Cleavable lipidsLiposomal delivery of nucleic acids has been used for site-specific delivery of therapies based on: encapsulated plasmid DNA, antisense oligonucleotides, short interfering RNAs, and micrornas. However, efficient delivery of nucleic acids to target cells and tissues and subsequent transfection of such target cells and tissues remains a technical challenge. Although multiple liposome-based systems and vehicles are available that facilitate the delivery of therapeutic agents to target cells and tissues, there are still a number of problems in vivo and in vitro applications. For example, a significant disadvantage of liposome delivery systems relates to the construction of liposomes that have sufficient cell culture or in vivo stability to reach the desired target cell and/or intracellular compartment, and such liposome delivery systems are capable of effectively releasing their encapsulated substances to such target cells. Furthermore, many of the cationic lipids employed for the construction of such liposome-based vehicles are generally toxic to target cells and accordingly are of limited use, particularly in large quantities necessary to successfully deliver the encapsulated substance to such target cells.
Despite the above limitations, and because of the ability of liposome-based vehicles to protect and facilitate delivery of encapsulated substances to one or more target cells, they are considered attractive carriers and therapeutic agents and efforts continue to be developed. Although liposome-based vehicles comprising cationic lipid components have shown promising results with respect to encapsulation, stability and site localization, there is still a need for improved liposome-based delivery systems. In particular, there remains a need for improved cations and lipids that are capable of delivering macromolecules, such as nucleic acids, to a variety of cell types and tissue types with enhanced efficiency. There also remains a particular need for novel lipids that incorporate multifunctional pathways for delivering encapsulated nucleic acids and polynucleotides.
Accordingly, the present invention provides novel compounds, pharmaceutical compositions comprising such compounds, and related methods of use thereof. In some embodiments, the compounds described herein are used as a liposome composition or component of a liposome composition that facilitates delivery to and subsequent transfection of one or more target cells. In certain embodiments, the compositions disclosed herein are cationic and/or ionizable lipids. In some embodiments, the compounds described herein have been designed based on desired characteristics or properties, for example, to enhance transfection efficiency or to promote specific biological outcomes. Furthermore, in certain embodiments, the compounds described herein employ a multifunctional strategy to facilitate delivery of the encapsulated substance (e.g., one or more polynucleotides) to and subsequent transfection of one or more target cells. For example, in certain embodiments, the compounds described herein are characterized by having one or more fusogenic, endosomal or lysosomal disrupting and/or strippable properties that provide advantages to such compounds over other similar classes of lipids.
The compounds described herein generally comprise R having two or more functional groups or moieties (e.g., hydrophobic) bonded (e.g., covalently bonded) thereto1Radical and hydrophilic R2Group) or one or more cleavable (e.g., cleavable enzyme or by reduction, oxidation or hydrolysis) functional groups. For example, disclosed herein are compounds comprising a cleavable disulfide (S-S) functional group. Also contemplated are compounds comprising any functional group capable of cleaving, for example, upon exposure to biological conditions, and such groups may include, but are not limited to, esters and ethers for this purpose (cleavable). In certain embodiments, inclusion of two or more functional groups (e.g., a head group and a tail group) of a compound renders such compounds amphiphilic. For example, in certain embodiments, at least one functional group is a non-polar, lipophilic, or hydrophobic tail group (e.g., a naturally occurring lipid such as cholesterol or C)6-C20Alkyl groups). In certain embodiments, at least one functional group is a polar or hydrophilic head group (e.g., imidazole).
