, tobacco mosaic virus, human IL-8, human actin, human GAPDH, human tubulin, hibiscus chlorotic linsgspot virus, woodchuck hepatitis virus post translationally regulated element, sindbis virus, turnip crinkle virus, tobacco etch virus, or Venezuelan equine encephalitis virus.
In some embodiments, the linker sequence comprises a 5’ UTR. In some embodiments, the 5’ UTR can be the 5’ UTR from human beta globin, Xenopus laevis beta globin, human alpha globin, Xenopus laevis alpha globin, rubella virus, tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein 70 kDa protein 1A, tobacco alcohol dehydrogenase, tobacco etch virus, turnip crinkle virus, or the adenovirus tripartite leader.
In some embodiments, the linker sequence comprises a polyA region. In some embodiments the polyA region is at least 30 nucleotides or at least 60 nucleotides in length.
8.2.5 Sequence optimization
CircRNAs provided herein comprise a protein-coding (Z) sequence. In some embodiments, the Z sequence can be codon optimized. In some embodiments, the VEGF sequence is codon optimized. Codon optimization methods are known in the art. Codon optimization, in some embodiments, can be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase stability or reduce secondary structures; minimize tandem repeat codons or base runs that can impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites) ; add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art -non-limiting examples include services from GeneArt (Life Technologies) , DNA2.0 (Menlo Park CA) and/or proprietary methods. In some embodiments, the Z sequence is optimized using optimization algorithms. In some embodiments, the VEGF sequence is optimized using optimization algorithms.
8.2.6 Modified nucleosides
In some embodiments, the circRNAs provided herein are not chemically modified and comprise the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some embodiments, the circRNAs provided herein comprise nucleotides and/or nucleosides that are modified as is known in the art. In some embodiments, nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art.
In some embodiments, a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.
In some embodiments, a non-naturally occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos. PCT/US2012/058519; PCT/US2013/075177; PCT/US2014/058897; PCT/US2014/058891; PCT/US2014/070413; PCT/US2015/36773; PCT/US2015/36759; PCT/US2015/36771; or PCT/IB 2017/051367 all of which are incorporated by reference herein.
As such, circRNAs disclosed herein can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof. In some embodiments, circRNAs disclosed herein comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides. In some embodiments, a particular region of a circRNA contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.
In some embodiments, modified circRNAs disclosed herein, introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified circRNAs comprising standard nucleosides.
In some embodiments, modified circRNAs disclosed herein, introduced into a cell or organism, exhibit reduced immunogenicity in the cell or organism (e.g., a reduced innate response) relative to an unmodified circRNA comprising standard nucleosides.
In some embodiments, circRNAs disclosed herein comprise non-natural modified nucleosides that are introduced during synthesis or post-synthesis to achieve desired functions or properties. The modifications can be present on internucleotide linkages, purine or pyrimidine bases, or sugars. The modification can be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain.
In some embodiments, the circRNAs provided herein provided herein have modified RNA nucleotides and/or modified nucleosides. In some embodiments, the modified nucleoside is m5C (5-methylcytidine) . In another embodiment, the modified nucleoside is m5U (5-methyluridine) . In another embodiment, the modified nucleoside is m6A (N6-methyladenosine) . In another embodiment, the modified nucleoside is s2U (2-thiouridine) . In another embodiment, the modified nucleoside is Y (pseudouridine) . In another embodiment, the modified nucleoside is Um (2 '-O-methyluridine) . In other embodiments, the modified nucleoside is m! A (1-methyladenosine) ; m2A (2-methyladenosine) ; Am (2’-0-methyladenosine) ; ms2 m6A (2-methylthio-N6-methyladenosine) ; i6A (N6-isopentenyladenosine) ; ms2i6A (2-methylthio-N6 isopentenyladenosine) ; io6A (N6- (cis-hydroxyisopentenyl) adenosine) ; ms2io6A (2-methylthio-N6- (cis-hydroxyisopentenyl) adenosine) ; g6A (N6-glycinylcarbamoyladenosine) ; t6A (N6-threonylcarbamoyladeno sine) ; ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine) ; m6t6A (N6-methyl-N6-threonylcarbamoyladenosine) ; hn6A (N6-hydroxynorvalylcarbamoyladenosine) ; ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine) ; Ar (p) (2’-0-ribosyladenosine (phosphate) ) ; I (inosine) ; m1I (1-methylinosine) ; m1hn (1, 2’-O-dimethylinosine) ; m3C (3-methylcytidine) ; Cm (2’-0-methylcytidine) ; s2C (2-thiocytidine) ; ac4C (N4-acetylcytidine) ; (5-formylcytidine) ; m5Cm (5 , 2 '-O-dimethylcytidine) ; ac4Cm (N4-acetyl-2’-O-methylcytidine) ; k2C (lysidine) ; m! G (1-methylguanosine) ; m2G (N2-methylguanosine) ; m7G (7-methylguanosine) ; Gm (2'-0-methylguanosine) ; m2 2G (N2, N2-dimethylguanosine) ; m2Gm (N2, 2’-O-dimethylguanosine) ; m2 aGm (N2, N2, 2’-O-trimethylguanosine) ; Gr (p) (2’-0-ribosylguanosine (phosphate) ) ; yW (wybutosine) ; oayW (peroxywybutosine) ; OHyW (hydroxy wybutosine) ; OHyW* (undermodified hydroxywybutosine) ; imG (wyosine) ; mimG (methylwyosine) ; Q (queuosine) ; oQ (epoxyqueuosine) ; galQ (galactosyl-queuosine) ; manQ (mannosyl-queuosine) ; preQo (7-cyano-7-deazaguanosine) ; preQi (7-aminomethyl-7-deazaguanosine) ; G+ (archaeosine) ; D (dihydrouridine) ; m5Um (5, 2’-0-dimethyluridine) ; s4U (4-thiouridine) ; m5s2U (5-methyl-2-thiouridine) ; s2Um (2-thio-2’-0-methyluridine) ; acp3U (3- (3-amino-3-carboxypropyl) uridine) ; ho5U (5-hydroxyuridine) ; mo5U (5-methoxyuridine) ; cmo5U (uridine 5-oxy acetic acid) ; mcmo5U (uridine 5-oxy acetic acid methyl ester) ; chm5U (5- (carboxyhydroxymethyl) uridine) ) ; mchm5U (5- (carboxyhydroxymethyl) uridine methyl ester) ; mcm5U (5-methoxycarbonylmethyluridine) ; mcm5Um (5-methoxycarbonylmethyl-2’-0-methyluridine) ; mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine) ; nm5S2U (5-aminomethyl-2-thiouridine) ; mnm5U (5-methylaminomethyluridine) ; mnm5s2U (5-methylaminomethyl-2-thiouridine) ; mnm5se2U (5-methylaminomethyl-2-selenouridine) ; ncm5U (5-carbamoylmethyluridine) ; ncm5Um (5-carbamoylmethyl-2 '-O-methyluridine) ; cmnm5U (5-carboxymethylaminomethyluridine) ; cmnm5Um (5-carboxymethylaminomethyl-2'-0-methyluridine) ; cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine) ; m6 2A (N6, N6-dimethyladenosine) ; Im (2’-0-methylinosine) ; m4C (N4-methylcytidine) ; m4Cm (N4, 2’-0-dimethylcytidine) ; hm5C (5-hydraxymethylcytidine) ; m3U (3-methyluridine) ; cm5U (5-carboxymethyluridine) ; m6Am (N6, 2’-O-dimethyladenosine) ; m6 2Am (N6, N6, 0-2’-trimethyladenosine) ; m2, 7G (N2, 7-dimethylguanosine) ; m2, 2, 7G (N2, N2, 7-trimethylguanosine) ; m3Um (3, 2’-0-dimethyluridine) ; m5D (5-methyldihydrouridine) ; f5Cm (5-formyl-2’-0-methylcytidine) ; m'Gm (l, 2’-0-dimethylguanosine) ; m'Am (l, 2’-0-dimethyladenosine) ; rm 5U (5-taurinomethyluridine) ; τm5s2U (5-taurinomethyl-2-thiouridine) ) ; imG-14 (4-demethylwyosine) ; imG2 (isowyosine) ; or ac6A (N6-acetyladenosine) .
In some embodiments, the modified nucleoside can include a compound selected from the group of: pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-m ethoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6- (cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6- (cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6, N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2, N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2, N2-dimethyl-6-thio-guanosine. In another embodiment, the modifications are independently selected from the group consisting of 5-methylcytosine, pseudouridine and 1-methylpseudouridine.
The circRNAs of the present disclosure can be partially or fully modified along the entire length of the molecule. The circRNAs of the present disclosure can contain from about 1%to about 100%modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1%to 20%, from 1%to 25%, from 1%to 50%, from 1%to 60%, from 1%to 70%, from 1%to 80%, from 1%to 90%, from 1%to 95%, from 10%to 20%, from 10%to 25%, from 10%to 50%, from 10%to 60%, from 10%to 70%, from 10%to 80%, from 10%to 90%, from 10%to 95%, from 10%to 100%, from 20%to 25%, from 20%to 50%, from 20%to 60%, from 20%to 70%, from 20%to 80%, from 20%to 90%, from 20%to 95%, from 20%to 100%, from 50%to 60%, from 50%to 70%, from 50%to 80%, from 50%to 90%, from 50%to 95%, from 50%to 100%, from 70%to 80%, from 70%to 90%, from 70%to 95%, from 70%to 100%, from 80%to 90%, from 80%to 95%, from 80%to 100%, from 90%to 95%, from 90%to 100%, and from 95%to 100%) . It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.
8.2.7 Preparation of circRNAs
Provided herein are immunogenic compositions comprising circRNA. CircRNAs are single-stranded RNAs that are joined head to tail, which can be prepared using methods known in the art. In some embodiments, methods of circRNA synthesis in vitro comprise ligating the ends of linear RNA precursor to produce a covalently closed circle. Linear RNA can be produced by chemical synthesis or enzymatic strategies (Müller S. (2017) , RNA Biol. 14, 1018-1027; Obi P. (2021) . Methods S1046-2023, 00065-00067) . The advantage of chemical synthesis is that the 5’ monophosphate can be directly introduced during the synthesis process for future cyclization. However, limited by the high cost of purification and low yield, chemical synthesis can only produce RNA of less than 50 to 70 nucleotides in length. Therefore, enzymatic strategy is the primary linear RNA synthesis method at present. Enzymatic strategy is usually realized through an in vitro transcription (IVT) reaction (Beckert B. (2011) . Methods Mol. Biol. 703, 29–41) , which includes DNA template, reaction buffer, and phage RNA polymerase. The phage RNA polymerase usually derives from the T7, SP6, or T3 bacteriophages, and T7 RNA polymerase is the most common phage RNA polymerase. IVT reaction allows for longer RNA synthesis at a lower cost. In some embodiments, mutated phage polymerases with improved transcription quality and reduced side reactions are used.
Circularization is achieved when the linear RNA precursor is ligated in vitro. The ligation methods include chemical ligation, enzymatic ligation, and ribozyme method. In some embodiments, the linear RNA precursors are circularized by chemical ligation using, for example cyanogen bromide (BrCN) or 1-ethyl-3- (3′-dimethylaminopropyl) carbodiimide to link DNA-RNA hybrids (Sokolova et al., (1988) . FEBS Lett. 232, 153–155) .
In some embodiments, the linear RNA precursors are circularized by enzymatic ligations by catalytic reactions of several enzymes from the bacteriophage T4, including T4 DNA ligase (T4 Dnl) , T4 RNA ligase 1 (T4 Rnl 1) , and T4 RNA ligase 2 (T4 Rnl 2) . As a linear RNA precursor needs a 3’-OH on the acceptor substrate and a 5’ monophosphate on the donor substrate for enzymatic ligation, if the linear RNA precursor is produced by chemical synthesis, a 5’ monophosphate can be incorporated during the synthesis or added after the synthesis using ATP and T4 polynucleotide kinase. If the linear RNA precursor is synthesized by IVT reaction, it usually starts with 5’-pppG, and the 5’-terminus needs to be dephosphorylated prior using calf intestinal (CIP) enzyme or other phosphatases. Then, 5’ monophosphate can be added using ATP and T4 polynucleotide kinase. In some embodiments, GMP is added to the IVT reaction mixture for phosphorylating the transcript at its 5’ end. In some embodiments, the linear RNA precursors are circularized by T4 Dnl ligation. T4 Dnl can help ligate double-stranded duplexes, such as DNA/RNA hybrids, and therefore requires a complementary DNA (cDNA) template or bridge to achieve RNA ligation. In some embodiments, the linear RNA precursors are circularized by T4 Rnl 1 ligation. T4 Rnl 1 catalyzes the nucleophilic attack of the 3’-OH terminus onto the activated 5’-terminus to form a covalent 5’, 3’-phosphodiester bond and produces circRNAs. In some embodiments, the linear RNA precursors are circularized by T4 Rnl 2 ligation. Similar to T4 Rnl 1, T4 Rnl 2 also catalyzes the nucleophilic attack of the 3’-OH terminus onto the activated 5’-terminus to form a covalent 5’, 3’-phosphodiester bond. However, T4 Rnl 2 is much more active at joining nicks in double-stranded RNA (dsRNA) than at ligating the ends of ssRNA. T4 Rnl 2 is more suitable for linear RNA precursor with the ligation junction in a double-stranded region.
In some embodiments, the linear RNA precursors are circularized by enzymatic ligations by ribozyme methods. Modified group I intron self-splicing system can be used, which is also called permuted introns and exons (PIE) method (Wesselhoeft et al. (2018) . Nat. Commun. 9, 2629; Rausch et al. (2021) Nucleic Acids Res. 49, E35) . PIE method requires only the addition of GTP and Mg2+ as cofactors and shows great potential for protein synthesis. This method realized RNA ligation through a regular group I intron self-splicing reaction, including two transesterifications at defined splice sites. PIE method can be used for RNA cyclization in vitro and in vivo. Compared to chemical ligation and enzymatic ligation, PIE method could be applied for the cyclization of larger linear RNA precursor, and the reaction condition and purification method of PIE method is simpler.
Group II introns can also be used for circRNA synthesis, which involves an inverse splicing reaction (Mikheeva S. et al., (1997) . Nucleic Acids Res. 25, 5085–94) . This splicing reaction involves the joining of the 5’ splice site at the end of an exon to the 3’ splice site at the beginning of the same exon. Compared to the group I introns, all exon sequences are dispensable for group II intron-catalyzed inverse splicing. Therefore, this method can enable more accurate linear RNA precursor ligation. Methods of preparing circRNAs using Group II introns are described in PCT Application Nos. PCT/CN2022/095749, PCT/CN2022/095949, PCT/CN2022/104527, PCT/CN2022/129435, PCT/CN2022/134803, PCT/CN2022/135585, all hereby incorporated by reference in their entireties.
Hairpin ribozyme (HPR) can produce circRNAs through rolling circle reaction and the self-splicing reaction from circular single-strand DNA template (Dallas et al. (2008) . Nucleic Acids Res. 36, 6752-66; Petkovic and Müller (2013) FEBS Lett. 587, 2435-40) . The linear RNA precursor with HPR will fold into two alternative cleavage-active conformations to remove the 3’-end and the 5’-end. As a result, the intermediate will contain a 5’-OH and a 2’, 3’-cyclic phosphate to produce the target circRNA. In this method, small circRNAs can be produced from long repeating RNAs transcribed by RNA polymerase through a rolling circle mechanism in vitro.
For illustrative purposes, in some embodiments, circRNAs provided herein can be prepared using Group II intron self-splicing as follows. First, vectors containing nucleotide sequences encoding the precursor RNA are designed and cloned, which comprise the following operably linked elements from 5’ to 3’: (1) a 3’ intron fragment; (2) a target sequence consisting of (i) a 3’ target sequence fragment and (ii) a 5’ target sequence fragment, from 5’ to 3’; and (3) a 5’ intron fragment; wherein the RNA has group II intron activity and, upon self-splicing, can form a circRNA that comprises both the 5’ and 3’ target sequence fragments with the 3’-end of the 5’ target sequence fragment linked to the 5’-end of the 3’ target sequence fragment. In some embodiments, the vectors are DNA vectors.
Many vectors can be used, including, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Examples of categories of animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus) , adenovirus, adeno-associated virus (AAV) , herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40) . Examples of expression vectors are pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DESTTM, pLenti6/V5-DESTTM, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. Exemplary AAV serotypes include AAV1, AAV2, AAV4, AAV5, AAV6, AAV9 AAV8, and AAV9. In some embodiments, the vector is an episomal vector or a vector that is maintained extrachromosomally. The vector is engineered to harbor the sequence coding for the origin of DNA replication or “ori” from a lymphotrophic herpes virus or a gamma herpesvirus, an adenovirus, SV40, a bovine papilloma virus, or a yeast, specifically a replication origin of a lymphotrophic herpes virus or a gamma herpesvirus corresponding to oriP of EBV. In some embodiments, the lymphotrophic herpes virus may be Epstein Barr virus (EBV) , Kaposi's sarcoma herpes virus (KSHV) , Herpes virus saimiri (HS) , or Marek's disease virus (MDV) . Epstein Barr virus (EBV) and Kaposi's sarcoma herpes virus (KSHV) are also examples of a gamma herpesvirus.
Additionally, vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. “Expression control sequences, ” “control elements, ” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector-origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, a polyadenylation sequence, 5'and 3'untranslated regions-which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters can be used.
Illustrative ubiquitous expression control sequences that can be used in present disclosure include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus 40 (SV40) promoter (e.g., early or late) , a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, an elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1) , ferritin H (FerH) , ferritin L (FerL) , Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) , eukaryotic translation initiation factor 4A1 (EIF4A1) , heat shock 70kDa protein 5 (HSPA5) , heat shock protein 90kDa beta, member 1 (HSP90B1) , heat shock protein 70kDa (HSP70) , β-kinesin (β-KIN) , the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477 -1482 (2007) ) , a Ubiquitin C promoter (UBC) , a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirus enhancer/chicken β-actin (CAG) promoter, and a β-actin promoter.
Illustrative examples of inducible promoters/systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone) , metallothionine promoter (inducible by treatment with various heavy metals) , MX-1 promoter (inducible by interferon) , the “GeneSwitch” mifepristone-regulatable system (Sirin et al., 2003, Gene, 323: 67) , the cumate inducible gene switch (WO 2002/088346) , tetracycline-dependent regulatory systems, etc.
The vectors provided herein can be made using standard techniques of molecular biology. For example, the various elements of the vectors provided herein can be obtained using recombinant methods, such as by screening cDNA and genomic libraries from cells, or by deriving the polynucleotides from a vector known to include the same. The various elements of the vectors provided herein can also be produced synthetically, rather than cloned, based on the known sequences. The complete sequence can be assembled from overlapping oligonucleotides prepared by standard methods and assembled into the complete sequence. See, e.g., Edge, Nature (1981) 292: 756; Nambair et al., Science (1984) 223 : 1299; and Jay et al., J. Biol. Chem. (1984) 259: 631 1.
Thus, particular nucleotide sequences can be obtained from vectors harboring the desired sequences or synthesized completely, or in part, using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques where appropriate. One method of obtaining nucleotide sequences encoding the desired vector elements is by annealing complementary sets of overlapping synthetic oligonucleotides produced in a conventional, automated polynucleotide synthesizer, followed by ligation with an appropriate DNA ligase and amplification of the ligated nucleotide sequence via PCR. See, e.g., Jayaraman et al., Proc. Natl. Acad. Sci. USA (1991) 88: 4084-4088. Additionally, oligonucleotide-directed synthesis (Jones et al., Nature (1986) 54: 75-82) , oligonucleotide directed mutagenesis of preexisting nucleotide regions (Riechmann et al., Nature (1988) 332: 323-327 and Verhoeyen et al., Science (1988) 239: 1534-1536) , and enzymatic filling-in of gapped oligonucleotides using T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86: 10029-10033) can be used.
Second, the linear RNA precursors can be generated by incubating a vector provided herein under conditions permissive of transcription of the RNAs encoded by the vector. For example, in some embodiments, the linear RNA precursors provided herein can be synthesized by incubating a vector provided herein that comprises an RNA polymerase promoter upstream of its 5’ duplex forming region and/or expression sequence with a compatible RNA polymerase enzyme under conditions permissive of in vitro transcription. In some embodiments, the vector is incubated inside of a cell by a bacteriophage RNA polymerase or in the nucleus of a cell by host RNA polymerase P. In some embodiments, the linear RNA precursors can be generated by performing in vitro transcription using a vector provided herein as a template (e.g., a vector provided herein with an RNA polymerase promoter positioned upstream of the 5’ homology region) .
Third, the linear RNA precursors, which have group II intron activity, can be used to generate circular RNA by self-splicing. In some embodiments, the linear RNA precursors are incubated under conditions suitable for circularization (self-splicing) . Self-splicing of group II introns needs to be accomplished under high-salinity conditions, and does not require the introduction of GTP. As such, in some embodiments, the buffer used in the self-splicing reaction comprises 10 mM to 100 mM, such as 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM divalent magnesium ions, such as MgCl2. The self-splicing buffer can also comprise 10 mM to 100 mM, such as 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM NaCl.