In certain embodiments, the compounds described herein (e.g., HGT4001, HGT4002, HGT4003, HGT4004, and/or HGT4005) are cationic or ionizable lipids that can be used as components of liposomal compositions that facilitate or enhance delivery and release of encapsulated substances (e.g., one or more therapeutic agents) to one or more target cells (e.g., by penetrating or fusing with the lipid membrane of such target cells). In certain embodiments, inclusion of one or more cleavable functional groups (e.g., disulfides) of such compounds allows, for example, a hydrophilic functional head group to dissociate from a lipophilic functional tail group of the compound (e.g., upon exposure to oxidative, reductive, or acidic conditions), thereby promoting phase transition in the lipid bilayer of one or more target cells. For example, when a liposome composition (e.g., a lipid nanoparticle) includes one or more compounds described herein, a phase transition in the lipid bilayer of one or more target cells facilitates delivery of an encapsulated substance (e.g., one or more therapeutic polynucleotides encapsulated in a lipid nanoparticle) into one or more target cells. Similarly, enrichment of the liposome composition with one or more compounds described herein can facilitate fusogenicity of the liposome composition, thereby enhancing the ability of such compounds to deliver substances (e.g., polynucleotides) encapsulated therein intracellularly.
In certain embodiments, the compounds have the structure of formula (I),
wherein R is1Selected from the group consisting of: imidazole, guanidine, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl; wherein R is2Selected from the group consisting of formula II and formula III;
wherein R is3And R4Each independently selected from the group consisting of: optionally substituted, variably saturated or unsaturated C6-C20Alkyl and optionally substituted, variably saturated or unsaturated C6-C20An acyl group; and wherein n is zero or any positive integer (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or greater). In certain embodiments, R3And R4Each being optionally substituted, polyunsaturated C18Alkyl, and in other embodiments, R3And R4Each being unsubstituted, polyunsaturated C18An alkyl group. In certain embodiments, oneOr a plurality of R3And R4Is (9Z, 12Z) -octadeca-9, 12-diene.
Also disclosed herein are pharmaceutical compositions comprising compounds of formula I, wherein R1Selected from the group consisting of: imidazole, guanidine, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl; wherein R is2Is of formula II; and wherein n is zero or any positive integer. Further disclosed herein are pharmaceutical compositions comprising compounds of formula I, wherein R1Selected from the group consisting of: imidazole, guanidine, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl; wherein R is2Is of formula III; wherein R is3And R4Each independently selected from the group consisting of: optionally substituted, variably saturated or unsaturated C6-C20Alkyl and optionally substituted, variably saturated or unsaturated C6-C20An acyl group; and wherein n is zero or any positive integer; in certain embodiments, R3And R4Each being optionally substituted, polyunsaturated C18Alkyl, and in other embodiments, R3And R4Each being unsubstituted, polyunsaturated C18Alkyl (e.g., octadeca-9, 12-diene).
In certain embodiments, R1The group or head group is a polar or hydrophilic group (e.g., one or more imidazole, guanidine, and amino groups) and is bonded to R, for example, as described in formula I, via a disulfide (S-S) cleavable linking group2A lipid group. Other contemplated cleavable linking groups include those comprising a linkage (e.g., covalent linkage) to, for example, an alkyl group (e.g., C)1To C10Alkyl) groups, or one or more disulfide (S-S) linking groups. In certain embodiments, R1Radical passing through C1-C20The alkyl group (e.g., where n is one to twenty) is covalently bonded to the cleavable linking group, or alternatively can be directly bonded to the cleavable linking group (e.g., where n is zero). In some embodiments of the present invention, the substrate is,the disulfide linker is cleavable in vitro and/or in vivo (e.g., enzymatically cleavable or cleavable upon exposure to acidic or reducing conditions).
In certain embodiments, the present invention relates to compound 5- (((10, 13-dimethyl-17- (6-methylhept-2-yl) -2, 3, 4,7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yl) disulfanyl) methyl) -1H-imidazole (referred to herein as "HGT 4001") having the structure of formula IV.
In certain embodiments, the invention relates to a compound having the structure of formula V- (2- (((3S, 10R, 13R) -10, 13-dimethyl-17- ((R) -6-methylhept-2-yl) -2, 3, 4,7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yl) disulfanyl) ethyl) guanidine (referred to herein as "HGT 4002").
In certain embodiments, the present invention relates to the compound 2- ((2, 3-bis ((9Z, 12Z) -octadec-9, 12-dien-1-yloxy) propyl) disulfanyl) -N, N-dimethylethylamine (referred to herein as "HGT 4003") having the structure of formula VI.