In some embodiments, the self-splicing reaction is performed in vitro for about 5 min to about 1 h, such as about 5 min, about 10 min, about 15 min, about 20 min, about 25 min, about 30 min, about 35 min, about 40 min, about 45 min, about 50 min, about 55 min, and about 1 h.
In some embodiments, the self-splicing reaction is performed at a temperature between 20 and 60 ℃, between 20 and 50 ℃, between 20 and 40 ℃, between 20 and 30 ℃, between 30 and 40 ℃, between 40 and 50 ℃, or between 50 and 60 ℃.
In some embodiments, the precursor RNAs are capable of achieving a circularization rate of at least 30%, such as a circularization rate of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95%.
Fourth, the circRNAs prepared by self-splicing the linear RNA precursors are purified. Purification includes, but is not limited to, the removal of non-circularized linear RNAs, dsRNAs, and other unwanted components. In some embodiments, the circRNAs disclosed herein are purified before being transfected into cells. The phosphate groups at both ends of a linear RNA and some dsRNAs might activate the RIG-1 signaling pathway, and the immune response resulted from RIG-1 signaling can lead to the degradation of exogenous RNAs, thus affecting the function of circular RNAs.
For illustrative purposes, the methods of purification can include any of the following: enzymatic treatment; chromatography, including but not limited to affinity column chromatography, reversed-phase silica gel column liquid chromatography, gel filtration chromatography, and gel exclusion liquid chromatography; and electrophoresis, including but not limited to gel electrophoresis such as agarose gel electrophoresis, and capillary electrophoresis; and any combination thereof. Methods for removing linear RNAs, for example, include enzymatic treatment, such as treatment with RNase R; and chromatography, such as high performance liquid chromatography (HPLC) . Methods for removing terminal phosphate groups, for example, include treatment with alkaline phosphatases, such as calf intestinal alkaline phosphatase (CIP) .
In some embodiments, purification comprises one or more of the following steps: phosphatase treatment, HPLC size exclusion purification, and RNase R digestion. In some embodiments, purification comprises the following steps in order: RNase R digestion, phosphatase treatment, and HPLC size exclusion purification. In some embodiments, purification comprises reverse phase HPLC. In some embodiments, a purified composition contains less double stranded RNA, DNA splints, triphosphorylated RNA, phosphatase proteins, protein ligases, capping enzymes and/or nicked RNA than unpurified RNA.
In some embodiments, the linear RNA precursors provided herein comprise a purification tag that is removed during self-splicing, which can be used for negative selection of the circRNAs. Specifically, a sample containing the circRNAs, such as the product of splicing reaction starting from the tagged linear precursors, can be mixed with a probe that is immobilized on a solid surface, wherein the tag-containing precursors or any other tag-containing impurities can bind to the probe and be removed from the solution, resulting in a circRNA solution substantially free from the precursor, intron and any other tag-containing impurities. In some embodiments, the purification tag is a 15-40 nt polynucleotides. A purification matrix includes, for example, magnetic resin or beads, silicone resin, Sephadex resin, affinity resin, nanoparticles, and nanomaterial surface or coated surfaces.
As such, in some embodiments, the circRNAs prepared by methods disclosed herein can have a functional half-life of at least 5 hours, 10 hours, 15 hours, 20 hours. 30 hours, 40 hours, 50 hours, 60 hours, 70 hours or 80 hours. In some embodiments, the circRNAs provided herein provided herein have a functional half-life of 5-80, 10-70, 15-60, and/or 20-50 hours. In some embodiments, the circRNAs provided herein provided herein have a functional half-life greater than (e.g., at least 1.5-fold greater than, at least 2-fold greater than) that of an equivalent linear RNAs encoding the same protein. In some embodiments, functional half-life can be assessed through the detection of functional protein synthesis.
In some embodiments, the circRNAs provided herein comprise one or more expression sequences and are configured for persistent expression in a cell of a subject in vivo. In some embodiments, the circRNAs is configured such that expression of the one or more expression sequences in the cell at a later time point is equal to or higher than an earlier time point. In such embodiments, the expression of the one or more expression sequences can be either maintained at a relatively stable level or can increase over time. The expression of the expression sequences can be relatively stable for an extended period of time. For instance, in some cases, the expression of the one or more expression sequences in the cell over a time period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days does not decrease by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In some cases, in some cases, the expression of the one or more expression sequences in the cell is maintained at a level that does not vary by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%for at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days.
In some embodiments, the circRNAs provided herein provided herein have a higher magnitude of expression than equivalent linear mRNA, e.g., a higher magnitude of expression 24 hours after administration of RNA to cells. In some embodiments, the circRNAs provided herein provided herein have a higher magnitude of expression than mRNA comprising the same expression sequence, 5moU modifications, an optimized UTR, a cap, and/or a polyA tail. In some embodiments, the circRNAs provided herein provided herein have higher stability than an equivalent linear mRNA. In some embodiments, this can be shown by measuring receptor presence and density in vitro or in vivoi post electroporation, with time points measured over 1 week. In some embodiments, this can be shown by measuring RNA presence via qPCR or ISH.
In some embodiments, the circRNAs disclosed herein can be of any length or size. In some embodiments the circRNA is between 300 and 10000, 400 and 9000, 500 and 8000, 600 and 7000, 700 and 6000, 800 and 5000, 900 and 5000, 1000 and 5000, 1100 and 5000, 1200 and 5000, 1300 and 5000, 1400 and 5000, and/or 1500 and 5000 nucleotides in length.
In some embodiments, the circRNAs disclosed herein can be at least 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, or 5000 nt in length. In some embodiments, the circRNA is no more than 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt in length.
In some embodiments, circRNAs disclosed herein can be about 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt in length.
In some embodiments, circRNAs disclosed herein can be at least 500 nucleotides in length, at least 1000 nucleotides in length, or at least 1500 nucleotides in length.
The practice of the invention employs, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described in the references cited herein and are fully explained in the literature. See, e.g., Maniatis et al. (1982) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989) , MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons (1987 and annual updates) ; CURRENT PROTOCOLS IN IMMUNOLOGY, John Wiley & Sons (1987 and annual updates) Gait (ed. ) (1984) OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, IRL Press; Eckstein (ed. ) (1991) OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, IRL Press; Birren et al. (eds. ) (1999) GENOME ANALYSIS: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Borrebaeck (ed. ) (1995) ; each of which is incorporated herein by reference in its entirety.
8.3 Pharmaceutical Formulations
Provided herein are immunologic compositions for prevention or treatment of diseases in humans and other mammals. In some embodiments, the compositions containing circRNAs as described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject) . In some embodiments, the provided herein are pharmaceutical compositions that can be used as therapeutic or prophylactic agents.
The term “pharmaceutical composition” as used herein refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. A “pharmaceutically acceptable carrier, ” after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be “acceptable” also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in REMINGTON'S PHARMACEUTICAL SCIENCES. Compositions can be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents, such as compositions, can be found, for example, in Remington: THE SCIENCE AND PRACTICE OF PHARMACY 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety) .
The immunogenic compositions provided herein can further include other prophylactic or therapeutic compounds. As a non-limiting example, a prophylactic or therapeutic compound can be an adjuvant or a booster.
The circRNA compositions described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous) .
Provided herein are pharmaceutical compositions including circRNA described herein in combination with one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions provided herein can further comprise other components including, but not limited to, adjuvants. In some embodiments, the pharmaceutical compositions provided herein do not include an adjuvant (they are adjuvant free) .
In some embodiments, circRNA compositions comprise at least one additional active substance, such as, for example, a therapeutically-active substance, a prophylactically-active substance, or a combination of both.
Formulations of the compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., circRNAs) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single-or multi-dose unit. Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition can comprise between 0.1%and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
In some embodiments, a circRNA composition is formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation) ; (4) alter the biodistribution (e.g., target to specific tissues or cell types) ; (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein (e.g., VEGF) in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the RNA (e.g., for transplantation into a subject) , hyaluronidase, nanoparticle mimics and combinations thereof.
A range of carrier systems have been described which encapsulate or complex RNA in order to facilitate RNA delivery and consequent expression of encoded antigens as compared to RNA which is not encapsulated or complexed. The present compositions can utilize any suitable carrier system. Particular carrier systems of note are further described below.
In embodiments, the circRNAs used herein are complexed, encapsulated, partially encapsulated, or associated with one or more lipids (e.g., cationic lipids and/or neutral lipids) , thereby forming lipid-based carriers such as liposomes, LNPs, lipoplexes, and/or nanoliposomes, suitably lipid nanoparticles.
In some embodiments, the circRNAs used herein are formulated in a LNP, either separately or together.
In some embodiments, the circRNAs used herein are formulated separately (in any formulation or complexation agent defined herein) , suitably wherein circRNAs used herein are formulated in separate liposomes, LNPs, lipoplexes, and/or nanoliposomes.
In some embodiments, the circRNAs used herein are co-formulated, i.e., formulated together.
The term “lipid nanoparticle” , also referred to as “LNP” , is not restricted to any particular morphology, and include any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g. in an aqueous environment and/or in the presence of a nucleic acid, e.g. an RNA. For example, a liposome, a lipid complex, a lipoplex and the like are within the scope of a lipid nanoparticle (LNP) .
LNPs are non-virion liposome particles in which RNA can be encapsulated. The incorporation of a nucleic acid into LNPs is also referred to herein as “encapsulation” wherein the nucleic acid, e.g. the RNA is contained within the interior space of the liposomes, LNPs, lipoplexes, and/or nanoliposomes. LNP delivery systems and methods for their preparation are known in the art.
In some embodiments, the circRNAs disclosed herein are complexed with one or more lipids thereby forming LNPs, liposomes, nanoliposomes, lipoplexes, suitably LNPs. In some embodiments, LNPs are suitable for intramuscular and/or intradermal administration.
In embodiments, at least about 80%, 85%, 90%, 95%of lipid-based carriers, suitably the LNPs, have a spherical morphology, suitably comprising a solid core or partially solid core.
LNPs typically comprise a cationic lipid and one or more excipients selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g. PEGylated lipid) . The circRNAs can be encapsulated in the lipid portion of the LNP or an aqueous space enveloped by some or the entire lipid portion of the LNP. The circRNAs or a portion thereof can also be associated and complexed with the LNP. An LNP can comprise any lipid capable of forming a particle to which the nucleic acids are attached, or in which the one or more nucleic acids are encapsulated. In some embodiments, the LNP comprising circRNAs comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and PEGylated lipids.
In some embodiments, the LNP comprises a PEG-modified lipid, a non-cationic lipid, a sterol, and a cationic lipid.
LNP can, for example, be formed of a mixture of (i) a PEG-modified lipid (ii) a noncationic lipid (iii) a sterol (iv) an ionisable cationic lipid. Alternatively, LNP can for example be formed of a mixture of (i) a PEG-modified lipid (ii) a non-cationic lipid (iii) a sterol (iv) a non-ionisable cationic lipid.
In some embodiments, the non-cationic lipid is a neutral lipid. In some embodiments, the cationic lipid is ionizable.
In vivo characteristics and behavior of LNPs can be modified by addition of a hydrophilic polymer coating, e.g. polyethylene glycol (PEG) , to the LNP surface to confer steric stabilization. Furthermore, LNPs (or liposomes, nanoliposomes, lipoplexes) can be used for specific targeting by attaching ligands (e.g. antibodies, peptides, and carbohydrates) to its surface or to the terminal end of the attached PEG chains (e.g. via PEGylated lipids or PEGylated cholesterol) .
In some embodiments, the LNPs comprise a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a PEGylated lipid. The term “PEGylated lipid” or “PEG-modified lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethyleneglycol) -2, 3-dimyristoylglycerol (PEG-s-DMG) and the like. The terms “PEGylated lipid” and “PEG-modified lipid” are used interchangeably herein.
In certain embodiments, the LNP comprises a stabilizing-lipid which is a polyethylene glycol-lipid (PEGylated lipid) . Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g. PEG-CerC14 or PEG-CerC20) , PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N- [ (methoxy poly (ethylene glycol) 2000) carbamyl] -1 , 2-dimyristyloxlpropyl-3-amine (PEG-c-DMA) . In some embodiments, the polyethylene glycol-lipid is PEG-2000-DMG. In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG) . In other embodiments, the LNPs comprise a PEGylated diacylglycerol (PEG-DAG) such as 1- (monomethoxy-polyethyleneglycol) -2, 3-dimyristoylglycerol (PEG-DMG) , a PEGylated phosphatidylethanoloamine (PEG-PE) , a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O- (2’, 3’-di (tetradecanoyloxy) propyl-1-O- (w-methoxy (polyethoxy) ethyl) butanedioate (PEG-5-DMG) , a PEGylated ceramide (PEG-cer) , or a PEG dialkoxypropylcarbamate such as w-methoxy (polyethoxy) ethyl-N- (2, 3di (tetradecanoxy) propyl) carbamate or 2, 3-di (tetradecanoxy) propyl-N- (w-methoxy (polyethoxy) ethyl) carbamate.
In some embodiments, the PEG-modified lipid comprises PEG-DMG or PEG-cDMA. In embodiments, the PEGylated lipid is suitably derived from formula (IV) of published PCT patent application W02018078053A1. Accordingly, PEGylated lipids derived from formula (IV) of published PCT patent application W02018078053A1, and the respective disclosure relating thereto, are herewith incorporated by reference.
Further examples of PEG-lipids suitable in that context are provided in WO2024068545A1, US20150376115A1 and WO2015199952, each of which is incorporated by reference in its entirety.
In some embodiments, LNPs include less than about 3, 2, or 1 mole percent of PEG or PEG-modified lipid, based on the total moles of lipid in the LNP.
In embodiments, the LNP comprises one or more additional lipids, which stabilize the formation of particles during their formulation or during the manufacturing process (e.g. neutral lipid and/or one or more steroid or steroid analogue) .
In embodiments, the circRNA is complexed with one or more lipids thereby forming lipid nanoparticles, wherein the LNP comprises one or more neutral lipid and/or one or more steroid or steroid analogue. Suitable stabilizing lipids include neutral lipids and anionic lipids. The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.
In some embodiments, the non-cationic lipid is a neutral lipid, such as 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) , 1 , 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) , 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) , 1 , 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or sphingomyelin (SM) , preferably the neutral lipid is DSPC.
In embodiments, the LNP (or liposome, nanoliposome, lipoplex) comprises one or more neutral lipids, wherein the neutral lipid is selected from the group comprising distearoylphosphatidylcholine (DSPC) , dioleoylphosphatidylcholine (DOPC) , dipalmitoylphosphatidylcholine (DPPC) , dioleoylphosphatidylglycerol (DOPG) , dipalmitoylphosphatidylglycerol (DPPG) , dioleoyl-phosphatidylethanolamine (DOPE) , palmitoyloleoylphosphatidylcholine (POPC) , palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1 carboxylate (DOPE-mal) , dipalmitoyl phosphatidyl ethanolamine (DPPE) , dimyristoylphosphoethanolamine (DM PE) , distearoyl-phosphatidylethanolamine (DSPE) , 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1-stearioyl-2-oleoylphosphatidyethanol amine (SOPE) , and 1 , 2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE) , or mixtures thereof.
The cationic lipid of an LNP can be ionizable, i.e., it becomes protonated as the pH is lowered below the pK of the ionizable group of the lipid but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.
Such cationic lipids (for liposomes, LNPs, lipoplexes, and/or nanoliposomes) include, but are not limited to, DSDMA, N, N-dioleyl-N, N-dimethylammonium chloride (DODAC) , N, N-distearyl-N, N-dimethylammonium bromide (DDAB) , 1 , 2-dioleoyltrimethyl ammonium propane chloride (DOTAP) (also known as N- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride and 1 , 2-Dioleyloxy-3-trimethylaminopropane chloride salt) , N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTMA) , N, N-dimethyl-2, 3-dioleyloxy) propylamine (DODMA) , ckk-E12, ckk, 1 , 2-DiLinoleyloxy-N, N-dimethylaminopropane (DLinDMA) , 1 , 2-Dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA) , 1 , 2-di-y-linolenyloxy-N, N-dimethylaminopropane (y-DLenDMA) , 98N12-5, 1 , 2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP) , 1 , 2-Dilinoleyoxy-3-(dimethylamino) acetoxypropane (DLin-DAC) , 1, 2-Dilinoleyoxy-3-morpholinopropane (DLin-MA) , 1 , 2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP) , 1 , 2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA) , 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP) , 1 , 2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA. CI) , ICE (Imidazol-based) , HGT5000, HGT5001 , DMDMA, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, XTC (2, 2-Dilinoleyl-4-dimethylaminoethyl- [1 , 3] -dioxolane) HGT4003, 1 , 2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP. CI) , 1 , 2-Dilinoleyloxy-3- (N-methylpiperazino) propane (DLin-MPZ) , or 3- (N, N-Dilinoleylamino) -1 , 2-propanediol (DLinAP) , 3- (N, N-Dioleylamino) -1 , 2-propanedio (DOAP) , 1 , 2-Dilinoleyloxo-3- (2-N, N-dimethylamino) ethoxypropane (DLin-EG-DM A) , 2, 2-Dilinoleyl-4-dimethylaminomethyl- [1 , 3] -dioxolane (DLin-K-DMA) or analogs thereof, (3aR, 5s, 6aS) -N, N-dimethyl-2, 2-di ( (9Z, 12Z) -octadeca-9, 12-dienyl) tetrahydro-3aH-cyclopenta [d] [1 , 3] dioxol-5-amine, (6Z, 9Z, 28Z, 31Z) -heptatriaconta-6, 9, 28, 31-tetraen-19-yl-4- (dimethylamino) butanoate (MC3) , ALNY-100 ( (3aR, 5s, 6aS) -N, N-dimethyl-2, 2-di ( (9Z, 12Z) -octadeca-9, 12-dienyl) tetrahydro-3aH-cyclopenta [d] [1 , 3] dioxol-5-amine) ) , 1 , 1’- (2- (4- (2- ( (2- (bis (2-hydroxydodecyl) amino) ethyl) (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethylazanediyl) didodecan-2-ol (C12-200) , 2, 2-dili noleyl-4- (2-dimethylaminoethyl) - [1 , 3] -dioxolane (DLin-K-C2-DMA) , 2, 2-dilinoleyl-4-dimethylaminomethyl- [1 , 3] -dioxolane (DLin-K-DMA) , NC98-5 (4, 7, 13-tris (3-oxo-3- (undecylamino) propyl) -N , N 16-diundecyl-4, 7, 10, 13-tetraazahexadecane-l, 16-diamide) , (6Z, 9Z, 28Z, 31Z) -heptatriaconta-6, 9, 28, 31-tetraen-19-yl 4- (dimethylamino) butanoate (DLin-M-C3-DMA) , 3- ( (6Z, 9Z, 28Z, 31Z) -heptatriaconta-6, 9, 28, 31-tetraen-19-yloxy) -N, N-dimethylpropan-1 -amine (MC3 Ether) , 4- ( (6Z, 9Z, 28Z, 31Z) -heptatriaconta-6, 9, 28, 31-tetraen-19-yloxy) -N, N-dimethylbutan-1 -amine (MC4 Ether) , (commercially available cationic liposomes comprising DOTMA and 1 , 2-dioleoyl-sn-3phosphoethanolamine (DOPE) , from GIBCO/BRL, Grand Island, N. Y. ) ; (commercially available cationic liposomes comprising N- (1- (2, 3dioleyloxy) propyl) -N- (2- (sperminecarboxamido) ethyl) -N, N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE) , from GIBCO/BRL) ; and (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis. ) or any combination of any of the foregoing. Further suitable cationic lipids for use in the compositions and methods of the invention include those described in international patent publications WO2010053572 (and particularly, Cl 2-200 described at paragraph [00225] ) and WO2012170930, both of which are incorporated herein by reference, HGT4003, HGT5000, HGTS001 , HGT5001 , HGT5002 (see US20150140070A1) .
In embodiments, the cationic lipid of the liposomes, LNPs, lipoplexes, and/or nanoliposomes can be an amino lipid.
Representative amino lipids include, but are not limited to, 1 , 2-dilinoleyoxy-3- (dimethylamino) acetoxypropane (DLin-DAC) , 1 , 2-dilinoleyoxy-3morpholinopropane (DLin-MA) , 1 , 2-dilinoleoyl-3-dimethylaminopropane (DLinDAP) , 1 , 2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA) , 1-linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2-DMAP) , 1 , 2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA. CI) , 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP. CI) , 1 , 2-dilinoleyloxy-3- (N-methylpiperazino) propane (DLin-MPZ) , 3- (N, Ndilinoleylamino) -1 , 2-propanediol (DLinAP) , 3- (N, N-dioleylamino) -1 , 2-propanediol (DOAP) , 1 , 2-dilinoleyloxo-3- (2-N, N-dimethylamino) ethoxypropane (DLin-EG-DMA) , and 2, 2-dilinoleyl-4-dimethylaminomethyl- [1, 3] -dioxolane (DLin-K-DMA) , 2, 2-dilinoleyl-4- (2-dimethylaminoethyl) - [1 , 3] -dioxolane (DLin-KC2-DMA) ; dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) ; MC3 (US20100324120) .
In embodiments, the cationic lipid of the liposomes, LNPs, lipoplexes, and/or nanoliposomes can be an aminoalcohol lipidoid. Aminoalcohol lipidoids may be prepared by the methods described in U.S. Patent No. 8,450,298, herein incorporated by reference in its entirety. Suitable (ionizable) lipids can also be the compounds as disclosed in Tables 1, 2 and 3 and as defined in WO2017075531 A1, hereby incorporated by reference.
Other suitable (cationic or ionizable) lipids are disclosed in W02009086558, W02009127060, WO2010048536, WO2010054406, WO2010088537, WO2010129709, WO2011153493, WO 2013063468, US20110256175, US20120128760, US20120027803, US8158601 , WO2016118724, WO2016118725, W02017070613, W02017070620, WO2017099823, W02012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865, W02008103276, WO2013086373, WO2013086354, US Patent Nos. 7,893,302, 7,404,969, 8,283,333, 8,466,122 and 8,569,256 and US Patent Publication No. US20100036115, US20120202871 , US20130064894, US20130129785, US20130150625, US20130178541 , US20130225836, US20140039032 and WO2017112865. In that context, the disclosures of W02009086558, W02009127060, W02010048536, W02010054406, W02010088537, W02010129709, WO2011153493, WO 2013063468, US20110256175, US20120128760, US20120027803, US8158601 , WO2016118724, WO2016118725, W02017070613, W02017070620, WO2017099823, W02012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865, W02008103276, WO2013086373, WO2013086354, US Patent Nos. 7,893,302, 7,404,969, 8,283,333, 8,466,122 and 8,569,256 and US Patent Publication No. US20100036115, US20120202871 , US20130064894, US20130129785, US20130150625, US20130178541 , US20130225836 and US20140039032 and WO2017112865 specifically relating to (cationic) lipids suitable for LNPs (or liposomes, nanoliposomes, lipoplexes) are incorporated herewith by reference.
In embodiments, amino or cationic lipids as defined herein have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4) , and neutral at a second pH, suitably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of lipids have to be present in the charged or neutral form. Lipids having more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded and may likewise suitable in the context of the present invention. In some embodiments, the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11, e.g., a pKa of about 5 to about 7. LNPs (or liposomes, nanoliposomes, lipoplexes) can comprise two or more (different) cationic lipids as defined herein. Cationic lipids may be selected to contribute to different advantageous properties. For example, cationic lipids that differ in properties such as amine pKa, chemical stability, half-life in circulation, half-life in tissue, net accumulation in tissue, or toxicity can be used in the LNP (or liposomes, nanoliposomes, lipoplexes) . In particular, the cationic lipids can be chosen so that the properties of the mixed-LNP are more desirable than the properties of a single-LNP of individual lipids.
In some embodiments, the LNP compositions can comprise an ionizable cationic lipid, a neutral lipid, a cholesterol, and a PEG. In some embodiments, the ionizable cationic lipid can be MC3, SM-102, ALC-0315, or E-1-0635. In some embodiments, the neutral lipid can be DSPC or DOPE. In some embodiments, the PEG can be DMG-PEG2000 or ALC-0159. The different components can be mixed at different ratios. In some embodiments, the ionizable cationic lipid, neutral lipid, cholesterol, and PEG are mixed at molar ratios of about 50: about 10: about 40: about 2. In some embodiments, the ionizable cationic lipid, neutral lipid, cholesterol, and PEG are mixed at molar ratios of 50±10: 10±2: 40±5: 2±1. In some embodiments, the ionizable cationic lipid, neutral lipid, cholesterol, and PEG are mixed at molar ratios of 50: 10: 38.5: 1.5. In some embodiments, the ionizable cationic lipid, neutral lipid, cholesterol, and PEG are mixed at molar ratios of 46.3: 9.4: 42.7: 1.6. In some embodiments, the ionizable cationic lipid, neutral lipid, cholesterol, and PEG are mixed at molar ratios of 46.3: 12: 39.2: 2.5.
In some embodiments, the following LNP compositions with specific components and ratios are used for preparation of the immunogenic compositions containing circRNAs provided herein. The ratios are molar ratios.
WO2017/070620 provides general information on LNP compositions and is incorporated herein by reference. Other useful LNPs are described in the following references: WO2012/006376; WO2012/030901; WO2012/031046; W02012/031043; WO2012/006378; WO2011/076807; WO2013/033563; WO2013/006825; WO2014/136086; WO2015/095340; WO2015/095346; WO2016/037053, which are also incorporated herein by reference.
Some embodiments relate to a method for preparing a formulation, comprising combining the circRNA in accordance with this disclosure with a lipid-based complex (such as liposomes, lipoplexes, and lipid nanoparticles) , phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer. Some embodiments relate to a method for preparing a formulation, comprising combining the circRNA with citrate saline buffer, wherein the formulation is effective for treating a subject suffering from a disease responsive to VEGF-A therapy; modulating a physiological process in mammalian cell, tissue, or subject; expressing VEGF-A in a mammalian cell or tissue; and/or producing VEGF-A in a subject.
In some embodiments, a method for preparing a formulation comprises combining the circRNA in accordance with this disclosure with citrate saline buffer, wherein the formulation comprises a concentration of the circRNA formulated in citrate saline buffer of between 0, 1 and 10 μg/μL. in some embodiments, the formulation comprises a concentration of the circRNA formulated in citrate saline buffer of between 1 and 10 μg/μL. In some embodiments, the formulation comprises a concentration of the circRNA formulated in citrate saline buffer of between 10 and 50 μg/μL.
In some embodiments, a method for preparing a formulation comprises combining the circRNA with citrate saline buffer in accordance with this disclosure, wherein the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium,
In some embodiments, a method for preparing a formulation comprises combining the circRNA with citrate saline buffer in accordance with this disclosure, wherein the formulation comprises a citrate saline buffer and further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient is chosen from a solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, core-shell nanoparticles, polymer, peptide, protein, cell, hyaluronidase, and mixtures thereof. In some embodiments, the solvent is an aqueous solvent. Formulations can be administered to a subject via a number of routes in accordance with this disclosure. Special formulations suitable for intradermal, intracardiac, subcutaneous, and intramuscular injections are known in the art. For example, in order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from intramuscular injection. Delayed absorption of an intramuscularly administered drug form is accomplished by dissolving or suspending the drug (for example, a circRNA) in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug (for example, a circRNA) in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides) . For intradermal and subcutaneous injections, the formulations are preferably formulated so that they do not induce an immune response when delivered to the intradermal compartment. Additives may be used in the formulations for intradermal injections include for example, wetting agents, emulsifying agents, or pH buffering agents. The formulations for intradermal injections may also contain on or more other excipients such as saccharides and polyols. For intracardiac injections, the formulations are preferably formulated so that they do not cause additional damages to the heart muscle and coronary arteries after the use of an injection needle. Suitable formulations for intracardiac injection are provided herein.
8.4 Administration
Provided herein are immunizing compositions, methods, kits and reagents for prevention and/or treatment of diseases in humans and other mammals. Immunizing compositions can be used as therapeutic or prophylactic agents. In some embodiments, immunizing compositions are used to provide prophylactic protection from diseases. In some embodiments, immunizing compositions are used to treat a disease. In some embodiments, immunizing compositions are used in the priming of immune effector cells, for example, to activate peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re-infused) into a subject.
Thus, an “effective amount” or “therapeutically effective amount” of an immunogenic composition is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the circRNA (e.g., length, nucleotide composition, and/or extent of modified nucleosides) , other components of the composition, and other determinants, such as age, body weight, height, sex and general health of the subject.
The effective amount of the circRNA, as provided herein, can be as low as 50 pg (total RNA) , administered for example as a single dose or as two 25 pg doses. A “dose” as used herein, represents the sum total of circRNA in the composition. In some embodiments, the effective amount is a total dose of 50 pg-300 pg, 100 pg -300 pg, 150 pg -300 pg, 200 pg -300 pg, 250 pg -300 pg, 150 pg -200 pg, 150 pg -250 pg, 150 pg -300 pg, 200 pg -250 pg, or 250 pg -300 pg. For example, the effective amount may be a total dose of 50 pg, 55 pg, 60 pg, 65 pg, 70 pg, 75 pg, 80 pg, 85 pg, 90 pg, 95 pg, 100 pg, 110 pg, 120 pg, 130 pg, 140 pg, 150 pg, 160 pg, 170 pg, 180 pg, 190 pg, 200 pg, 210 pg, 220 pg, 230 pg, 240 pg, 250 pg, 260 pg, 270 pg, 280 pg, 290 pg, or 300 pg. In some embodiments, the effective amount is a total dose of 66 pg. In some embodiments, the effective amount is a total dose of 67 pg. In some embodiments, the effective amount is a total dose of 68 pg. In some embodiments, the effective amount is a total dose of 132 pg. In some embodiments, the effective amount is a total dose of 133 pg. In some embodiments, the effective amount is a total dose of 134 pg. In some embodiments, the effective amount is a total dose of 266 pg. In some embodiments, the effective amount is a total dose of 267 pg. In some embodiments, the effective amount is a total dose of 268 pg. In some embodiments, the effective amount is a total dose of 100 pg. In some embodiments, the effective amount is a total dose of 200 pg. In some embodiments, the effective amount is a total dose of 300 pg.
8.5 Methods of treatment
Subjects with insufficient expression of VEGF-A can suffer from many vascular diseases including, without limitation, heart failure with reduced or preserved ejection fraction, kidney disease, a disease involving skin grafting and tissue grafting, post-MI cardiac dysfunction, ischemic heart disease, a vascular injury from trauma or surgery, a skin ulcer including a diabetic ulcer, diabetic foot, critical limb ischemia, lower limb ischemia, pulmonary hypertension, stable severe angina pectoris, refractory angina, congenital and acquired maxillofacial defects, alveolar bone atrophy, ligament or tendon lesions, ocular disease, and peripheral arterial disease. Provided herein are methods to treat subjects suffering from diseases responsive to VEGF-A therapy by direct administration of a circRNA described herein encoding VEGF-A polypeptides. In some embodiments, circRNA disclosed herein is administered to the subject in a citrate saline buffer without lipids and calcium.
In some embodiments, a circRNA described herein can be administered to a subject, wherein the circRNA is translated to produce a therapeutic protein of a VEGF-A polypeptide. Provided are methods for treatment of disease or conditions in humans and other mammals.
In some embodiments, compositions and particular formulations comprising a circRNA encoding VEGF-A polypeptides can be used for treatment of a disease such as heart failure with reduced or preserved ejection fraction, kidney disease, a disease involving skin grafting and tissue grafting, post-MI cardiac dysfunction, ischemic heart disease, a vascular injury from trauma or surgery, a skin ulcer including a diabetic ulcer, diabetic foot, critical limb ischemia, lower limb ischemia, pulmonary hypertension, stable severe angina pectoris, refractory angina, congenital and acquired maxillofacial defects, alveolar bone atrophy, ligament or tendon lesions, ocular disease, and peripheral arterial disease.
In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat heart failure with reduced or preserved ejection fraction. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat post-MI cardiac dysfunction. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat ischemic heart disease. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat a vascular injury from trauma or surgery. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat a skin ulcer including a diabetic ulcer. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat diabetic foot. In some embodiments, the formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used in wound healing, for example, in healing lacerations. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to critical limb ischemia. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat lower limb ischemia. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat pulmonary hypertension. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat peripheral arterial disease. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat kidney disease. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat a disease involving skin grafting and tissue grafting. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat stable severe angina pectoris. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat refractory angina. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat congenital and acquired maxillofacial defects. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat alveolar bone atrophy. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat ligament or tendon lesions. In some embodiments, formulations comprising a VEGF-A-encoding circRNA disclosed herein can be used to treat ocular disease.
Other aspects of the disclosure relate to administration of the formulations comprising circRNA to subjects in need thereof. Administration of the formulation can be intramuscular, intradermal, subcutaneous, intracranial, intrathecal, vitreous, and subretinal, muscular, cardiac, intracardiac, or epicardiac route, through a portal vein catheter, through a coronary sinus catheter, and/or by direct administration into the area to be treated.
However, the present disclosure also encompasses the delivery of naked circRNA molecules or circRNA complexes, and/or pharmaceutical, prophylactic, or diagnostic formulations thereof, by any appropriate route taking into consideration likely advances in the sciences of drug delivery.
As non-limiting examples, in some embodiments, formulations in accordance with the present disclosure are administered to the subject via intramuscular, intradermal, subcutaneous, intracardiac or epicardiac route, through a portal vein catheter, through a coronary sinus catheter, and/or by direct administration into the area to be treated. In some embodiments, the formulation is administered to the subject intramuscularly. In some embodiments, the formulation is administered to the subject intradermally. In some embodiments, the formulation is administered to the subject subcutaneously. In some embodiments, the formulation is administered to the subject intracranially. In some embodiments, the formulation is administered to the subject intrathecally. In some embodiments, the formulation is administered to the subject vitreously. In some embodiments, the formulation is administered to the subject subretinally. In some embodiments, the formulation is administered to the subject muscularly. In some embodiments, the formulation is administered to the subject cardiacly.
In some embodiments, the formulation is delivered to the subject via microneedles, hydrogels, or sprays.
In some embodiments, the composition or formulation is administered to the subject intracardially or epicardially, preferably at a fixed-dosage in multiple administrations. In some embodiments, the composition or formulation is administered to the subject through a portal vein catheter, preferably at a fixed-dosage in multiple administrations. In some embodiments, the composition or formulation is administered to the subject through a coronary sinus catheter, preferably at a fixed-dosage in multiple administrations. In some embodiments, the composition or formulation is administered to the subject by direct administration into the area to be treated, preferably at a fixed-dosage in multiple administrations.
In some embodiments, the formulation is administered to the subject intracardially. In some embodiments, the formulation is administered to the subject through a portal vein catheter. In some embodiments, the formulation is administered to the subject through a coronary sinus catheter. In some embodiments, the formulation is administered to the subject by direct administration into the area to be treated. For example, the formulation is administered to the subject by direct injection to the damaged area during open heart surgery. In some embodiments, the formulation is administered to the subject pericardially. For example, in patients undergoing coronary artery by-pass grafting (CABG) , the formulation is administered to the patient from the external side of the heart.
The formulation can be administered to a subject using any amount of administration effective for treating a disease, disorder, and/or condition. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular formulation, its mode of administration, its mode of activity, and the like. It will be understood, however, that the total daily usage of the formulations may be decided by the attending physician within the scope of sound medical judgment. The specific pharmaceutically effective, dose level for any particular patient will depend upon a variety of factors including the disease being treated and the severity of the disease; the activity of the specific compound employed: the specific formulation employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rale of excretion of the specific compound employed; the duration of the treatment: drugs (for example, a circRNA) used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts,
In some embodiments, formulations in accordance with the present disclosure are administered to a subject, wherein the formulations comprise a lipid-based complex (such as liposomes, lipoplexes, and lipid nanoparticles) . In other embodiments, naked circRNA is administered in phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer. In other embodiments, naked circRNA is administered in the absence of divalent cations, including calcium. In preferred embodiments, formulations for intracardiac or intradermal administration comprising the circRNA are formulated in citrate saline buffer containing no calcium or magnesium.
In some embodiments, formulations in accordance with the present disclosure are administered to a subject, wherein the formulation comprising a VEGF-A-encoding circRNA disclosed herein formulated in citrate saline buffer is less toxic to the subject than a lipid-based formulation. The assessments of toxicity due to RNA delivery in formulations in accordance with the present disclosure can be evaluated by methods well-known in the art. For example, cationic lipids are typically included in lipid formulations of RNA therapeutics to improve RNA encapsulation and stability. However, some cationic lipids may disrupt the integrity of a membrane structure, cause cell lysis and necrosis, and/or alter the expression of multiple genes in undesirable manner (Xue FLY, , Curr Pharm Des, , (2015) 21 (22) : 3140-7; its entirety is incorporated herein by reference) , Accordingly, examples of toxicity can be assessed by measuring the degree of cell lysis and necrosis, and/or alteration to the expression of multiple genes due to RNA delivery in formulations in accordance with the present disclosure. At preclinical and clinical levels, systemic dose-dependent toxicities of some lipoplexes have been well-documented. Capture of some lipoplexes by Kupffer cells in the liver can trigger inflammatory responses, which may inflict damages to liver and result in elevated levels in major liver function indicators. Leukopenia and thrombocytopenia may also occur (Zhang J., Adv Drug Deliv Rev., (2005) 57 (5) : 689-698; its entirety is incorporated herein by reference) . Accordingly, examples of toxicity can be assessed by measuring the inflammatory responses due to RNA delivery in formulations in accordance with the present disclosure. In addition, examples of toxicity can be assessed by measuring infusion related reactions such as dyspnea, hypoxia, rigors, back pain, hypotension, and liver injury,
A subject suffering from a vascular disease is responsive to the treatment if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is working if the progression of a disease is reduced or halted. For example, heart failure (HF) occurs when the heart is weakened and is not filled with, or cannot pump, enough blood to meet the body's needs for blood and oxygen. Likewise, a patient with ischemic heart disease (IHD) has heart problems caused by narrowed heart arteries. When arteries are narrowed, less blood and oxygen reaches the heart muscle. As a consequence of microvascular dysfunction, loss of functional vessels, and/or loss of cardiac tissue, a patient usually develops cardiac dysfunction post-MI. Furthermore, vascular structures are most commonly injured by penetrating trauma or surgery. Diabetes impairs numerous components of wound healing, and a patient with diabetic wound healing generally has altered blood flow due to vascular dysfunction. Accordingly, a patient with skin ulcer including diabetic ulcers usually has decreased or delayed would healing, Critical limb ischemia (CL1) is a severe obstruction of the arteries which markedly reduces blood flow to the extremities (hands, feet and legs) and has progressed to the point of severe pain and even skin ulcers, sores, or gangrene. Pulmonary hypertension (PH or PHTN) is an increase of blood pressure in the pulmonary artery, pulmonary vein, or pulmonary capillaries, together known as the lung vasculature, leading to shortness of breath, dizziness, fainting, leg swelling and other symptoms. Peripheral artery disease (PAD) is a narrowing of the peripheral arteries to the legs, stomach, arms, and head -most commonly in the arteries of the legs. These conditions are routinely clinically diagnosed based on various physical examinations with confirmation by echocardiography, blood tests, magnetic resonance imaging (MRI) electrocardiography, and other suitable tests, For example, the diagnosis of significant vascular injury rests upon close physical examination and imaging tests. Accordingly, a treatment for any one of these conditions is working if the patient shows less severe symptoms by physical examinations and/or improvements in testing results from echocardiography, blood tests, MRI, electrocardiography, or any other suitable and/or routine tests. In some embodiments, the subject suffering from a vascular disease has suffered a myocardial infarction. Additionally, molecular measurements for vascular functions can be used to assess the improvements of the general pathophysiology. Examples of these measurements include enhanced nitric oxide (NO) availability, increased angiogenesis/arteriogenesis, and recruitment of stem cells to cardiac tissue. A patient suffering from a vascular disease is therefore responsive to the treatment if the patient shows improved molecular functions in these measurements.
8.6 Methods of modulating physiological processes
Another aspect of the present disclosure relates to the administration of a composition or a formulation comprising a VEGF-A-encoding circRNA disclosed herein for modulating a physiological process in a mammalian cell, tissue, or subject. In some embodiments, a method for modulating a physiological process in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure. As non-limiting examples, the modulation of a physiological process can include inducing angiogenesis, stimulating vascular cell proliferation, inducing the proliferation and migration of vascular endothelial cells, increasing proliferation and/or altering the fate of epicardial derived progenitor cells, upregulating endothelialization, inducing cardiac regeneration, increasing revascularization of tissue grafts for wound healing, improving vascular function, increasing tissue perfusion and new vessel formation, reducing scar tissue, promoting stent biofunctionality, inducing lymphangiogenesis, inducing bone repair, or improving cardiac function, or any combination thereof.
In some embodiments, a method for inducing angiogenesis in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA of present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer. In some embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. The effects of circRNA on inducing angiogenesis can be evaluated by methods well-known in the art (see, e.g., Lopez J J. et aL, Cardiovasc Res, (1998) 40, 272-281 ; Gaiiano R.D. et ah, Am J Pathol, (2004) 164, 1935-1947; Lin Y.D. et al., Sci Transl Med, , (2012. ) 4 (146) : 146ral 09; Zangi L, et al., Nat Biotcchnol., (2013) 10, 898-907; all of which are incorporated herein by reference) .
In some embodiments, a method for inducing lymphangiogenesis in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA of present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer. In some embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. The effects of circRNA on inducing lymphangiogenesis can be evaluated by methods well-known in the art.
In some embodiments, a method for stimulating vascular cell proliferation in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer. In preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. The effects of circRNA on stimulating vascular cell proliferation can be evaluated by methods well-known in the art (see, e.g., Galiano R.D. et al., Am J Pathol, (2004) 164, 1935-1947; its entirety is incorporated herein by reference) .
In some embodiments, a method for inducing the proliferation and migration of vascular endothelial cells in a mammalian tissue, or subject comprises contacting the mammalian tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer. In preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. The effects of circRNA on inducing the proliferation and migration of vascular endothelial cells can be evaluated by methods well-known in the art (see, e.g., Galiano R.D. et al., Am J Pathol, (2004) 164, 1935-1947; its entirety is incorporated herein by reference) .