In other embodiments, the invention relates to the compound 5- (((2, 3-bis ((9Z, 12Z) -octadec-9, 12-dien-1-yloxy) propyl) disulfanyl) methyl) -1H-imidazole (referred to herein as "HGT 4004") having the structure of formula VII.
In still other embodiments, the invention relates to the compound 1- (((2, 3-bis ((9Z, 12Z) -octadec-9, 12-dien-1-yloxy) propyl) disulfanyl) methyl) guanidine (referred to herein as "HGT 4005") having the structure of formula VIII.
In certain embodiments, the compounds described herein are cationic and/or ionizable lipids that can be used as, or alternatively as a component of, a liposome composition (e.g., a lipid nanoparticle). In certain embodiments, the compounds described herein are used to enrich liposomal compositions (e.g., lipid nanoparticles), thereby imparting improved properties to such enriched liposomal compositions (e.g., improved delivery of encapsulated polynucleotides to one or more target cells and/or reduced in vivo toxicity of the liposomal compositions). Accordingly, also encompassed are pharmaceutical compositions, and in particular liposome compositions comprising one or more of the compounds described herein. In certain embodiments, such drug and liposome compositions include one or more PEG-modified lipids, non-cationic lipids, and helper lipids (such as cholesterol). For example, contemplated are pharmaceutical and liposomal compositions (e.g., lipid nanoparticles) comprising one or more compounds described herein (e.g., HGT4001, HGT4002, HGT4003, HGT4004, and/or HGT4005) and one or more cationic lipids, non-cationic lipids, helper lipids/cholesterol, and PEG-modified lipid components. Also encompassed are pharmaceutical and liposomal compositions comprising one or more compounds described herein and further comprising one or more additional cationic lipids. Similarly, also encompassed are liposomal compositions and pharmaceutical compositions (e.g., lipid nanoparticles) comprisingOne or more HGT4001, HGT4002, HGT4003, HGT4004 and/or HGT4005 compounds and one or more C12-200、DLinDMA、DLinKC2-DMA, CHOL, DOPE, DMG-PEG-2000, ICE, DSPC, DODAP, DOTAP, and C8-PEG-2000. In certain embodiments, such pharmaceutical and liposome compositions are filled or otherwise encapsulated with a substance, such as one or more biologically active polynucleotides.
In certain embodiments, one or more of the drug and liposome compositions described herein (e.g., lipid nanoparticles) comprise one or more compounds disclosed herein and one or more additional lipids. For example, a lipid nanoparticle that includes or is otherwise enriched in one or more compounds described herein may further include one or more of DOTAP (1, 2-dioleyl-3-trimethylammonium propane), DODAP (1, 2-dioleyl-3-dimethylammonium propane), DOTMA (1, 2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA, DLin-KC2-DMA, C12-200, and ICE. In one embodiment, a pharmaceutical composition includes a lipid nanoparticle including HGT4001, DOPE, and DMG-PEG 2000. In another embodiment, a pharmaceutical composition comprises a lipid nanoparticle comprising HGT4003, DOPE, cholesterol, and DMG-PEG 2000.
In certain embodiments, one or more of the pharmaceutical compositions described herein can include one or more PEG-modified lipids. For example, a lipid nanoparticle comprising or otherwise enriched in one or more compounds described herein can further comprise a PEG-modified lipid comprising a covalent attachment to a polymer comprising one or more C6-C20The lipids of the alkane have polyethylene glycol chains up to 5 kDa.
Similarly, the pharmaceutical compositions disclosed herein (e.g., lipid nanoparticles) may include or may be additionally enriched with one or more compounds described herein and may further include one or more helper lipids selected from the group consisting of: DSPC (1, 2-distearoyl-sn-glycero-3-phosphocholine, DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1, 2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE (1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (, 2-dioleoyl-sn-glycero-3-phosphate- (1' -rac-glycerol)), DOPE (1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine), DSPE (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine), DLPE (1, 2-dilauroyl-sn-glycero-3-phosphoethanolamine), DPPS (1, 2-dipalmitoyl-sn-glycero-3-phospho-L-serine), ceramides, sphingomyelines and cholesterol.