In some embodiments, a method for increasing proliferation and/or altering the fate of epicardial derived progenitor cells in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure, in some embodiments, the composition is formulated in a phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer. In preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. The effects of circRNA on increasing proliferation and/or altering the fate of epicardial derived progenitor cells can be evaluated by methods well-known in the art (see, e.g., Zangi L. et al., Nat Biotechnol., (2013) 10, 898-907; its entirety is incorporated herein by reference) .
In some embodiments, a method for upregulating endothelializaiion in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, ′I′HΑΜ buffer, or citrate saline buffer, in preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially tree of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. The effects of circRNA on upregulating endotheliaiization can be evaluated by methods well-known in the art (see, e.g., Galiano R.D. et a! ., Am J Pathol, (2004) 164, 1935-1947; its entirety is incorporated herein by reference) .
In some embodiments, a method for inducing cardiac regeneration in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, ′I′HΑΜ buffer, or citrate saline buffer. In preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium, in some embodiments, the citrate saline buffer contains no calcium or magnesium. The effects of circRNA on inducing cardiac regeneration can be evaluated by methods well-known in the art (see, e.g., Zangi L. et al., Nat Biotechnol., (2013) 10, 898-907; Lin Y.D. et al., Sci Transl Med., (2012) 4 (146) : 146ral09; both of which are incorporated herein by reference) .
In some embodiments, a method for increasing revascularization of tissue grafts for wound healing in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer. In preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer, in some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. The effects of circRNA on increasing revascularization of tissue grafts for wound healing can be evaluated by methods well-known in the art (see, e.g., Tohyama H. et al., Chang Gung Med J, (2009) 32, 133-139; Chen J. et al, Exp Ther Med, (2012) 4, 430-434; both of which are incorporated herein by reference) .
In some embodiments, a method for improving vascular function in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer. In preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. The effects of circRNA on improving vascular function can be evaluated by methods well-known in the art (see, e.g., Lopez J.J. et al., Cardiovasc Res, (1998) 40, 272-281 ; Tio R.A, et al., Hum Gene Ther., (1999) 10, 2953-2960; Sato K. et aL J Am Coll Cardiol., (2001 ) 37, 616-23; all of which are incorporated herein by reference) ,
In some embodiments, a method for increasing tissue perfusion and new vessel formation in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer, in preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. The effects of circRNA on increasing tissue perfusion and new vessel formation can be evaluated by methods well-known in the art (see, e.g., Chiappini C, et ah, Nat Mater., (2015) 14, 532-539; Lin Y.D, et al,, Sci Transl Med., (2012) 4 (146) : 146ral 09; Zangi L. et al,, Nat. Biotechnol, (2013) 10, 898-907; all of which are incorporated herein by reference) .
In some embodiments, a method for reducing scar tissue in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer. In preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. The effects of circRNA on reducing scar tissue can be evaluated by methods well-known in the art (see, e.g., Rosano J.M. et al., Cardiovasc Eng Technol., (2012) 3, 237-247; Galiano R.D. et al., Am J Pathol, (2004) 164, 1935-1947; Lin Y.D, et al., Sci Transl Med., (2012) 4 (146) : 146ral09; Zangi L. et al, Nat Biotechnol. (2013) 10, 898-907; all of which are incorporated herein by reference) .
In some embodiments, a method for improving cardiac function in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer, in preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. The effects of circRNA on improving cardiac function can be evaluated by methods well-known in the art (see, e.g., Rosano J.M. et al., Cardiovasc Eng Technol., (2012) 3, 237-247; Lin Y.D. et al,, Sci Transl Med., (2012) 4 (146) : 146ral 09; Zangi L. et al,, Nat Biotechnol., (2013) 10, 898-907; all of which are incorporated herein by reference) .
In some embodiments, a method for reducing infarct size in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer, in preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. In some embodiments, the infarct size is reduced. In some embodiments, the infarct is eliminated. The effects of circRNA on infarct size can be evaluated by methods well-known in the art, such as left ventriculography, coronary angiography, echocardiography, MRI scans, CT scans, gated myocardial SPEC scans or gated myocardial. PET scans.
In some embodiments, a method for reducing fibrosis in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, Τ′HΑΜ buffer, or citrate saline buffer. In preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. In some embodiments, the fibrosis is reduced. In some embodiments, the fibrosis is eliminated. The effects of circRNA on fibrosis can be evaluated by methods well-known in the art, such as left ventriculography, coronary angiography, echocardiography, MRI scans, CT scans, gated myocardial SPEC scans or gated myocardial PET scans.
In some embodiments, a method for reducing scar tissue in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, Τ′HΑΜ buffer, or citrate saline buffer. In preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. In some embodiments, the scar tissue is reduced. In some embodiments, the scar tissue is eliminated. The effects of circRNA on scar tissue can be evaluated by methods well-known in the art, such as left ventriculography, coronary angiography, echocardiography, MRI scans, CT scans, gated myocardial SPEC scans or gated myocardial PET scans.
In some embodiments, a method for inducing bone repair in a mammalian cell, tissue, or subject comprises contacting the mammalian cell, tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, Τ′HΑΜ buffer, or citrate saline buffer. In preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. In some embodiments, bone repair is induced. The effects of circRNA on fibrosis can induce bone repair can be evaluated by methods well-known in the art.
In some embodiments, a method for promoting stent biofunctionality in a mammalian tissue, or subject comprises contacting the mammalian tissue, or subject with a composition comprising the circRNA in accordance with the present disclosure. In some embodiments, the composition is formulated in a phosphate-buffered saline buffer, Τ′HΑΜ buffer, or citrate saline buffer. In preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium. In some embodiments, the stent biofunctionality is enhanced. The effects of circRNA on stent biofunctionality can be evaluated by methods well-known in the art.
8.7 Methods of expressing VEGF-A
Another aspect of the present disclosure relates to the administration of a composition or a formulation comprising a circRNA encoding a VEGF-A polypeptide for in vivo, in vitro, in situ, or ex vivo protein expression in a mammalian cell or tissue.
Some embodiments relate to a method for expressing VEGF-A in a mammalian cell or tissue, comprising contacting the mammalian cell or tissue in vivo, in vitro, or ex vivo with a composition comprising the circRNA. In some embodiments, a method for expressing VEGF-A in a mammalian cell or tissue comprises contacting the mammalian cell or tissue with a composition comprising the circRNA in accordance with the present disclosure.
In some embodiments, a method for expressing VEGF-A in a mammalian cell or tissue comprises contacting the mammalian cell or tissue with a composition comprising the circRNA in accordance with the present disclosure, wherein the composition is formulated in a phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer. In preferred embodiments, compositions comprising the circRNA comprise a citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium.
In some embodiments, a method for expressing VEGF-A in a mammalian cell or tissue comprises contacting the mammalian cell or tissue with a composition comprising the circRNA in accordance with the present disclosure.
In some embodiments, a method for expressing VEGF-A in a mammalian cell or tissue comprises contacting the mammalian cell or tissue with a composition comprising the circRNA in accordance with the present disclosure, wherein the composition is formulated in a lipid-based complex (such as liposomes, lipoplexes, and lipid nanoparticles) .
In some embodiments, a method for expressing VEGF-A in a mammalian cell or tissue comprises contacting the mammalian cell or tissue with a composition comprising the circRNA formulated in citrate saline buffer, wherein the composition is less toxic to the subject than a lipid-based formulation.
Some embodiments relate to a method of producing VEGF-A in a subject, comprising administering to the subject a composition or a formulation comprising the circRNA in accordance with this disclosure.
As non-limiting examples, in some embodiments, a method of producing VEGF-A in a subject comprises administering to the subject a formulation comprising the circRNA in accordance with this disclosure. In some embodiments, formulations are formulated in citrate saline buffer. In some embodiments, the formulation further comprises a pharmaceutically acceptable excipient. In some embodiments, the formulation further comprises a pharmaceutically acceptable excipient chosen from a solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, core-shell nanoparticles, polymer, peptide, protein, cell, hyaluronidase, and mixtures thereof. In some embodiments, the solvent is an aqueous solvent.
In some embodiments, the formulation further comprises a pharmaceutically acceptable excipient selected from lipid, lipidoid, liposome, lipid nanoparticle, lipoplex, and mixtures thereof. In some embodiments, a method of producing VEGF-A in a subject comprises administering to the subject a formulation comprising the circRNA in accordance with this disclosure, wherein the subject is suffering from a disease responsive to VEGF-A therapy. In some embodiments, the subject is suffering from a disease chosen from reduced or preserved ejection fraction, kidney disease, a disease involving skin grafting and tissue grafting post-MI cardiac dysfunction, ischemic heart disease, a vascular injury from trauma or surgery, a skin ulcer including a diabetic ulcer, critical limb ischemia, pulmonary hypertension, stable severe angina pectoris, congenital and acquired maxillofacial defects, alveolar bone atrophy, ligament or tendon lesions, ocular disease, and peripheral arterial disease.
In some embodiments, the disease is heart failure with reduced or preserved ejection fraction. In some embodiments, the disease is post-MI cardiac dysfunction. In some embodiments, the disease is ischemic heart disease. In some embodiments, the disease is a vascular injury from trauma or surgery. In some embodiments, the disease is a skin ulcer including a diabetic ulcer. In some embodiments, the disease is diabetic foot. In some embodiments, the disease is critical limb ischemia. In some embodiments, the disease is lower limb ischemia. In some embodiments, the disease is pulmonary hypertension. In some embodiments, the disease is severe angina pectoris. In some embodiments, the disease is refractory angina. In some embodiments, the disease is maxillofacial defects. In some embodiments, the disease is congenital or acquired maxillofacial defects. In some embodiments, the disease is alveolar bone atrophy. In some embodiments, the disease is ligament or tendon lesions. In some embodiments, the disease is ocular disease. In some embodiments, the disease is peripheral arterial disease. In some embodiments, the disease is kidney disease, in some embodiments, the disease is a disease involving skin grafting and tissue grafting.
In some embodiments, a method of producing VEGF-A in a subject comprises administering to the subject a formulation comprising the circRNA in accordance with this disclosure, wherein the formulation is administered to the subject via intramuscular, intradermal, subcutaneous, intracranial, intrathecal, vitreous, and subretinal, muscular, cardiac, intracardiac, or epicardiac route, through a portal vein catheter, through a coronary sinus catheter, and/or by direct administration into the area to be treated. In some embodiments, the formulation is administered to the subject intramuscularly, in some embodiments, the formulation is administered to the subject intradermally. in some embodiments, the formulation is administered to the subject subcutaneously. In some embodiments, the formulation is administered to the subject intracardially, preferably in multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more) administrations at a fixed-dosage. In some embodiments, the formulation is administered to the subject through a portal vein catheter, in some embodiments, the formulation is administered to the subject through a coronary sinus catheter. In some embodiments, the formulation is administered to the subject by direct administration into the area to be treated.
In some embodiments, a method of producing VEGF-A in a subject comprises administering to the subject a formulation comprising the circRNA in accordance with this disclosure, wherein the formulation comprises a lipid-based complex (such as liposomes, lipoplexes, and lipid nanoparticles) , phosphate-buffered saline buffer, THAM buffer, or citrate saline buffer. In preferred embodiments, formulations comprising the circRNA are formulated in citrate saline buffer. In some embodiments, the citrate saline buffer is substantially free of calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium.
In some embodiments, the citrate saline buffer has 10 mM citric acid and 130 M sodium chloride, with a pH range 6-7.5.
In some embodiments, the method expresses VEGF-A in heart cell or heart.
In some embodiments, a method of producing VEGF-A in a subject comprises administering to the subject a formulation comprising the circRNA in accordance with this disclosure, wherein the formulation comprises a concentration of the circRNA formulated in citrate saline buffer of between 0.1 and 1 μg/μL, In some embodiments, the formulation comprises a concentration of the circRNA formulated in citrate saline buffer of between 1 and 10 μg/μL. In some embodiments, the formulation comprises a concentration of the circRNA formulated in citrate saline buffer of between 10 and 50 μg/μL.
In some embodiments, a method of producing VEGF-A in a subject comprises administering to the subject a formulation comprising the circRNA in accordance with this disclosure, wherein the formulation comprises a in citrate saline buffer and is less toxic to the subject than a lipid-based formulation.
8.8 Experimental
The examples provided below are for purposes of illustration only, which are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
8.8.1 Example 1. IRES-like sequence-based vectors
Coding sequences of an expression protein with IRES-like sequence were designed and used to construct circRNA vectors. RNAs were in vitro transcribed ( “IVT” ) from the XbaI digested linearized plasmid DNA template using T7 RiboMAXTM Large-Scale RNA Production System (PromegaTM, P1320) in the presence of unmodified NTPs. After DNase I treatment, the RNA products were column purified with RNA Clean and Concentrator Kit (ZYMO research, R1013) to remove excess NTP and other salt in IVT buffer, as well as the possible small RNA fragments generated during IVT. In some experiments, the purified RNA was further circularized in a new circularization buffer. The RNAs were first heated to 75℃ for 5 min and quickly cooled down to 45℃, after which a buffer including indicated magnesium and sodium was added to a final concentration: 50mM Tris-HCl at pH 7.5, 50/100mM NaCl, 0-40mM MgCl2, and was then heated at 53℃ for indicated time for circularization. The best optimized reaction condition including concentration of magnesium and sodium, and incubation time at 53℃, was selected for further experiments.
8.8.2 Example 2. Expression from the circRNA of the present disclosure
The expression of circular RNA products of different target sequences after transfection of cells was further tested. To minimize immunodegradation caused by linear RNAs, the RNA products were treated in the following three steps prior to transfection.
(1) RNase R treatment was performed to digest linear RNAs. The reaction conditions were at 37℃ for 30 min.
(2) CIP treatment was performed to remove phosphate groups at both ends of the linear RNAs. The reaction conditions were to add quick CIP (NEB) , and react at 37℃ for 30 min.
(3) HPLC purification was performed to remove small linear RNAs. HPLC conditions: gel exclusion chromatographic column: WatersBEH450A, column temperature: 40℃, flow rate: 1 min/ml, and elution conditions: 0 to 30 min, 100%buffer A (prepared with 10 mM Tris, 0.5 mM EDTA, and DEPC-treated water) .
The target RNAs were transfected using lipo RNAmaxunder the transfection conditions according to the supplier’s instructions, for 24 hours.
Using the construction methods described in the Examples above, different target sequences were tested. To facilitate detection of protein expression, constructs comprising a fluorescent protein coding sequence together with the IRES sequences disclosed herein were constructed. The addition of IRES may initiate cap-independent non-canonical translation, enabling the translation of coding sequences in circular RNAs into proteins.
For different target sequences, different methods were used to detect protein expression.
In the case that the translation product was GFP, the fluorescence was observed under a microscope. In addition, the cells were lysed with RIPA lysis solution (Beyotime) , and then the protein expression was detected by Western blotting. In the case that the translation product was luciferase, the cells were first lysed with Passive lysis solution (PromegaTM) and then the protein expression was detected by a microplate reader using a luciferase detection kit (PromegaTM) . It was confirmed that the expression of Gluc protein was obtained by the method of the present disclosure.
8.8.3 Example 3. Administration of circRNAs into mice
The circular RNA was encapsulated in a lipid nanoparticle via the NanoAssemblr Ignite system as previously described (Corbett K.S. et al., Nature 586, 567 (2020) , Polack F.P., et al., N. Engl. J. Med. 383, 2063 (2020) . In brief, an aqueous solution of circRNA at pH 4.0 was rapidly mixed with a lipid mixture dissolved in ethanol, which contained different ionizable cationic lipid, distearoylphosphatidylcholine (DSPC) , DMG-PEG2000, and cholesterol. The ratios for the lipid mixture were MC3: DSPC: Cholesterol: PEG-2000=50: 10: 38.5: 1.5 for formulation 1, SM-102: DSPC: Cholesterol: PEG-2000 = 50: 10: 38.5: 1.5 for formulation 2, and ALC-0315: DSPC: Cholesterol: ALC-0159 = 46.3: 9.4: 42.7: 1.6 for formulation 3
. The resulting LNP mixture was then dialyzed against PBS and stored at -80 ℃ at a concentration of 0.5 μg/μl for further application.
Female BALB/C, C57BL/6J, and/or B6C3F1/J mice aged 8 weeks were purchased from Shanghai Model Organisms Center. 20 μg of CircRNA-LNPs in PBS were administrated into mice intramuscularly with 3/10 insulin syringes (BD biosciences) . The serum was collected 24 hours after the administration of the LNP, and 50 μl serum was used for Luciferase activity assay in vitro. Bioluminescence imaging was performed with an IVIS Spectrum (Roper Scientific) . 24 hours after Gluc-LNP injection, 2 mg/kg of Coelenterazine (MedChemExpress, MCE) was administrated to mice intraperitoneally. Mice were then anesthetized after receiving the substrate in a chamber with 2.5%isoflurane (RWD Life Science Co. ) and placed on the imaging platform while being maintained on 2%isoflurane via a nose cone. Mice were imaged 5 minutes post substrate injection with 30 seconds exposure time to ensure the signal were effectively and sufficiently acquired.
8.8.4 Example 4. circRNAs mediate prolonged protein production
A major advantage of circular mRNAs is their superb stability because of lacking the free ends, therefore the circRNA should have good shelf life for protein expression compared to linear counterparts. To directly test this, both linear and circular mRNA encoding the Gaussia luciferase (Gluc) are synthesized and stored parallelly in pure water at room temperature for different days before being transfecting into 293 T cells and/or A549 cells. The activity of the circRNA to direct protein translation is tested in comparison to that of the linear mRNA. The prolonged protein translation is expected for the circRNA.
A major advantage of circular mRNAs is their superb stability because of lacking the free ends, therefore the circRNA should have good shelf life for protein expression compared to linear counterparts. To directly test this, both linear and circular mRNA encoding VEGF in Table 2 are synthesized and stored parallelly in pure water at room temperature for different days before being transfecting into 293 T cells and/or A549 cells. The activity of the circRNA to direct protein translation is tested in comparison to that of the linear mRNA. The prolonged protein translation is expected for the circRNA.
8.8.5 Example 5. Purified circRNAs direct robust translation of target proteins
The mRNA purity was found to be a key factor for the protein production and induction of innate immunity, as the removal of dsRNA by HPLC can eliminate immune activation and improves translation of linear nucleoside-modified mRNA (Kariko, K. et al., (2011) . To examine if the circRNAs produced as disclosed herein can induce innate immune response and cell toxicity, the circRNAs are purified with gel purification or HPLC and measured if the circRNAs can induce cellular immune response upon transfection of circRNAs by testing cell survival and cytokine production (e.g., IL-10, IL-6, TNFα, MCP-1, IP-10, RIG-I, IFN-α2 and IFN-B1) . In-life examinations are monitored, such as viability, clinical observations, local tolerance, necropsy observations, cytokine detection. PK serum sample are collected at pre-dose (0 h) , 6 h, 1 day (24 h) , 2 day (48 h) , 3 day (72 h) , 5 day (120 h) , 7 day (168 h) , 10 day (240 h) . IVIS are collected at pre-dose (0 h) , 6 h, 1 day (24 h) , 2 day (48 h) , 3 day (72 h) , 5 day (120 h) , 7 day (168 h) , 10 day (240 h) .
8.8.6 Example 6. In vitro evaluation of a linear mRNA or circular RNA for protein expression
An RNA encodes a reporter protein (e.g., a luciferase) is employed to determine the in vitro protein expression efficiency of the RNA encapsulated in an LNP. Exemplary expression RNAs include, but are not limited to, SEQ ID NOs: 14-39, 49-61 or a nucleic acid sequence encodes an amino acid sequence selected from SEQ ID NOs: 1-13. Exemplary circular RNAs include those described in PCT Appl. Nos. PCT/CN2020/077026, filed February 27, 2020; PCT/CN2022/095749, filed May 27, 2022; and PCT/CN2022/095949, filed May 30, 2022; the disclosure of each of which is incorporated herein by reference in its entirety.
Briefly, a luciferase-coding linear mRNA or circular RNA is encapsulated in an LNP comprising a compound provided herein as described in Example 17. Cells are seeded into a 24-well plate and incubated with the LNP-encapsulated luciferase-coding mRNA or circular RNA at 37 ℃ for 24 h. The cell lysis and supernatant are separately collected and analyzed in a luminescence assay using the Reporter Assay System.
8.8.7 Example 7. In vivo evaluation of a linear mRNA or circular RNA for protein expression
An RNA encodes a reporter protein (e.g., a luciferase) is employed to determine the in vivo protein expression efficiency of the RNA encapsulated in an LNP, according to the procedures as described in WO 2017/049245 A2 or WO 2018/081480 A1, the disclosure of each of which is incorporated herein by reference in its entirety. Exemplary expression RNAs include, but are not limited to, SEQ ID NOs: 14-39, 49-61 or a nucleic acid sequence encodes an amino acid sequence selected from SEQ ID NOs: 1-13. Exemplary circular RNAs include those described in PCT Appl. Nos. PCT/CN2020/077026, filed February 27, 2020; PCT/CN2022/095749, filed May 27, 2022; and PCT/CN2022/095949, filed May 30, 2022; the disclosure of each of which is incorporated herein by reference in its entirety.
Briefly, a luciferase-coding mRNA or circular RNA is encapsulated in an LNP comprising a compound provided herein as described in Example 17. The LNP-encapsulated luciferase-coding mRNA or circular RNA is systemically administered to 6-8 week old female C57BL/6 mice by tail vein injection at predetermined doses. The mice are euthanized at predetermined time points (e.g., 4 h, 6 h, 12 h, …14 days) post-administration. The liver, spleen, brain, lung, heart, kidney, pancreas, and muscle tissues of the mice are collected, immediately snap frozen in liquid nitrogen, and stored at -80 ℃ for analysis. The collected tissues of each type are homogenized. The homogenate is analyzed using the STEADY-GLO luciferase assay system according to the manufacturer’s protocol. The amount of proteins assayed is determined using a BCA protein assay kit. Relative luminescence units (RLU) can be normalized to total μg protein assayed and compared with a standard curve.
Briefly, a luciferase-coding mRNA or circular RNA is encapsulated in an LNP comprising a compound provided herein as described in Example 17. The LNP-encapsulated luciferase-coding mRNA or circular RNA is systemically administered to 6-8 week old female C57BL/6 mice by tail vein injection at predetermined doses. The mice are euthanized at predetermined time points (e.g., 4h, 6h, 12h, …14 days) post-administration and placed on an imaging platform while being maintained on isoflurane via a nose cone. Bioluminescence imaging is performed with an IVIS Spectrum (Roper Scientific) . Mice are imaged 5 minutes post injection with 30 seconds exposure time to ensure the signal is effectively and sufficiently acquired.
8.8.8 Example 8. Immunogenicity evaluation of a linear mRNA or circular RNA
Immunogenicity is assessed in six-week-old female BALB/cJ, C57BL/6J, and/or B6C3F1/J mice by two intramuscular immunizations with a linear mRNA or circular RNA LNP at predetermined doses (e.g., 0.01, 0.1, or 1 μg) , separated by 2-week interval or 3-week interval. Sera are collected 2 weeks post-prime and 2 weeks post-boost and assessed for a target-specific IgG by an enzyme-linked immunosorbent assay (ELISA) . Mouse spleens are also harvested at the end point (2 weeks after boost or 3 weeks after boost) for immunostaining and flow cytometry. Time points are compared within each dose level by statistical method (e.g., two-sided Wilcoxon signed-rank test) , and doses are compared post-boost (via e.g., Kruskal–Wallis ANOVA with Dunn’s multiple comparisons test) .
8.8.9 Example 9. Immunization with a linear mRNA or circular RNA
Protective immunity is assessed in young adult BALB/cJ C57BL/6J, and/or B6C3F1/J mice challenged with a linear mRNA or circular RNA LNP at predetermined doses. Mononuclear single-cell suspensions from each immunized mouse are generated using RWD tissue dissociator, followed by 70 μM filtration and density gradient centrifugation using the FICO/LITE-LM medium. Spleenocytes are cryopreserved for further analysis. Before a T cell flow cytometry analysis, spleenocytes are cultured in R10 media and incubated for 6 h at 37 ℃ with an mRNA or circular RNA LNP. The cells are then washed before staining with a ZOMBIE NIRTM solution. The cells are washed and permeabilized using a BD CYTOFIX/CYTOPERM fixation/permeabilization solution according to the manufacturer’s instructions. The cells are washed in a permeabilization/wash solution and resuspended in FC BLOCK before staining with an antibody cocktail containing one or more antibodies selected from the following: I-A/I-E PE, CD4 BV421, CD8a BUV510, CD44 PE-Cy7, CD62L BB515, CD3e AF700, IFN-γ PE/Dazzle, TNF BB700, IL-2 BV605, IL-4 BV650, and IL-5 APC and in 1 × permeabilization/wash solution diluted with a brilliant stain buffer. The cells are washed in permeabilization/wash solution and resuspended in the FC buffer (PBS supplemented with 2%heat-inactivated FBS and 0.05%NaN3) before running on a full spectrum flow cytometry system. Analysis is performed using FLOWJO software.
8.8.10 Example 10. In vitro transfection
Different doses of VEGF-A circular RNA are transfected into primary myocardial cells of neonatal rat or cardiomyocytes IHC-SV40 (MeisenCTCC) and HL-1 (Procell Life Science&Technology) , using the transfection reagent MessengerMAX (Life Technologies) . Dilute transfection reagent and VEGF-A circular RNA in Opti-MEM Medium (Life Technologies) respectively, and incubate the mixture for 10 minutes after mixing the diluted components. A prepared circular RNA-lipid complex is evenly added into the cells cultured in complete medium. Then, 24 hours post-transfection, cells and supernatant is collected to evaluate the efficiency of transfection or protein expression.
8.8.11 Example 11. In vitro detection of VEGF-A expression according to western blot
Cellular supernatant is centrifugated and concentrated using Ultracel-10 regenerated cellulose membrane (Millipore) . The concentration of the total protein is quantified using BCA method (Beyotime) . Forty micrograms of total protein thermal denaturing with reduced or nonreduced loading buffer are loaded on a 4–20%Bis-Tris precast gel (Genscript) , and electrophoresis is carried out using formulated Tris-MOPS running buffer (Genscript) . Fractionated protein is electroblotted onto a polyvinyl difluoride (PVDF) membrane (Millipore) . Membranes are blocked in block buffer (5%BSA in TBS-Tween (0.1%) ) and incubated with primary VEGF-A antibodies (Rabbit mAb, Cell Signaling Technology) at room temperature for an hour. Subsequently, the membranes are incubated with a HRP (horseradish-peroxidase) labeled secondary antibody (Cell Signaling Technology) . The immunoreactions between VEGF-A circular expressed protein and the antibody in the membranes are detected using the western ECL substrate (Bio-rad) . Chemiluminescent signals were visualized and analyzed using an Ultra-sensitive multi-functional CCD imager (Amersham ImageQuant 800, Cytiva) .
8.8.12 Example 12. Assessment of the activation of angiogenic signaling after stimulated by VEGF circular RNA expressed protein
Activation of VEGF receptor 2 (VEGFR2, KDR, Flk-1) is an initial condition for VEGF-induced signal transduction in endothelial cells and angiogenesis. After binding with VEGF-A, VEGFR2 is activated by self-phosphorylated and subsequently activating intracellular signaling with phosphorylation. To assess the activation of angiogenesis, HUVECs are seeded into 6-well plates and cultured in endothelial basal medium (EBM) . After cells starved for 18 hours, 20 ng/mL recombinant VEGF-A protein (Acrobiosystems, CN) and VEGF-A protein translated from VEGF circular RNA are respectively added into the supernatant as conditioned media. After stimulated 7.5 min and 15 min, total protein is extracted from stimulated cells immediately by added RIPA lysis buffer I containing protease and phosphatase inhibitors (Sangon Biotech (Shanghai) ) . Protein concentrations are measured using the BCA method (Beyotime) . Forty micrograms of total protein thermal denaturing with reduced loading buffer are loaded on a 4–20%Bis-Tris precast gel (Genscript) , and electrophoresis is carried out using formulated Tris-MOPS running buffer (Genscript) . Fractionated protein is electroblotted onto a polyvinyl difluoride (PVDF) membrane (Millipore) and blocked the membrane in 5%BSA in TBS-Tween (0.1%) . To detect the total protein and the phosphorylation, the membranes are incubated in primary antibodies: VEGF Receptor 2 antibody (Cell Signaling Technology) , Phospho-VEGF Receptor 2 (Tyr1175) antibody (p-VEGFR2, Cell Signaling Technology) , endothelial nitric oxide synthase (eNOS) antibody (Affinity Biosciences) ; Phospho-eNOS (Ser1177) antibody (Affinity Biosciences) , PI3K (p85 alpha) antibody (Affinity Biosciences) and Phospho-PI3K p85 alpha (Tyr607) antibody (Affinity Biosciences) . Membranes are then incubated with HRP-labeled secondary antibody (CST) and immunoreactions are detected using the ECL western blotting substrate (Bio-rad) . Chemiluminescent signals are visualized and analyzed using an Ultra-sensitive multi-functional CCD imager (Amersham ImageQuant 800, Cytiva) .
8.8.13 Example 13. In vitro quantification of VEGF-A protein expression according to ELISA
Human VEGF-A protein produced from VEGF-A circular RNA translation is measured with a human VEGF-A ELISA kit (Elabscience) according to the manufacturer’s instructions. Cellular supernatants are collected and centrifugated (2000 rpm, 5 min) to remove cell fragments. Dilute the standards (as the specification) and the samples (100x-1000x) and set three duplicates. Add 100 μL of each sample (or standard) into precoated wells with cover and incubated for 1.5 hours at 37℃ with gentle shaking. Remove the liquid and subsequently add biotinized VEGF-A antibodies incubated for 1 hour at 37℃ with gentle shaking. After the last wash, completely remove liquid by aspirating or decanting. Add 100 μL of HRP-Streptavidin solution to each well and incubate for at 37℃ with gentle shaking. Discard the solution and wash the plate thoroughly. Then add 90 μL of ELISA Colorimetric TMB Reagent (Item H) to each well and incubate for 15 minutes at 37℃ in the dark with gentle shaking. After add 50 μL of Stop Solution, determine the optical density (OD value) of each well at 450 nm immediately according to a multimode microplate reader (BioTek Synergy H1, Agilent) . For calculation, average the duplicate readings for each standard and samples and subtracte the average zero standard optical density. Plot a four parameters logistic curve on log-log axis, with standard concentration on the x-axis and OD values on the y-axis. The actual concentration is the calculated concentration multiplied by the dilution factor.
8.8.14 Example 14. In vitro quantification of VEGF-A circular RNA according to qPCR
The transfection effect of VEGF-A circular RNA in vitro is essential to evaluate the expressional efficiency of VEGF-A circular RNA and the immune activation. The cells are seeded in 12-wells plate and transfected with 200 ng/well of VEGF-A circular RNA using MessengerMAX (Life Technologies) . After transfection for 24 hours, the total RNAs of cells are extracted according to the instruction of FastPure cell/tissue total RNA isolation kit V2 (Vazyme, CN) , and the concentration of total RNAs are determined by NanoDrop One micro-UV-VIS spectrophotometer (Thermo Scientific) . For Real Time RT-PCR (qPCR) , the total RNAs are reverse-transcribed using PrimeScript RT reagent Kit (Takara, CN) , according to the manufacturer’s instructions. Realtime qPCR analyses are performed on a real-time PCR amplification using TAKARA TB Green Premix Ex Taq II Kit (QIAGEN) and detect the amplified signal in real-time PCR instrument (LightCycler 480 instrument ll, Roche) . Fold changes in gene expression are determined by the vvCT method. The amplifications of RIG-1 and INF-B1 are indicated the activation of immune, and the amplifications of specific region in circular RNA are indicated the transfection effect of VEGF-A circular RNA. GAPDH expression is presented relative to an internal control to normalize the detection value. PCR primer sequences are as follows: for RIG-1, forward primer (SEQ ID NO. 68) and reverse primer (SEQ ID NO. 69) ; for INF-B1, forward primer (SEQ ID NO. 70) and reverse primer (SEQ ID NO. 71) ; for VEGF-A circular RNA, forward primer (SEQ ID NO. 72) and reverse primer (SEQ ID NO. 73) ; and for GAPDH, forward primer (SEQ ID NO. 74) and reverse primer (SEQ ID NO. 75) .
8.8.15 Example 15. VEGFR2 reporter Cell-based assay to assess the bioactivity of VEGF-A circular RNA
The Reporter gene assay is a bioluminescent cell-based assay that measures VEGF stimulation and inhibition of KDR (VEGFR-2) using luciferase as a readout. This assay could be used for assess the bioactivity of VEGF-A protein translated from VEGF-A circular RNA, and could be supplement to HUVECs assay. VEGFR2/NFAT reporter HEK293 cells (Sanyou Bio) stably express human VEGFR2, and simultaneously express the firefly luciferase gene under the control of NFAT response elements, based on the activated VEGF-VEGFR-NFAT pathway with VEGF stimulation. VEGFR2/NFAT reporter HEK293 is seeded in the 96 well transparent base plate with white border (Corning) . After cells seeded for 16 hours, 10 ng/mL recombinant VEGF-A protein (VE5-H4210, Acrobiosystems, CN) and expressed VEGF-A protein translated from VEGF circular RNA are respectively added into the HEK293 supernatant. Each tested sample and reference standard is set in triplicate. The assay plates are incubated in a 5%CO2 incubator at 37 ℃ for 6 h. The levels of firefly luciferase activity are measured using Rapid Detection of Firefly Luciferase Activity per manufacturer’s instructions (Promega, Madison, WI, USA) . The bioactivity of VEGF-A circular RNA is evaluated according to levels of firefly luciferase detection.
8.8.16 Example 16. Cell Function Assay to assess the bioactivity of VEGF-A circular RNA
The interaction between VEGF-A and VEGFR2 is an important mediator of angiogenesis as well as endothelial cell proliferation and migration. To assess proliferation, HUVECs are cultured in HUVEC medium with a small serum concentration (mix HUVEC complete medium and HUVEC basal medium in a ratio of 1: 30) . Count the cells number/mL and adjust the cell density into 3x103/well in 50 μL medium. After cells seeded for 16 hours, 50 μL recombinant VEGF-A protein (VE5-H4210, Acrobiosystems, CN) and expressed VEGF-A protein translated from VEGF circular RNA with same concentration gradient are respectively added into the HUVECs supernatant. Incubate the treated cells for 96 h (37 ℃, 5%CO2) and subsequently add 100 μL of CellTiter-Glo 2.0 cell viability assay reagent (Promega) into each well. Mix the contents for 2 minutes on a mini shaker (500 rpm) and then record luminescence by multimode microplate reader (BioTek Synergy H1, Agilent) . The intensity of the luminescence is indicated the HUVECs proliferation with VEGF-A stimulation. In migration assay, motility of HUVECs is measured using Corning BioCoat multiwell Plate (Corning) with 3-μm pore size polycarbonate filter insert that divides the chamber into upper and lower portions. HUVECs are starved overnight in 2%FBS and subsequently resuspended in 0.1%FBS medium at a density of 4x105 cells/mL. 250 μL of cell suspension (1x105) is then added to the upper chamber of the insert and 750 μL medium with 0.1%FBS is added in the lower chamber. After 20 hours in the absence or presence of 10 ng/mL recombinant VEGF-A protein (Acrobiosystems, CN) and expressed VEGF-A protein translated from VEGF circular RNA, cells on the top of the filter are removed by gentle swabbing, the cells on the bottom side of the filter are fixed with anhydrous ethanol and stained using 0.25%cresyl violet. The images in multiple fields are captured using a light microscope (DMi8, Leica) . The number of cells cross the membrane represent motility of HUVECs.
8.8.17 Example 17. Characterization of lipid nanoparticles
A lipid nanoparticle (LNP) comprising a compound provided herein is prepared using a benchtop from PRECISION NANOSYSTEMS Inc., according to the procedures as described in Roces et al., Pharmaceutics 2020, 12, 1095; the disclosure of which is incorporated herein by reference in its entirety.
The particle size (Z-average diameter) , polydispersity index (PDI) , and zeta potential (ZP) of the LNP are determined by dynamic light scattering (DLS) using a ZETASIZER NANO ZS equipped with a 633 nm laser and a detection angle of 173°, according to the procedures as described in Roces et al., Pharmaceutics 2020, 12, 1095. Briefly, the LNP is diluted in filtered (0.22 μm) ultrapure water down to a LNP concentration of 0.2 mg/mL. The diluted LNP (1 mL) is then loaded into a 4-mL cuvette for measurements.
The surface pKa of the compound provided herein in the LNP is determined by measuring the fluorescence of 2- (p-toluidino) -6-napthalene sulfonic acid (TNS) in buffer solutions having pH from 3.5 to 9 with 0.5 pH unit increments (10 mM HEPES, 130 mM NaCl, 10 mM NH4OAc, and 10 mM MES) , according to the procedures as described in Roces et al., Pharmaceutics 2020, 12, 1095. Briefly, the LNP is diluted to 0.78 mM with MILLIQ-water and the diluted LNP (6.4 μL) is added to a black 96-well plate containing a pH buffer (233.6 μL) and a 25 μM TNS solution (10 μL) . Fluorescence was quantified at λem = 445 nm and λex = 321 nm. A pKa value, defined as pH at half-maximal fluorescence intensity, is determined.
The nucleic acid encapsulation efficiency (EE%) of the LNP is measured using a QUANT-ITTM RIBOGREENTM RNA assay kit from INVITROGENTM, according to the procedures as described in Roces et al., Pharmaceutics 2020, 12, 1095.
An RNA encodes a reporter protein (e.g., a luciferase) is employed to determine the nucleic acid encapsulation efficiency (EE%) of an LNP. Exemplary expression RNAs include, but are not limited to, SEQ ID NOs: 14-39, 49-61 or a nucleic acid sequence encodes an amino acid sequence selected from SEQ ID NOs: 1-13. Exemplary circular RNAs include those described in PCT Appl. Nos. PCT/CN2020/077026, filed February 27, 2020; PCT/CN2022/095749, filed May 27, 2022; and PCT/CN2022/095949, filed May 30, 2022; the disclosure of each of which is incorporated herein by reference in its entirety.
8.8.18 Example 18. in vitro self-splicing activity of cRANzymes
This example relates to a method for determining the in vitro self-splicing activity of cRANzymes provided herein.
A DNA sequence encoding a cRNAzyme provided herein is cloned into an expression vector, e.g., psiCHECK-2 (PromegaTM, C8021) , puc57.
The purified template DNAs are transcribed in vitro by T7 RNA polymerase. The transcripts are digested with DNase I at 37℃ for 10～60 min to degrade the PCR templates. The transcripts are then column purified to obtain high-purity RNAs.
Table. 8 Detailed IVT condition:
The column-purified transcript RNAs are added to a self-splicing buffer (10, 20, 50 or 100 mM MgCl2, 50 mM NaCl, 40 mM Tris-HCl, pH = 7.5) for self-splicing reaction. The reaction conditions are: 95℃ for 1 min, 75℃ to 45℃ (-0.5℃, 15 sec/cycle, for 60 cycles in total) , holding at 45℃ with a buffer added, 45℃ for NMT 60 min, and 53℃ for 15 to 30 min.
Table. 9 detailed IVC condition:
Table. 10 Unspliced RNAs and spliced RNAs can be separated by affinity chromatography.
8.8.19 Example 19. Dose-Dependent and Time-Dependent Course Expression of VEGF-A in Various Cell Types Across Different Species Following A00A Transfection
8.8.19.1 Summary
The study demonstrated dose-dependent and time-course expression of VEGF-A in cardiomyocytes from various species, including humans, rodents, and pigs (i.e., immortalized human cardiomyocytes, human iPS-derived cardiomyocytes, rat primary cardiomyocytes, mouse primary cardiomyocytes, rat primary cardiomyocytes, porcine cardiac fibroblasts and immortalized porcine aortic smooth muscle cells) following A00A transfection.
For dose-dependent expression, cells were seeded at specific densities, transfected with varying A00A doses (12.5 ng to 200 ng) , and subjected to ELISA after 24 hours to quantify VEGF-A levels. The ELISA involved standard sample dilution, incubation in pre-coated plates, and detection with labeled antibodies. For time-course expression, cells were treated similarly, with supernatant collected at intervals up to 168 hours for VEGF-A quantification. Data analysis included nonlinear fitting equations and statistical considerations using triplicate wells for robustness.
The results showed that the expression of VEGF-A could be sustained for over 5 days across all tested cardiomyocytes and associated cells, indicating a prolonged and consistent expression profile over time.
Overall, these findings highlight the robust and sustained expression of VEGF-A in cardiomyocytes and associated cells from various species, supporting its potential therapeutic utility and warranting further investigation into its biological effects and mechanisms of action. 8.8.19.2 Objective
To evaluate the dose-dependency and time course-dependency of VEGF-A expression in various cell types across different species following A00A transfection.
8.8.19.3 Background
Vascular Endothelial Growth Factor (VEGF) is an intracellular-translated signaling protein that promotes angiogenesis, primarily through paracrine and thereby stimulating vascular endothelial cells (Collén, A., Bergenhem, N., Carlsson, L. et al. VEGFA mRNA for regenerative treatment of heart failure. Nat Rev Drug Discov 21, 79–80 (2022) . https: //doi. org/10.1038/s41573-021-00355-6) .
Among the VEGF-A subtypes, VEGF165 (VEGF-165) and VEGF121 (VEGF-121) are the most abundant isoforms. VEGF165 is a predominate angiogenic factor known to stimulate endothelial cell proliferation, survival, angiogenesis, and increase vascular permeability.