In certain embodiments, compounds and pharmaceutical and liposomal compositions comprising such compounds (e.g., lipid nanoparticles) comprise one or more polynucleotides (e.g., encapsulated DNA or RNA). In other embodiments, one or more polynucleotides include at least one locked nucleic acid (e.g., two, three, four, five, six, seven, eight, nine, ten, twelve, fifteen, sixteen, eighteen, twenty or more locked nucleic acid residues or monomers). Wherein the one or more encapsulated polynucleotides comprise RNA, such RNA can comprise, for example, mRNA, siRNA, snoRNA, microrna, and combinations thereof.
In certain embodiments, the polynucleotide encapsulated in the drug and liposome compositions thereof comprises mRNA encoding, for example, a functional polypeptide, protein, or enzyme, and when expressed (i.e., translated) by one or more target cells, the polynucleotide produces a functional polypeptide product (e.g., a protein or enzyme), and in some cases, the functional polypeptide product is secreted by the target cells into the peripheral circulation of the subject. In certain embodiments, one or more polynucleotides comprising (or otherwise filled with or encapsulated with) the compounds and pharmaceutical and liposome compositions described herein encode a nucleic acid (e.g., a polypeptide) that is aberrantly expressed by the subject. In certain embodiments, one or more encapsulated polynucleotides comprising such compounds and liposomes or pharmaceutical compositions (e.g., lipid nanoparticles) encode functional enzymes such as urea cycle enzymes (e.g., Ornithine Transcarbamylase (OTC), carbamyl phosphate synthase 1(CPS1), argininosuccinate synthase (ASS1), argininosuccinate lyase (ASL), or arginase 1(ARG 1)). In certain embodiments, the one or more encapsulated polynucleotides comprise an mRNA encoding an enzyme associated with a lysosomal storage disorder (e.g., the encapsulated polynucleotide is an mRNA encoding one or more enzymes such as alpha galactosidase, iduronate 2-sulfatase, N-acetylglucosamine-1-phosphotransferase, beta-glucosidase, galactocerebrosidase, and acid alpha-glucosidase). In other embodiments, wherein the nucleic acid comprises an mRNA, such an mRNA can encode one or more proteins or enzymes, for example, proteins or enzymes that may be deficient in the subject (e.g., an enzyme or protein selected from the group consisting of cystic fibrosis transmembrane conductance regulator (CFTR), α -L-iduronidase, N-acetylglucosaminidase, α -glucosaminyl acetyltransferase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine-4-sulfatase, galactose-6-sulfatase, β -galactosidase, β -glucuronidase, glucocerebrosidase, heparan sulfamidase (heparan sulfamidase), and hyaluronidase).
Also encompassed herein are pharmaceutical and liposomal compositions (e.g., lipid nanoparticles) comprising one or more compounds described herein and one or more polynucleotides (e.g., antisense oligonucleotides), and in particular one or more chemically modified polynucleotides. For example, in certain embodiments where the polynucleotide is an mRNA, such chemical modifications make the mRNA more stable and may include, for example, terminal blocking modifications of the 5 'or 3' untranslated regions of the mRNA. In certain embodiments, the chemical modification comprises including a partial sequence of the CMV immediate early 1(IE1) gene into a 5' untranslated region of an mRNA such as, for example, the nucleotide sequence set forth in SEQ id no: 1:
XCAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAGACACCGGACCGAUCCAGCUCCGCGGGCCGGGAACGGUGCAUUGGACGGAUUCCCGGCCAGUGAGUGAGUGACUCCGUCUGACACAG, wherein X, if present, is GGA (SEQ ID NO: 1);
or with SEQ ID NO: 1 sequence which is at least 90% or at least 95% identical.