A00A, developed by CirCode, is a circular RNA encoding human VEGF165 protein. Upon delivery into myocardial cells, A00A utilizes the cell's own translation machinery to produce the VEGF-165 protein, functionally identical to endogenous protein. The A00A-coded VEGF-165 protein stimulates angiogenesis through paracrine and binding to the receptors on vascular endothelial cells. The production and secretion of VEGF165 protein are positively correlated with the transfection efficiency of A00A. After entering the myocardial cells or the cardiovascular cells (including vascular smooth muscle cells and fibroblasts) , circular RNA A00A enables to be continuously translated into VEGF165 protein, leading to a significantly longer half-life compared to recombinant proteins.
In vitro transfection of myocardial cells and cardiovascular cells from different species and quantitative analysis of protein products through Enzyme-Linked Immunosorbent Assay (ELISA) can simulate the expression of A00A in vivo, thereby evaluating the dose effects and time course effects of VEGF-A expression and providing pharmacological evidence to support its in vivo pharmacological efficacy.
8.8.19.4 Methods and experiment
Study design
Dose-Dependent Expression of A00A
Cell Culture and Seeding
In standard culturing procedures, adherent cells are detached through trypsin digestion, then suspended in culture medium, and the cell density in the suspension is assessed. Subsequently, the cell density is adjusted to 1×105 cells/mL, and cells are seeded into a 96-well cell culture plate at densities of 1×104 cells/well (100 μL) or 2×104 cells/well (200 μL) . The cells are cultured overnight to promote adherence to the surface and restoration of normal cell morphology.
Transfection
In a 96-well plate filled with an equal number of cells, different doses (as described below) of A00A molecules were transfected using commercial reagents. Each sample for a specific transfection dose should be plated in triplicate wells.
Table. 11 Transfection in Different types of cell lines
ELISA Detection of VEGF-A Expression
After 24 hours post-transfection, the expression levels of VEGF-A in the cell supernatant at different transfection doses were quantified using ELISA.
In the ELISA detection process, VEGF-A standard samples are initially diluted using a dilution buffer. Subsequently, the cell supernatant is diluted by a factor of 500×. Following dilution, 100 μL of both standard and test samples are added to a 96-well plate that has been pre-coated with anti-human VEGF-A capture antibody. The plate is then incubated at 37℃ for 90 minutes to facilitate the binding of antigens to antibodies.
After the completion of the incubation period, the supernatant is removed, and 100 μL of biotin-labeled anti-human VEGF-A detection antibody, at a working concentration, is added to each well. The plate is again incubated at 37℃ for 60 minutes to allow for the binding of the detection antibody to the captured VEGF-A.
After the incubation with the detection antibody, the plate undergoes washing three times with a washing buffer to remove any unbound antibodies. Following the washing steps, 100 μL of HRP-labeled streptavidin, at a working concentration, is added to each well, and the plate is incubated at 37℃ for 30 minutes to allow the binding of streptavidin to biotin.
Upon completion of the streptavidin incubation, the supernatant is once again removed, and the plate is washed five times with the washing buffer to eliminate any unbound streptavidin-HRP. Following the washing steps, 90 μL of TMB substrate is added to each well, and the plate is incubated at 37℃ in the dark for 5 minutes to initiate the colorimetric reaction.
After the incubation with the TMB substrate, 50 μL of stop solution is added to halt the reaction, and the optical density (OD450) of both standard and test samples is measured using an enzyme-linked immunosorbent assay reader.
Using the known standard concentrations and their corresponding OD450 values, a nonlinear fit equation is derived. Subsequently, the concentration of the test sample is calculated based on the OD450 value of the sample using this equation. The initial calculated result is then adjusted by the dilution factor to determine the final VEGF-A concentration in the corresponding sample well. During plotting and statistical analysis, the average of the detection results from triplicate wells is considered.
Time-Dependent Course of Expression of A00A Expression
Cell Culture and Seeding
Same procedure as detailed in 8.8.19.4 above.
Transfection
In a 96-well plate filled with an equal number of cells, different doses (as described below) of A00A molecules were transfected using commercial reagents. Each sample for a specific transfection dose should be plated in triplicate wells.
Table. 12 Transfection in Different types of cell lines
Cell supernatant (100 μL) was collected at 6h, 24h, 72h, 96h, 120h, 144h, and 168h intervals, with fresh 100 μL cell culture medium replenished after each collection. Upon completion of sample collection, the expression levels of VEGF-A in the cell supernatant at various specific transfection doses were quantified using ELISA (Leif Carlsson, Jonathan C Clarke, Christopher Yen, et al. Biocompatible, Purified VEGF-A mRNA Improves Cardiac Function after Intracardiac Injection 1 Week Post-myocardial Infarction in Swine. Mol Ther Methods Clin Dev. 2018 Apr 10: 9: 330-346. doi: 10.1016/j. omtm. 2018.04.003. ) .
ELISA Detection of VEGF-A Expression
Same procedure as detailed in Section 8.8.19.4 ‘Cell Culture and Seeding’a bove.
Cell line information
Table. 13 Cell Types Evaluated in In Vitro Expression of VEGF-A
Formulation Preparation
Transfection Mixture
Add 25/50/100/200/400 nL of transfection reagent to 5 μL of Opti-MEM, mix thoroughly and let it stand for 10 minutes. Add 12.5/25/50/100/200 ng of A00A sample to 5 μL of Opti-MEM, mix thoroughly, then combine with the transfection reagent Opti-MEM suspension after standing, and let it stand for another 10 minutes, ensuring that the ratio of transfection reagent to nucleic acid is 2: 1. The transfection mixture was prepared for added to the cell culture supernatant to complete transfection.
1x Antibody Working Solution
Dilute the biotin-labeled anti-human VEGF-A detection antibody or HRP-labeled streptavidin to the recommended dilution (1: 100) in corresponding dilution buffer (Biotinylated Detection Ab Diluent and HRP Conjugate Diluent) . Then the 100× Concentrated antibody were diluted to 1×working solution. The working solution should be prepared just before use.
Table. 14 Details of Test Articles Used in Study
Method
Cell Culture
Table. 15 Cell Cultures and Conditions
ELISA method
Table. 16 ELISA Method Used for Measuring Absorbance
8.8.19.5 Results
The dose-dependent expression of VEGF-A in the tested concentration range (25-400 ng) is observed in cardiomyocytes and cardiovascular cells across different species in human, rodent, and pig as shown in FIG. 5.
The time-dependent course expression of VEGF-A is studied in cardiomyocytes and cardiovascular cells across different species in human, rodent, and pig, and the expression of VEGF-A can be sustained for more than 5 days across all cardiomyocytes tested as shown in FIG. 6.
8.8.19.6 Conclusion
The dose-dependent and time-dependent course expression of VEGF-A sustained for more than 5 days in cardiomyocytes across different species in human, rodent, and pig (i.e., immortalized human cardiomyocytes, human iPS-derived cardiomyocytes, rat primary cardiomyocytes, mouse primary cardiomyocytes, immortalized porcine aortic smooth muscle cells and porcine cardiac fibroblast) .
8.8.20 Example 20. The Bioassay of A00A Expression Products from Various Cell
Types Across Different Species to Activate VEGF Receptor 2-Reporter Cell
8.8.20.1 Summary
A00A is a circRNA encoding human VEGF-A protein (VEGF165 subtype; amino acid sequence as set forth in SEQ ID NO: 76) . Specifically, the circRNA of A00A includes IRES sequence of SEQ ID NO: 41, linker sequence of SEQ ID NO: 46, and VEGF-A encoding sequence of SEQ ID NO: 77. The precursor molecule used to produce A00A is set forth in SEQ ID NO: 27, and the precursor backbone is set forth in SEQ ID NO: 40.
In various species of myocardial cells and cardiovascular cells (including vascular smooth muscle cells and fibroblasts) , circular RNA A00A can be continuously translated into human VEGF-A protein upon entering the cells. This translated expressed product is then secreted into the extracellular space, where it enables to binds to VEGFR2 on endothelial cells surrounding the myocardial cells, thereby promoting the formation of new blood vessels (Anna Collén. et al. VEGFA mRNA for regenerative treatment of heart failure. Nat Rev Drug Discov 2022 Jan; 21 (1) : 79-80) .
In this study, after transfecting the circular RNA A00A into myocardial cells and associated cardiovascular related cells of different species in vitro, its translated product, huVEGF-AVEGF165, demonstrates potential activity to stimulate the huVEGFR2 receptor and promote angiogenesis. To demonstrate its ability to bind and activate huVEGFR2, we collected A00A expression products from myocardial cells and cardiovascular cells of various species, including mouse, rat, pig, and human.
After quantifying the concentration of VEGF-A protein in the supernatant using ELISA, the activation status of the reporter cells overexpressing huVEGFR2 can be assessed with specific concentrations (0-100 ng/mL) of A00A expression products, reflecting the proangiogenic potential. Based on the numerical values of the activation signals of downstream pathways of huVEGFR2 in reporter cells, the activation status of huVEGFR2 by A00A expression products at different concentrations in different species of myocardial cells and cardiovascular cells can be simulated, thus mimicking the initial process of promoting angiogenesis in in vivo experiments using different species models.
By analyzing the numerical values of huVEGFR2 activation in reporter cells at different concentrations, the EC50 of A00A expression products activating huVEGFR2 in different species of myocardial cells and cardiovascular cells can be calculated. Additionally, by comparing with the EC50 of recombinant human VEGF-A (VEGF165) , we can observe that the expressed products of A00A in myocardial cells and cardiovascular related cells of different species exhibit similar biological activity to human-derived VEGF-A protein.
8.8.20.2 Objective
The objective of this study is to assess the ability of A00A to activate huVEGFR2 via the expressed product huVEGF-A in myocardial cells and cardiovascular cells from different species, utilizing HEK293-VEGFR2 reporter cell lines. This experimental approach aimed to simulate and validate the pharmacological effect of A00A on promoting angiogenesis by stimulating its associated specific receptor, VEGFR2, following drug administration.
8.8.20.3 Background
The VEGFR2/NFAT Reporter HEK293 Recombinant Cell Line is a specialized HEK293 cell line engineered to express the firefly luciferase reporter gene under the control of NFAT response elements. This cell line also features stable expression of human VEGFR2 (Human Vascular Endothelial Growth Factor Receptor 2; KDR, FLK1; ref seq. NM_002253.1) .
VEGF is a highly specific angiogenic factor that primarily regulates signaling pathways through two tyrosine kinase receptors, VEGFR1 and VEGFR2. Among these receptors, VEGFR2 plays a key role in angiogenesis. Activation of the tyrosine kinase receptor-mediated calcium ion signaling pathway promotes the generation of T cell transcription factors (NF-AT) (Takahashi, S. (2011) Vascular Endothelial Growth Factor (VEGF) , VEGF receptors and their inhibitors for anti-angiogenic tumor therapy. Biol. Pharm. Bull. 34, 1785–88) .
The VEGFR2-NFAT-HEK293 reporter system is designed to reflect the response mechanism of the VEGFR2 signaling pathway. It serves as a valuable complement to the conventional HUVEC assay and aids in validating the activation of VEGFR2 by cell-expressed VEGF-A products, thereby promoting angiogenesis (Wang L. et al. (2016) Development of a robust reporter-based assay for the bioactivity determination of antiVEGF therapeutic antibodies. J Pharm. Biomed. Anal. 125, 212–18) .
The Human VEGFR2 (Luc) HEK293 Reporter Cell is engineered to express both NFAT signaling response elements and the full length human VEGFR2 receptor (Gene ID: 3791) . When stimulated with human VEGF protein, the VEGF/VEGFR2 interaction drives NFAT-mediated luminescence. Inhibition of VEGF binding to VEGFR2 by either anti-VEGF or anti-VEGFR2 antibodies results in a decrease in luminescence. This reporter cell line serves a dual purpose: screening for anti-human VEGFR2 or anti-human VEGF neutralizing antibody and small molecule inhibitors, as well as evaluating the binding activity of VEGF-A protein produced from A00A in different cells.
VEGF-A, as a ligand for VEGFR2, elicits a dose-dependent response. The level of luminescence in the assay is directly proportional to the activity of VEGF-A. The dose-response relationship can be calculated using a four-parameter model to determine the EC50, which can then be compared with the activity of recombinant VEGF-A protein serving as a positive control.
8.8.20.4 Methods and Experiments
Study Design
Myocardial cells and cardiovascular related cells of different species were transfected with A00A and incubated for 48 hours. The cell supernatant containing A00A expression products, specifically the human VEGF-A protein (VEGF165 subtype-A) , was harvested and quantified by ELISA.
A00A (test article) expression products from different species and VEGF165 protein (a commercially available isoform of huVEGF-A) were diluted to concentrations between 0-100 ng/mL using the reporter cell assay culture medium (DMEM) . The test article and control solutions were then incubated for 6 hours in human VEGFR2 (Luc) HEK293 reporter cells, in which huVEGF-A binds to huVEGFR2 on the membrane of reporter cells and activates the downstream NFAT-Luciferase pathway (see FIG. 7 and 8.8.20.3) . The activation of huVEGFR2 is evaluated by measuring luminescence of the reporter cells, from which the EC50 values can be calculated.
Methods
Collection of A00A Expression Product
Different species of myocardial cells were seeded in a 12-well plate at a cell density of 2.5×105 cells/well. After 16 hours of incubation, RNA samples (A00A, Lot No. D23102301) were transfected into the cells using Lipofectamine Messenger MAX reagent at a transfection dose of 2 μg/well.
The transfection reagent and RNA were mixed at a ratio of 2: 1 and slowly added to the cell supernatant over 10 minutes. Details of the transfection formulation preparation can be found in paragraph [00679] . After 6 hours of transfection, the original myocardial cell culture medium (500 mL/well) was replaced. Following the medium change, the cells were further incubated for 48 hours. The supernatant was collected from the cell culture dish in a laminar flow hood and transferred to 1.5 mL centrifuge tubes. The tubes were centrifuged at 2000 rpm for 5 minutes to remove debris. The collected supernatant was used as the A00A expression product.
Quantification of A00A Expression Product
20 μL of the collected supernatant was taken and the concentration of A00A expression product was measured using a Human VEGF-A ELISA Kit. The samples were diluted in sample diluent at dilutions of 500×, 1000×, 2000×, and 4000×, respectively. Following the instructions provided in the kit, the VEGF-A concentration in the supernatant was determined.
Reporter Cell Experiment
Based on the ELISA results of A00A expression product from different cells, samples were diluted to concentrations ranging from 0-100 ng/mL. Negative control (NC) samples were diluted in the same proportion. Positive control VEGF165 protein was diluted in DMEM medium to a theoretical concentration of 2000 ng/mL. Then, based on the ELISA results, DMEM complete medium was further diluted to the same concentrations of 0-100 ng/mL.
HEK293-VEGFR2 cells were diluted to a concentration of 8×105 cells/mL. For each desired concentration of A00A expression product, three replicate wells were set up in a 96-well white bottom clear cell culture plate, with 50 μL added per well (equivalent to 4×104 cells/well) .
The diluted A00A expression products (50 μL) were added to the HEK293-VEGFR2 cell supernatants in the cell culture plate. The plate was then incubated at 37℃ with 5%CO2 for 6 hours. Subsequently, 50 μL of Bright-Lite Luciferase Assay System substrate was added to each well, and the plate was incubated at room temperature for 10 minutes. Details of the formulation preparation for Bright-Lite Luciferase Assay System substrate can be found inparagraph [00680] . Fluorescence readings were then taken using a multifunctional ELISA reader.
Based on the concentrations of VEGF-A in the A00A expression product and VEGF165 protein, as well as the corresponding fluorescence readings from the HEK293-VEGFR2 reporter cells, a four-factor fit was used to calculate the EC50 for each cell expression supernatant and VEGF165 recombinant protein.
Cell Line Information
Table. 17 Cell Types Evaluated in In Vitro Activation of VEGF Receptor 2-Reporter Cell
Formulation Preparation
Transfection
Add 2 μL of transfection reagent to 50 μL of Opti-MEM, mix thoroughly and let it stand for 10 minutes. Add 1 μg of A00A sample to 50 μL of Opti-MEM, mix thoroughly, then combine with the transfection reagent Opti-MEM suspension after standing, and let it stand for another 10 minutes before proceeding to the next step for transfection mentioned inparagraphs [00671] to
.
Fluorescence Detection
Thaw and equilibrate the Bright-Lite Luciferase Assay System substrate in the dark at 4℃ in advance. Prior to use, bring it to room temperature. Remove the 96-well cell culture plate from the incubator and allow it to equilibrate to room temperature. Add 50 μL of Bright-Lite Luciferase Assay System substrate to each well of the culture plate, gently tap or pipette mix to ensure thorough mixing, then proceed to fluorescence signal detection mentioned in paragraphs
to [00677] using the appropriate instrumentation.
Dose Verification
A00A Nucleic Acid Concentration
The solution was diluted 5×, 25×, and 125× with nuclease-free water. The RNA concentration of the undiluted and diluted samples was quantified using NanoDropTM One/UV-Vis spectrophotometer. A coefficient of variation (CV) <15%was considered acceptable for reliable concentration measurements.
A00A Product Protein Concentration
Different A00A expression product samples from supernatants of various cells were diluted 500×, 1000×, 2000×, and 4000× with a specialized diluent. The concentration of the diluted products was determined using a Human VEGF-A ELISA Kit. After back calculation, a coefficient of variation (CV) <15%was considered acceptable for reliable protein concentration measurements.
Bioanalysis
After the 6-hour stimulation of A00A expression products to VEGFR2 reporter cell line are over, add Bright-Lite Luciferase Assay System substrate and incubate at room temperature for 10 minutes. Measure luminescence corresponding to graded concentrations from 0 to 100 ng/mL using a luminescence plate reader (Biotek, Synergy HI) . Graph the data as RLU (y axis) versus Log10 [VEGF-A concentrations] (x axis) . Fit curves using nonlinear regression-analysis and determine the EC50 value of A00A-productions by calculating the dose-dependent response relationship based on a variable slope (four parameters) model using GraphPad software.
Table. 18 Fluorescence values from HEK293-VEGFR2 Reporter Cells
8.8.20.5 Conclusion
The translated products of A00A in human iPS-derived cardiomyocytes, immortalized human cardiomyocytes, porcine primary myocardial fibroblasts and aortic smooth muscle immortalized cells, rat primary cardiomyocytes, and mouse primary cardiomyocytes have a dose-dependent effect on the activation of HEK293-hVEGFR2-Luci cells, and EC50 values of those productions were close to the human VEGF165 recombinant protein. (FIG. 8)
8.8.21 Example 21. Activation Analysis of A00A Expression Products in Various Cell Types Across Species to Stimulate VEGF Receptor 2
8.8.21.1 Summary
Under physiological conditions in the human body, the vascular endothelial growth factor-A (VEGF-A) protein interacts with its receptor VEGFR2, initiating phosphorylation signaling cascades. This phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) triggers downstream pathways that facilitate endothelial cell functions such as proliferation, migration, invasion, and vascular formation. Once A00A enters into target cells, it is translated into human VEGF-A protein (VEGF165 subtype) , which is then secreted into the extracellular space. The VEGF165 produced by the A00A enables to binds to VEGFR2 on endothelial cell surfaces, activating downstream signaling pathways and promoting blood vessel formation. To confirm this pharmacological process, we stimulated human umbilical vein endothelial cells (HUVEC) with A00A expression products from myocardial cells and cardiovascular cells of various species in vitro. By assessing the phosphorylation levels of VEGFR2 and protein in the downstream pathway, we observed activation of VEGFR2 and its downstream pathway, consistent with previous findings. This study indicates that A00A expression products possess the capability to stimulate endothelial cell-mediated angiogenesis at a molecular level.
8.8.21.2 Objective
This study aimed to investigate the ability of A00A expression products from different species'myocardial cells and porcine cardiac fibroblast to activate huVEGFR2 via huVEGF-A using protein immunoblotting (Western Blot, WB) . This experimental approach aimed to elucidate and validate the molecular mechanism by which A00A translation products stimulate their corresponding receptor, VEGFR2, after administration, thus promoting angiogenesis.
8.8.21.3 Background
VEGFR2/KDR/Flk-1 serves as a primary receptor for transmitting VEGF-triggered signals within endothelial cells. Upon VEGF binding, VEGFR2 undergoes self-phosphorylation, initiating its activation. An essential site for this autophosphorylation lies within the kinase insert domain at Tyr1175. Once activated, the receptor swiftly recruits adaptor proteins such as Shc, GRB2, PI3 kinase, NCK, and the protein tyrosine phosphatases SHP-1 and SHP-2 (JKroll 1, J Waltenberger. The vascular endothelial growth factor receptor KDR activates multiple signal transduction pathways in porcine aortic endothelial cells. J Biol Chem. 1997 Dec 19; 272 (51) : 32521-7) . Phosphorylation at Tyr1175 facilitates binding of the intracellular domain to the p85 subunit of PI3 kinase and PLCγ, as well as Shb. This signaling cascade from VEGFR2 is indispensable for VEGF-induced cellular processes including proliferation, chemotaxis, sprouting, and the survival of endothelial cells both in vitro and in vivo, crucial for angiogenesis (Lorena Pérez-Gutiérrez & Napoleone Ferrara, Biology and therapeutic targeting of vascular endothelial growth factor A. Nature Reviews Molecular Cell Biology volume 24, pages816–834 (2023) ; Michael Simons, Emma Gordon & Lena Claesson-Welsh. Mechanisms and regulation of endothelial VEGF receptor signalling. Nature Reviews Molecular Cell Biology volume 17, pages611–625 (2016) ) .
HUVECs are primary cells isolated from the umbilical vein. They serve as a model system for studying endothelial cells and find application in various research areas such as hypoxia, inflammation, oxidative stress, infection responses, as well as normal and tumor-associated angiogenesis (Lorena Pérez-Gutiérrez & Napoleone Ferrara, Biology and therapeutic targeting of vascular endothelial growth factor A. Nature Reviews Molecular Cell Biology volume 24, pages816–834 (2023) ; Laurent Lamalice, Houle, Guillaume Jourdan & Jacques Huot. Phosphorylation of tyrosine 1214 on VEGFR2 is required for VEGF-induced activation of Cdc42 upstream of SAPK2/p38. Oncogene volume 23, pages434–445 (2004) ; Arlinda Ljoki 1, Tanzila Aslam 1, Tina Friis 1, Ragnhild G Ohm 2, Gunnar Houen. In Vitro Angiogenesis Inhibition and Endothelial Cell Growth and Morphology. Int. J. Mol. Sci. 2022, 23 (8) , 4277) .
By stimulating HUVECs with A00A expressed products in vitro, and subsequently detecting the phosphorylation of VEGFR2 and downstream proteins in angiogenesis-related pathways on HUVECs, this method can simulate and evaluate the activity of A00A in promoting angiogenesis in vivo (Hong Xin, Cuiling Zhong, Eric Nudleman, Napoleone Ferrara. Evidence for Pro-angiogenic Functions of VEGF-Ax. Cell. 2016 Sep 22; 167 (1) : 275-284. e6) .
8.8.21.4 Methods and experiments
Methods
Collection of A00A Expression Products
Specific cells from different species were seeded at a density of 2.5×105 cells/well in a 12-well plate. 16 hours after plating, RNA samples (A00A, Lot No. D2312002) were transfected into the cells at a dosage of 2 μg/well using Lipofectamine MessengerMAX reagent. The transfection reagent was mixed with the RNA samples at a ratio of 2: 1 and slowly added to the cell culture supernatant over a period of 10 minutes. Following transfection for 6 hours, the original culture medium was replaced with the original cell’s culture medium at 500 μL/well. After medium replacement, cells were further cultured for 48 hours. Cell supernatant was collected from the cell culture dishes within a laminar flow hood and transferred to 1.5 mL centrifuge tubes. Cell debris was removed by centrifugation at 2000 rpm for 5 minutes. The resulting supernatant was collected as the A00A cell expression product for each different species.
Quantification of A00A Expression Products
20 μL of the collected supernatant was collected and concentration was detected by using the Human VEGF-A ELISA Kit (Elabscience; Catalog No. E-EL-H0111) . The samples were diluted with sample dilution buffer at dilutions of 500×, 1000×, 2000×, and 4000×. VEGF-A concentration in the supernatant was determined by following the instructions provided in the ELISA kit manual. Before stimulating HUVEC cells, the diluted supernatant was added to the HUVEC cell culture medium to achieve a final concentration of 20 ng/mL, according to the quantified concentration.
Cultivation and Stimulation of HUVEC
After HUVECs in logarithmic growth phase were digested with trypsin, they were neutralized and resuspended in complete medium containing serum, then counted. 1×106 cells/well were seeded in a 6-well cell culture plate and allowed to fully attach by incubating them in complete medium for 6 hours. Subsequently, the existing culture medium was replaced with serum-free endothelial cell culture medium, and the cells were starved for 16 hours. The A00A expression product from cells of different species was added to achieve a final concentration of 20 ng/mL. After stimulating the cells for 15 minutes, they were washed once with pre-chilled PBS and immediately lysed by adding 100 μL of lysis buffer (containing protease inhibitors and phosphatase inhibitors) . The plate was then placed on ice for 20 minutes to extract total proteins.
Detection of VEGFR2 and its Downstream Phosphorylated Molecules
After adding 20 μL of 5× SDS-PAGE protein loading buffer and thoroughly mixing, the samples were heated at 95℃ for 5 minutes. Upon cooling to room temperature, equal amounts of protein were loaded into the wells of SurePAGE gels in parallel for two sets within the same gel. One set was used to detect total protein, while the other set was used to detect phosphorylated protein. After electrophoresis, the semi-dry transfer method was employed with 25V for 10 minutes to transfer the proteins onto PVDF membranes. The membranes were then blocked with 5%BSA/0.5%TBS-Tween-20 for 1 hour. Human VEGFR2 antibody (diluted 1: 1000) and phosphorylated VEGFR2 antibody (diluted 1: 1000) were incubated overnight at 4℃. After washing for 10 minutes, HRP-conjugated anti-rabbit IgG antibody (diluted 1: 2000) was added and incubated at room temperature for 45 minutes. Following another 10-minute wash, ECL staining was applied, and the image data were saved using a Cytiva AIQ800 imager.
After staining, the PVDF membranes were subjected to Stripping Buffer on a horizontal shaker at 80 rpm for 20 minutes to remove the antibodies. The membranes were then re-blocked and incubated with human ERK1/2 antibody (diluted 1: 1000) and phosphorylated ERK1/2 antibody (diluted 1: 1000) , followed by staining and recording of image data using the same method as for the detection of total VEGFR2 and phosphorylated VEGFR2.
Study Design
1. Transfection of A00A into Cardiomyocytes of Different Species
2. Collection and ELISA Quantification of A00A Expression Products (huVEGF-A protein) from Cardiomyocytes of Different Species
3. Stimulation of HUVECs Cells with A00A Expression Products and Collection of HUVECs Cell Lysates
4. Detect the levels of total protein and phosphorylated protein of huVEGFR2 and ERK1/2 in the cell lysates of HUVECs using Western Blot
Cell Line Information
Table. 19 Cell Line Information
Formulation Preparation
A00A Expression Products in Cardiomyocytes of Different Species
For the expression of A00A in cardiomyocytes of different species, the following steps were performed:
1. Transfection Reagent Preparation: 4 μL of transfection reagent were added to 50 μL of Opti-MEM medium, thoroughly mixed, and incubated for 10 minutes at room temperature.
2. A00A Sample Preparation: 2 μg of A00A sample were added to 50 μL of Opti-MEM medium, thoroughly mixed, and incubated for 10 minutes at room temperature.
3. Transfection Complex Preparation: After the incubation period, the A00A sample in Opti-MEM was combined with the transfection reagent/Opti-MEM suspension, thoroughly mixed, and allowed to stand for an additional 10 minutes before proceeding to the next step of the transfection process.
HUVEC Complete Culture Medium
To prepare 500 mL complete medium for HUVECs culture, 465 mL endothelial cell base medium adds 25 mL fetal bovine serum; 5mL endothelial cell growth factor and 5mL penicillin/streptomycin solution.
1x Antibody Working Solution
Dilute the primary antibody or secondary antibody to the recommended dilution (1: 1000) in fresh blocking solution (Western Blocking and Antibody Dilution Buffer) .
Chromogenic (Colorimetric) Substrate Solution
According to the commercial reagent instructions, Mix Clarity Western Peroxide Reagent and Clarity Western Luminol/Enhancer Reagent in a ratio of 1: 1.
Dose Verification
A00A Nucleic Acid Concentration
The concentration of A00A nucleic acid was determined by diluting the solution 5x, 25x, and 125x with nuclease-free water. The diluted samples, along with the undiluted solution, were quantified for RNA concentration using the NanoDropTM One/UV-Vis spectrophotometer. A coefficient of variation (CV) of <15%was considered indicative of reliable concentration.
A00A Nucleic Acid Concentration
The concentration of A00A nucleic acid was determined by diluting the solution 5x, 25x, and 125x with nuclease-free water. The diluted samples, along with the undiluted solution, were quantified for RNA concentration using the NanoDropTM One/UV-Vis spectrophotometer. A coefficient of variation (CV) of <15%was considered indicative of reliable concentration.
A00A Protein Concentration
Samples of A00A expression products from different cells were diluted 500x, 1000x, 2000x, and 4000x with specific dilution buffer. The diluted samples were then subjected to quantification of protein concentration using the Human VEGF-A ELISA Kit. Protein concentration calculations were performed, and a coefficient of variation (CV) of <15%was considered indicative of reliable protein concentration.
Bioanalysis
The intensity of bands observed in the Western blot (WB) results serves as a measure of both total protein expression and the level of protein phosphorylation. Evaluation of the phosphorylated protein/total protein ratio allows for the assessment of the activation levels of specific proteins, including VEGFR2 and ERK1/2. This analytical approach facilitates the emulation of the molecular biology process by which A00A exerts its pharmacological effects in vivo, thus enabling the evaluation of the bioactivity of A00A expression products.
8.8.21.5 Results
By stimulating human umbilical vein endothelial cells (HUVEC) , the expression products of A00A in human iPS-derived cardiomyocytes, immortalized human cardiomyocytes, mouse cardiomyocytes, rat cardiomyocytes and porcine cardiac fibroblast can activate VEGFR2 and its downstream vascular generation-related pathways. In porcine cardiac fibroblasts, the expression products of A00A do not activate VEGFR2 and its downstream vascular generation-related pathways (FIG. 9) .
8.8.21.6 Conclusions
After stimulating HUVECs, A00A expression products obtained from human and rat cardiomyocytes and porcine cardiac fibroblast were capable of activating VEGFR2 and its downstream pathway involved in angiogenesis. Immortalized porcine aortic smooth muscle cells were also able to activate VEGFR2 and its downstream vascular generation-related pathways but not for porcine cardiac fibroblasts.
8.8.22 Example 22. The transfection efficiency and protein translation of VEGF-A circRNA in mouse heart were dose-dependent (24h)
VEGF-A circRNA citrate solution was administered to the 8-week-old, male, C57BL/6J mice via left ventricular myocardial injection (Dosage range: 60 μg/mouse, volume: 30 μL, 6 mice per group) . 24 hours after the administration, the left ventricular tissue homogenate was collected and then tissue RNA and tissue protein was extracted, respectively. By using the VEGF-A circRNA sequence specific-primer (SSP) , the level of the VEGF-A circRNA in the extracted RNA sample was quantified so that the transfection efficiency of VEGF-A circRNA at different dosages were compared. The expression of VEGF-A in the tissue was quantified using MSD V-PLEX Human VEGF Kit so that the protein translation of VEGF-A circRNA in left ventricle at different dosages were demonstrated. As shown in FIGs. 10A and 10B, both the circRNA level and the protein expression level of VEGF-A in the mouse left ventricle exhibited consistent dose-dependent patterns.
8.8.23 Example 23. The expression of VEGF-A circRNA and VEGF-A mRNA in mouse myocardial tissue were at comparable level and dose-dependent
VEGF-A circRNA citrate solution and synthetic VEGF-A mRNA were administered to the 8-week-old, male, C57BL/6J mice via left ventricular myocardial injection (Dosage range: 0-150 μg/mouse, volume: 30 μL, 6 mice per group) . 24 hours after the administration, the left ventricular tissue homogenate was collected and then the tissue protein was extracted. By using the MSD V-PLEX Human VEGF Kit, the expression of the VEGF-A in the left ventricular tissue was quantified so that the translation levels of VEGF-A circRNA and VEGF-A-mRNA in the left ventricular tissue were compared. The result demonstrated that the expression levels of VEGF-A in the mouse left ventricular tissue were comparable when using the same dosage of VEGF-A circRNA and VEGF-A mRNA. Additionally, the protein translation were both dose-dependent and followed a similar pattern with dosage escalation. (FIG. 11)
8.8.24 Example 24: The expression of VEGF-A circRNA in mouse myocardial tissue had long-term effects
VEGF-A circRNA citrate solution and synthetic VEGF-A mRNA were separately administered to the 8-week-old, male, C57BL/6J mice via left ventricular myocardial injection (Dosage range: 60 μg/mouse, volume: 30 μL, 4 mice per group) . Respectively, the left ventricular tissue homogenate was collected 24h, 48h, 72h, 120h, 168h after the administration. By using MSD V-PLEX Human VEGF Kit, the expression of VEGF-A was quantified so that the translation levels of VEGF-A circRNA and VEGF-A mRNA in the left ventricular tissue at different time points after the administration were compared. The result showed that, 7 days after the administration, the area under the curve (AUC) of VEGF-A circRNA was larger compared to that of VEGF-A-mRNA (AUC: 2933 vs 1727) and VEGF-A circRNA had longer half-life (T1/2: 457 h vs 10.45 h) , which demonstrated that the expression and metabolic cycle of VEGF-A circRNA lasted longer in vivo than that of mRNA (FIG. 12) .
8.8.25 Example 25. Pharmacodynamic Evaluation of A00A on Ischemic Myocardial Infarction Model of Bama Miniature Pig
8.8.25.1 Abstract
Objective: To evaluate the pharmacodynamic effects of A00A (the HM2002 test article) on ischemic myocardial infarction model of the Bama miniature pig.
Methods: Based on baseline ejection fraction (EF) and body weight (BW) , 13 animals were randomly assigned into 4 groups: G1-Sham (N=1) , G2-Vehicle (N=4) , G3-A00A_1mg (N=4) , and G4-A00A_4mg (N=4) . Except for the animals of the sham group, the animals in other groups were subjected to establish a model of ischemic myocardial infarction (MI) by ligation of the anterior descending branch of the left coronary artery when performed a thoracotomy operation. Seven days after ischemic myocardial infarction modeling, the second thoracotomy was undergone while multi-point myocardial injection was performed in the infarcted and peri-infarcted areas. Echocardiography was performed to evaluate the cardiac function before modeling, at 7 days after modeling (before dosing) , and 7, 14, 21, and 28 days after dosing. Myocardial TTC and Masson staining were performed to evaluate myocardial infarction and fibrosis at 28 days after dosing, respectively.
Results: During the study, no obvious abnormal clinical symptoms were observed in the test animals, and their body weight were increasing slightly.
During the study, EF and fractional shortening (FS) values in sham group (G1) were stable and no significant changes were observed. Compared with before modeling, EF and FS values of each modeling group decreased significantly at 7 days after modeling (i.e., Day 0) , indicating that the model was established successfully. EF and FS values in the vehicle control group (G2) continued to decrease, and reached the lowest level at 21 days after dosing (28 days after modeling) .
Compared with before dosing (Day 0) , EF and FS values in all dosing groups (G3-G4) were significantly increased at 7 days after dosing (EF values were increased by 6.4%, 12.9%, respectively, and p<0.01, G3 vs G4; FS value increased by 8.8%, 17.1%, respectively, and p<0.01, G3 vs G4) , and maintain a continuous upward trend until the end of study period (Day 28) . At the end of study period (Day 28) , EF and FS values were significantly increased in each dosing group (G3-G4) (EF values were increased by 12.5%, 22.7%, respectively, and p<0.05, G3 vs G4; FS values were increased by 18.1%, 31.3%, respectively) . Compared with the vehicle control group, EF and FS values increased significantly at all time points after dosing in each dosing group (G3-G4) .
During the study, left ventricular end diastolic volume (LVEDV) and left ventricular end systolic volume (LVESV) values in sham group were stable and no significant changes were observed. Compared with before modeling, LVEDV values of each modeling group increased to different degrees after modeling, and no significant difference was observed among the groups. Compared with before modeling, LVESV values increased significantly in all modeling groups at 7 days after modeling, and no significant differences were observed among the groups. Compared with before dosing (i.e., Day 0) , LVESV values of the vehicle group increased significantly at 7 days after dosing and maintained this level until the end of the study period (Day 28) , LVESV values in G3 remained at the pre-dosing level until the end of the study period, LVESV values in G4 decreased significantly. Compared with the vehicle control group, LVESV values in each dosing group (G3-G4) was lower than that in the vehicle control group at all time points after dosing.
At the end of the study period, myocardial TTC staining showed that no MI was observed in the sham group, and there were different degrees of MI in other modeling groups. Compared with the vehicle control group, the MI weight and ratio of MI in all treatment groups were significantly reduced (the ratio of MI in G2-G4 were 10.6%, 4.5%, 3.4%, respectively) . Myocardial Masson staining showed that myocardial fibrosis (%) in the vehicle control group was 2.9 times higher than that in the sham group. Compared with the vehicle control group, myocardial fibrosis (%) in each treatment group was reduced in different degrees (the ratio of myocardial fibrosis in G2-G4 were 6.2%, 4.4%, 1.8%, respectively) . The degree of myocardial fibrosis in G4_A00A_4mg group was significantly reduced.
Conclusion: Under the conditions of this study, A00A (the HM2002 test article) showed positive effects on cardiac function (EF and FS values) , myocardial infarction and fibrosis in Bama miniature pigs with ischemic myocardial infarction. A00A showed a certain degree of dose dependency, and the lower dose 1mg has a good efficacy.
8.8.25.2 Background
A00A is a VEGF-A-coded (Vascular Endothelial Growth Factor A) circular RNA. In preliminary pharmacological studies, A00A demonstrated continuously dose-dependent expression of VEGFA in human, pig, rat, and mouse cells inc vitro, which activated VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) and its downstream signaling pathways. VEGF-A was reported to induce the proliferation and migration of vascular endothelial cells and promote angiogenesis. This process is critical in cardiovascular diseases management, where improved blood supply can alleviate symptoms and enhance tissue repair in conditions like ischemic heart disease, refractory angina, and peripheral artery disease. The further in vivo efficacy of A00A is needed to be studied. Previous studies of mRNA-8601 (AZD-8601) , a modified VEGF-A-coded messenger RNA, have shown that permanent occlusion myocardial infarction models in small-and large-animal, VEGFA mRNA improves systolic ventricular function and limits myocardial damage. Following a single administration a week post-infarction in mini pigs, left ventricular ejection fraction, inotropy, and ventricular compliance improved, border zone arteriolar and capillary density increased, and myocardial fibrosis decreased at 2 months post-treatment (Carlsson L, Clarke JC, Yen C, et al. Biocompatible, purified VEGF-A mRNA improves cardiac function after intracardiac injection 1 week post-myocardial infarction in swine. Mol Ther Methods Clin Dev. 2018; 9: 330-346) . Furthermore, the studies were transferred to Clinical Phase IIa trial in subjects with heart failure undergoing coronary artery bypass grafting (CABG) surgery via epicardial injection.
The permanent occlusion myocardial infarction model in mini pig used in the preclinical study of AZD0861 is one of the most common preclinical models of MI currently employed in the researches on ischemic heart disease, which has the similar pathophysiology of post-MI remodeling to humans. Myocardial ischaemia in the mini pig MI model or patients with ischemic heart disease, which starves cardiomyocytes of vital oxygen leading to acute cardiomyocyte death and necrosis within the infarct zone (IZ) . The surviving cardiomyocytes immediately surrounding the IZ create a transcriptomically and functionally distinct border zone (BZ) , where tissue can be salvaged or die, leading to expansion of the initial infarct (Martin TP, MacDonald EA, Elbassioni AAM, et al. Preclinical models of myocardial infarction: from mechanism to translation. Br J Pharmacol. 2022; 179 (5) : 770-791) . Angiogenesis and lymphangiogenesis in the BZ permit cardiomyocyte salvage and promotion of vessel maturation and can prevent infarct expansion beyond the IZ (Klaourakis K, Vieira JM, Riley PR. The evolving cardiac lymphatic vasculature in development, repair and regeneration. Nat Rev Cardiol. 2021; 18 (5) : 368-379) . Molecular and cellular changes in the heart after MI lead to ventricular contractile dysfunction and changes to left ventricular (LV) architecture including wall stress, dilation, myocardial stiffness, infarct expansion and thinning of the myocardium (collectively referred to as adverse cardiac remodelling) . Contractile dysfunction and thinning of the LV posterior wall can be observed as early as 1 day after MI (Fattah C, Nather K, McCarroll CS, et al. Gene therapy with angiotensin- (1-9) preserves left ventricular systolic function after myocardial infarction. J Am Coll Cardiol. 2016; 68 (24) : 2652-2666; Sicklinger F, Zhang Y, Lavine KJ, et al. A minimal-invasive approach for standardized induction of myocardial infarction in mice. Circ Res. 2020; 127 (9) : 1214-1216) . Activation of multiple intracellular signaling cascades (Prabhu SD, Frangogiannis NG. The biological basis for cardiac repair after myocardial infarction: from inflammation to fibrosis. Circ Res. 2016; 119 (1) : 91-112) and adverse cardiac remodeling leads to the initiation of asymmetrical LV remodeling that includes the key hallmarks of human MI including thinning of the LV free wall, thickening of the septal non ischaemic LV wall (Lindsey ML, Kassiri Z, Virag JAI, et al. Guidelines for measuring cardiac physiology in mice. Am J Physiol Heart Circ Physiol. 2018; 314 (4) : H733-H752) , increased wall stress leading to progressive dilation, expansion of the scar and deterioration of cardiac function. Infarct size, dilation and impaired contractility (e.g., reduced EF) are major indicators of consequent adverse cardiovascular outcomes including HF and mortality (Konstam MA, Kramer DG, Patel AR, et al. Left ventricular remodeling in heart failure: current concepts in clinical significance and assessment. JACC Cardiovascular Imaging. 2011; 4 (1) : 98-108) .
However, pigs may not reproduce all aspects of remodelling in human hearts. For example, a recent study has shown that pig cardiomyocytes grow primarily by multi-nucleation and longitudinal hypertrophy, distinct from humans (Velayutham N, Alfieri CM, Agnew EJ, et al. Cardiomyocyte cell cycling, maturation, and growth by multinucleation in postnatal swine. J Mol Cell Cardiol. 2020; 146: 95-108) . Moreover, permanent ligation and resultant large infarcts as produced in this model are less frequently observed in humans. [2]
Importantly, however, no single preclinical model can perfectly reproduce the complexity of MI in humans. Indeed, a major advantage of using the permanent ligation method is that the extensive infarct induced provides more distinct differences in cardiac function between sham and MI animals. [2] Furthermore, the heart size, heart rate, vascular anatomy and BP of mini pig are more akin to humans, as well as the low collateral flow (Silva KAS, Emter CA. Large animal models of heart failure: a translational bridge to clinical success. JACC Basic Transl Sci. 2020; 5 (8) : 840-856) . Cardioprotective signalling in pigs is thought to be closer to that in humans, making it a likely more predictive model of translation for agents designed to protect cardiomyocytes from death following ischaemia (Heusch G, Skyschally A, Schulz R. The in-situ pig heart with regional ischemia/reperfusion-ready for translation. J Mol Cell Cardiol. 2011; 50 (6) : 951-963) .
In conclusion, an ischemic myocardial infarction model would be established by permanent ligation of the left anterior descending (LAD) coronary artery in mini pigs to evaluate the pharmacodynamic effects of A00A. Gold standard primary end point is myocardial infarct size (TTC) , potential secondary end points are measures of LV function (Lindsey ML, Bolli R, Canty JM Jr, et al. Guidelines for experimental models of myocardial ischemia and infarction. Am J Physiol Heart Circ Physiol. 2018; 314 (4) : H812-H838) . Considering animal welfare, the second thoracotomy for A00A administration was performed without the 1-week surgical nursing period after the first thoracotomy. Changes that occur over the first week provide information on myocyte cell death and infarct development, inflammation and leukocyte physiology, extracellular matrix turnover and fibroblast activation, and the role of endothelial cells in neovascularization. [12] Furthermore, according to the experience of our testing facility and AZD8601, timing of A00A administration would be a week post-infarction, when EF value is supposed to be significantly reduced and clear infarct area formed. Clear IZ and BZ would be visible to deliver A00A via intracardiac injections across the peri-infarct and infarct area, targeting BZ. A00A is supposed to mainly distribute in the BZ, express VEGF-A, stimulate angiogenesis in the BZ permit cardiomyocyte salvage to improve LV function and prevent infarct expansion beyond the IZ, indicating its potential in the treatment of ischemic heart disease.
8.8.25.3 Objective
To evaluate the pharmacodynamic effects of A00A (the HM2002 test article) on ischemic myocardial infarction model of Bama miniature pig.
8.8.25.4 Experimental Declaration
This is a non-GLP compliance study. The project was performed at TaiPu MedTech (Suzhou) Co., Ltd. The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of TaiPu MedTech (Suzhou) Co., Ltd. The animal experimentation process complies with the “China Laboratory Animal Welfare Guidelines” .
8.8.25.5 Study Schedule
Animal Adaptation Start Date: 2023-07-04
Animal Dosing Start Date: 2023-07-18
Experimental Completion Date: 2023-08-16
Study Completion Date: 2024-XX-XX
8.8.25.6 Dose Formulation Preparation and Disposition Preparation of Dose Formulation
The vehicle provide by the sponsor was used for G1-Sham and G2-Vehicle.
The mother liquor provided by the sponsor was used for G4-A00A_4mg group.
Took out the vehicle to dilute the corresponding stock solution (5 mg/ml) to obtain the dose
formulation with concentration of 1.25 mg/ml, which was used for G3-A00A_1mg group.
Storage and Disposal of Remaining Dose Formulation
The remaining dose formulation were stored at -60℃ or below.
At the end of the study, all the remaining dose formulation had been sent back to the sponsor.
8.8.25.7 Animal Housing and Monitoring
Test Animals
Species/Strain: Bama miniature pig
Age: 3-5 months
Body Weight: 10-14 kg
Number and Sex: 17 animals (including 4 backup animals) , male
Animal Supplier: Pizhou Dongfang Breeding Co., Ltd.
Housing Condition
Test animals were housed individually in stainless steel cages equipped with automatic drinking valves to provided uninterrupted drinking water 24 hours a day. The animal room implements temperature and humidity monitoring and control. The target range for humidity is 40%to 70%, and the target range for temperature is 18℃ to 26℃. Any exceedance of this range for a period greater than 2 hours will be recorded as a deviation. The air in the room is refreshed at least 10 times per hour. A time-controlled lighting system (lighting from 7 am. to 7 pm. ) provides a 12-hour light/12-hour dark cycle. During the study, the cages are cleaned regularly according to facility SOPs to maintain hygiene.
Feed and Water
The experimental animals are given 150 g of normal feed twice a day, in the morning and afternoon. The nutritional composition and contaminants in the feed are routinely tested by the supplier /or an independent laboratory. Analysis reports and feed batch numbers are kept within the experimental facility. Animal drinking water is the organic chlorine disinfection reverse osmosis water provided by the automatic drinking water system, which is freely used. The drinking water is regularly tested for contaminants by an independent laboratory. The amounts of contaminants in drinking water should not reach a range that may affect the results of the experiment. Analysis reports for the drinking water are kept in the veterinary department of the experimental facility.
8.8.25.8 Study Design
Groups and Doses Design
After animals were entered the facility, a 7-day acclimation period began. The animals were randomly divided into 4 groups according to the EF and body weight during the adaptation period. Specific information is shown in FIG. 13:
Model Preparation
The animals were fasted overnight and weighed on the day of surgery. Anesthesia was induced with Zoletil 50 (5 mg/kg) via intramuscular (IM) injection. Endotracheal intubation was performed, and the animals were connected to a respiratory anesthesia machine. Anesthesia was maintained using 1.5-2.0%isoflurane. Connected the vital signs monitor, established vein access, and disinfected the surgical area. The surgical site on the left chest wall was exposed, made an incision along the 4-5 intercostals, exposed the heart, located the LAD coronary artery, and ligated the LAD with surgical suture 4-0 between the 1st and 2nd diagonal branches (near to the 2nd one) at the lower margin of the left aurcle. The success of the ligation was confirmed based on ST-segment changes on the electrocardiogram (ECG) . Animals in the sham group were not undergone ligation.
After the ligation procedure, the incision was closed. Animals were continuously monitored until fully awake. Preoperative and postoperative subcutaneous (SC) injection of meloxicam (0.4 mg/kg) to pain relief for three consecutive days, ceftriaxone sodium (50 mg/kg) was administrated via IM injection for 7 days after operation to prevent infection.
Administration
Timing of Administration
On the 7th day after modeling, the second thoracotomy was performed, and the test article was dosing by multipoint injection in and around the infarcted area, which were consistent with those of the patient during thoracotomy.
Route of Administration and Justification
Intramyocardial injection via epicardium was performed because it is the intended clinical route of administration in humans.
Injection Sites
The test articles were administered equally to the myocardial infarction area and surrounding area (peri-infarct area) , which is the intended clinical injection sites.
Injection Method
The dose formulation was administered by an insulin syringe with a needle (～50μl/site, ～16 delivery sites) .
Study Period
28 days (28 days after administration as endpoint, which was 35 days after modeling; and timing of administration was on the 7th day after modeling) .
According to literature reports [2] , chronic evaluation at time points 4–8 wk post-MI provides information on long-term remodeling. And according to the experience of our testing facility, under the condition of this model, which was basically stable after 2～3 weeks, while obvious myocardial infarction can be noted, and there is enough window between the model and sham groups to evaluate the impact of myocardial infarction after administration of A00A. In addition, combined with the pharmacological and biodistribution characteristics of A00A, early results suggest that VEGF-A, the active product of A00A, can be only detected in the heart within 1 week, which means it has been cleared within 1 week, and 4 weeks as the primary pharmacodynamic endpoint is sufficient to evaluate the efficacy of A00A. Therefore, myocardial infarction model of Bama miniature pig was selected as the first experimental study of pharmacodynamic efficacy, and 28 days after administration as the study endpoint.
Clinical Observation
Cage side observation was performed twice a day (once in the morning and once in the afternoon) throughout the entire study period.
Body Weight Measurement
During the whole study period, the weight was weighed once a week. (FIG. 14)
Echocardiography
Echocardiography was performed before modeling, 7 days after modeling (before dosing) , 7, 14, 21, and 28 days after dosing. Animals were fasted overnight, and on the day of the examination, the cardiac function was performed after anesthesia with Zoletil 50 (5 mg/kg) via IM injection.
Histopathological Evaluation
At the end of the study period, the experimental animals were euthanized and the heart tissues were collected.
1) TTC staining: the hearts were sliced, weighed, quickly immersed in 1.5%TTC staining solution, and stained in oven at 37℃ in the dark for 15 min, turned over, and then continued to dye for 15 min. After staining, the sections were washed with PBS, then photographed, and analyzed by ImageJ software to calculate the infarct ratio. The calculation formula is as follows:
a. Infarct weight per piece = infarct size per piece/total area per piece x weight per piece
b. Infarction ratio = sum of infarct weight per piece/sum of weight per piece x100
2) Before TTC staining, a small piece of tissue was taken from the same part of the area around the infarction, fixed with 10%formalin, dehydrated, transparent, embedded in paraffin, sectionalized, and Masson staining was performed for histopathological observation. Each section of myocardium sample was photographed under the OlympusBX53 microscope, and three 200-fold field of view were selected isometrically. The collagen fiber area was measured by Image Pro Plus6.0.
The Masson dyeing process was as follows:
a. Pruning: The initial fixed tissue was trimmed into a tissue block of appropriate size
(thickness of 2-3mm) . Put the tissue into encapsulation box immersed in the new fixing
solution, and put it in the LeicaASP200S automatic vacuum dehydrator waiting for
dehydration.
b. Dehydration: The tissue blocks were dehydrated successively in an automatic dehydrator by 70%, 80%, 95%, 95%, 100%, and 100%gradient alcohol for 30min each.
c. Transparent: After dehydration, the tissue is transparent through xylene for 15min in the dehydrator.
d. Wax dipping: After transparency, the tissue was immersed in liquid paraffin wax at 60℃in a dehydrator for 90 min.
e. Embedding: The wax-impregnated tissue was removed from the dehydrator and placed in the EG1150H tissue embedding machine. Use pointed pliers to pick up the tissue in the embedding box, and make the tissue plane of the repaired block face down on the table surface of the embedding machine.
f. Slice: The wax block wrapped in tissue was sliced on the LeicaRM2245 hand microtome with a slice thickness of 3 μm.
g. Exhibition: Use small tweezers to gently pick up the cut wax sheet and place it on the surface of the spreading machine at 45℃.
h. Scoop pieces: Completely flattened wax sheet, using a slide to lift. Use a pencil to mark the slide and place it vertically to dry for a while.
i. Baked slices: The collected tissue sections were baked in a baking machine or an incubator at 60 ℃ for 3hr.
j. Dyeing and sealing: Insert the section into the dyeing rack and place it in the Leica automatic section dyeing machine for dyeing.
k. Dyeing time is as follows: xylene I 10 min--xylene II 10 min--anhydrous alcohol 5 min--95%alcohol 2 min--95%alcohol 2 min--85%alcohol 2 min--75%alcohol 2 min-tap water 5 min--weigert iron hematoxylin 8 min--running water wash slightly --1%hydrochloric acid alcohol separation 5 s--running water rinse 5 min--ponceau acid Fuchsin-colored solution 8 min--rinse slightly with running water –phosphomolybdic acid solution 5 min--pour phosphomolybdic acid solution on the slide (no need water washing) --aniline blue dye 5 min--pour the dye solution on the slide (no need water washing) --1%acetic acid solution rinse the section --anhydrous alcohol 5 min--95%alcohol 5 min--95%alcohol 5 min --anhydrous alcohol 5 min--anhydrous alcohol 5 min--xylene I 5 min--xylene II 5 min--removed from the automatic dyeing machine, and sealed by neutral gum.
8.8.25.9 Statistical Analysis
Statistical analysis was performed using GraphPad Prism 8 software. The data were analyzed by one-way ANOVA, two-way ANOVA, and unpaired t test for between two groups, as Mean ± SE, and P<0.05 indicated statistically significant differences.
8.8.25.10 Study Results
During surgical modeling, one animal in G2 (ID: 2306129) died of intraoperative ventricular fibrillation and was replaced with a backup animal (ID: 2306120) .
Clinical Observation
During the study, no obvious abnormal clinical symptoms was found in the animals.
Body Weight
During the study period, the body weight of the animals mainted an increasing trend, and there was no significant difference among the groups. The changes of body weight in each group are shown in FIG. 14 and FIG. 15.
Echocardiographic
During the study, EF values of sham group was stable and no significant change was observed. Compared with before modeling, EF values of each group decreased significantly at 7 days after modeling (i.e., Day 0) . The EF values of the vehicle control group (G2) continued to decrease and reached the lowest level 21 days after dosing (i.e., 28 days after modeling) . Compared with before dosing (Day 0) , EF values in all treatment groups were significantly increased at 7 days after dosing (G3-G4 increased by 6.4%, 12.9%, respectively) , and maintained a continuous upward trend until the end of study period (Day 28) . At 28 days after dosing, EF values in all dosing groups were significantly increased (G3-G4 increased by 12.5%, 22.7%, respectively) . Compared with the vehicle control group, EF values increased significantly at all time points after dosing. The changes of EF values in each group are shown in FIG. 16 and FIG. 17.
During the experiment, FS values of sham group was stable and no significant change was observed. Compared with before modeling, FS values of all model group decreased significantly at 7 days after modeling (i.e., Day 0) . The FS values of vehicle control group (G2) continued to decrease and reached the lowest level 21 days after dosing (i.e., 28 days after modeling) . Compared with before dosing (Day 0) , the FS values of each dosing group increased significantly at 7 days after administration (G3-G4 increased by 8.8%, 17.1%, respectively) , and maintained a continuous upward trend until the end of study period (Day28) . At 28 days after administration, FS values of each dosing group significantly increased (G3-G4 increased by 18.1%, 31.3%, respectively) . Compared with the vehicle control group, FS values increased significantly at all time points after dosing. The changes of FS values in each group are shown in FIG. 18 and FIG. 19.
During the study, LVEDV values of sham group was stable and no significant change was observed. Compared with before modeling, LVEDV values of each module has an increasing trend in different degrees after modeling. There were no significant differences among the groups. The changes of LVEDV in each group are shown in FIG. 20 and FIG. 21.
During the study, LVESV values of sham group was stable and no significant changes were observed. Compared with before modeling, LVESV values increased significantly in all modeling groups 7 days after modeling, and no significant differences were observed among all groups. Compared with before dosing (i.e., Day 0) , LVESV values in the vehicle control group increased significantly at 7 days after dosing and maintained this level until the end of the study period (Day 28) , LVESV values of G3 was maintained at the pre-dosing level until the end of the study, LVESV values of G4 was decreased obviously. Compared with the vehicle control group, LVESV values in the dosing group (G3-G4) was lower than that in the vehicle control group at all time points after the treatment. The LVESV changes in each group are shown in FIG. 22 and FIG. 23.
Evaluation of Myocardial Infarction
At the end of the study period, there was no MI noted in the sham group. The remaining groups had varying degrees of MI. Compared with the vehicle control group, there was a significant decrease in MI weight and ratio of MI in all treatment groups (the ratio of MI in G2-G4 was 10.6, 4.5%, 3.4%, respectively) . The results of MI weight and ratio of MI in each group are shown in FIGs. 24-26.
Evaluation of Myocardial Fibrosis
At the end of the study period, the myocardial fibrosis (%) in the vehicle control group was 2.9 times higher than that in the sham group. Compared with the vehicle control group, the myocardial fibrosis (%) in each dosing group was reduced in different degrees (G2-G4: 6.2%, 4.4%, 1.8%, respectively) . The degree of myocardial fibrosis in G4-A00A_4mg group was significantly reduced. The results of myocardial fibrosis in each group are shown in FIGs. 27-29. 8.8.25.11 Discussion
EF and FS values in all dosing groups were significantly increased at 7 days after dosing (G3-G4: EF values were increased by 6.4%, 12.9%, respectively; FS values increased by 8.8%, 17.1%, respectively) , and maintained a continuous upward trend until the end of study period (Day 28) . At the end of study period (Day 28) , in each dosing group (G3-G4) , EF values were increased by 12.5%, 22.7%, respectively; and FS values were increased by 18.1%, 31.3%, respectively. There were no significant differences in EF and FS values of each dosing group (G3-G4) at all time points after dosing. However, significant differences of EF and FS values in G3 and G4 can be noted on Day 0 (after modeling while before dosing) , therefore, the relative changes (vs Day0) of EF and FS were further analyzed, and the results presented in FIG. 30 shows that there were significant differences in each dosing group (G3-G4) at all time points after dosing, indicating that the impact of A00A on cardiac function may be dose-dependent at dosage range of 1-4 mg/animal.
Combined with the pharmacological and biodistribution characteristics of A00A, VEGF-A, the active product of A00A, will be cleared within 1 week, suggesting that even after VEGF-A had been cleared, the biological activity -angiogenesis expressed by it can continue to improve cardiac function, and neovascularization can continue to improve cardiac function for more than 4 weeks. However, how long the improvement in cardiac function can be sustained and how long the pharmacodynamic effects can be last need further study. It is expected that how long the pharmacodynamic effects of generated blood vessels can impact on cardiac function and ventricular remodeling process is greatly related to the cardiovascular status of tested subject and the disease development itself. LAD MI is induced by permanent ligation, which is quite different from the cardiovascular status and disease development of the patients. The time-effect relationship of A00A in this model is only a proof of concept, and its time-effect relationship and durability of pharmacodynamic effects are recommended to be investigated in clinical studies.
In this study, due to unable to obtain the blood vessel-specific labeled antibody, thus the planned immunohistochemical study of angiogenesis was not performed. Although angiogenesis could not be evaluated as planned in this study, the pharmacodynamic effects is very clear from the results of the primary pharmacological indicator (myocardial infarction) and secondary pharmacological indicators (cardiac function and myocardial fibrosis) : Compared with the vehicle control group, the lower dose 1mg of A00A showed positive effects on myocardial infarction and fibrosis. EF and FS values in all dosing groups were significantly increased at 7 days after dosing, and maintain a continuous upward trend until the end of study (Day 28) , which showed a certain degree of dose dependency. Combined with preliminary pharmacological studies in our test facility, A00A can expressed target natural human VEGF-A protein in human and different species (including pig) cells, and it has the biological activity of natural human VEGF-A protein, which activated VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) and its downstream signaling pathways. VEGF-A is expected to promote angiogenesis and growth, thus in this study, A00A showed positive effects on cardiac function, myocardial infarction and fibrosis in Bama miniature pig with ischemic myocardial infarction. The clear pharmacodynamic effects of A00A and its mechanism is sufficient to suggest its potential in the treatment of ischemic heart disease.
8.8.25.12 Conclusion
Under the conditions of this study, A00A showed positive effects on cardiac function (EF and FS) , myocardial infarction and fibrosis in Bama miniature pig with ischemic myocardial infarction. A00A showed a certain degree of dose dependency, and the lower dose 1mg has a good efficacy.
Example 26: The expression of VEGF-A circRNA/MC3 in cells
This study compared the VEGF-A expression when delivered by DLin-MC3-DMA (referred to as MC3) having the original nitrogen-to-phosphorus ratio (N/P ratio) of: MC3_6/1 and the optimized N/P ratio: MC3_3/1 in 293T cells and immortalized human cardiomyocytes (IHC-SV40) . To investigate the expression-dose relationship of different LNPs, MC3_6/1 and MC3_3/1 were transfected at gradient doses of 25 ng, 50 ng, 100 ng, and 200 ng per well in 1E4 cells, followed by centrifugation at 1000 rpm for 1 hour. Expression levels in the cells were measured 24 hours after transfection. The results showed that after increasing the N/P ratio, the expression of VEGF-A circRNA/MC3_6/1 was significantly higher than that of VEGF-A circRNA/MC3_3/1 (FIG. 31, left panel) , and the expression increased more significantly with dose compared to VEGF-A circRNA/MC3_3/1 (FIG. 32, right panel) .
9. Sequence Listing and Tables
Table 1: Exemplary VEGF-A Amino Sequence
Table 2: Exemplary VEGF-A RNA Sequence
Table 3: Exemplary RNA sequence
*As to the SEQ ID NOs. 27-39, the nucleotides from precursor are represented by UNDERLINED LETTERS. The nucleotides from IRES are represented by underlined lower letters. The nucleotides from CDS are represented by ITALIC BOLD UNDERLINED UPPER LETTERS. The nucleotides from pUC57 are represented by UPPER LETTERS.
Table 4: Exemplary IRES sequence
Table 5: Exemplary other VEGF Amino Sequence
Table 6: Exemplary other VEGF RNA Sequence
Table 7: Exemplary PCR primer