In other embodiments, the chemical modification comprises inclusion of a poly a tail into the 3' untranslated region of the mRNA. Also contemplated are chemical modifications that include inclusion of the cap1 structure into the 5' untranslated region of the mRNA. In yet other embodiments, the chemical modification comprises incorporating a sequence from the human growth hormone (hGH) gene into the 3' untranslated region of the mRNA. The hGH sequence may comprise: SEQ ID NO: 2
CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC(SEQ ID NO：2)
Or with SEQ ID NO: 2 sequences that are at least 90% or at least 95% identical.
The compounds and pharmaceutical compositions described herein can be formulated to specifically target and/or transfect one or more target cells, tissues, and organs. In certain embodiments, such compounds and pharmaceutical compositions facilitate transfection of such target cells by one or more mechanisms (e.g., fusogenic-based release of the lipid bilayer membrane of the target cell and/or proton sponge-mediated disruption). Contemplated target cells include, for example, one or more cells selected from the group consisting of: hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, heart cells, adipocytes, vascular smooth muscle cells, cardiac muscle cells, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.
Further the present invention provides pharmaceutical compositions comprising a liposomal delivery vehicle and a liposomal formulation (e.g., a lipid nanoparticle) for accomplishing delivery of the encapsulated contents (e.g., polynucleotide) to one or more target cells, tissues or organs. The invention further provides related methods and processes for preparing such pharmaceutical compositions, and methods of treating one or more diseases or conditions by administering such pharmaceutical compositions to a subject in need thereof. It is also desirable that the lyophilized compositions described herein (e.g., lipid nanoparticles) have improved long-term stability when stored under refrigeration or at ambient temperatures (e.g., room temperature).
In certain embodiments, a pharmaceutical composition comprising a lyophilized nanoparticle or liposome (lyosol) delivery vehicle is characterized in that it is stable (e.g., as stable as a pharmaceutical composition comprising an equivalent amount of non-lyophilized vehicle). The stability of the lyophilized delivery vehicle can be determined, for example, with reference to the particle size of the lipid nanoparticles comprising such compositions. In certain embodiments, lyophilization of the lipid nanoparticles does not significantly alter or alter the particle size of the lipid nanoparticles after lyophilization and/or reconstitution. For example, disclosed herein are pharmaceutical compositions comprising a lyophilized lipid delivery vehicle, wherein the lipid nanoparticles do not flocculate or aggregate or alternatively exhibit limited or negligible flocculation or aggregation (e.g., as determined by the particle size of the reconstituted lipid nanoparticles) when reconstituted (e.g., with purified water). Accordingly, in certain embodiments, the lipid nanoparticles have a Dv of less than about 500nm when the lyophilized lipid nanoparticles are reconstituted50(e.g., less than about 300nm, 200nm, 150nm, 125nm, 120nm, 100nm, 75nm, 50nm, 25nm, or less). Similarly, in certain embodiments, the lipid nanoparticles have a Dv of less than about 750nm when the lyophilized lipid nanoparticles are reconstituted90(e.g., less than about 700nm, 500nm, 300nm, 200nm, 150nm, 125nm, 100nm, 75nm, 50nm, 25nm, or less).
In other embodiments, the pharmaceutical composition comprising the lyophilized lipid delivery vehicle is characterized by having a polydispersity index of less than 1 (e.g., less than 0.95, 0.9, 0.8, 0.75, 0.7, 0.6, 0.5, 0.4, 0.3, 0.25, 0.2, 0.1, 0.05 or less). In addition, in other embodiments, pharmaceutical compositions comprising the lyophilized lipid delivery vehicle exhibit a reduced tendency to flocculate or otherwise aggregate (e.g., during lyophilization or upon reconstitution). For example, when reconstituted, the lipid delivery vehicle may have an average particle size (Z) of less than 500nmave) (e.g., less than about 400nm, 300nm, 200nm, 175nm, 150nm, 125nm, 100nm, 75nm, 50nm, 25nm, or less in PBS solution).
The stable lyophilized lipid delivery vehicles (e.g., lipid nanoparticles) provided by the present invention are also characterized by their improved storage properties. For example, in certain embodiments, the lyophilized lipid delivery vehicle can be stored refrigerated and remain stable (e.g., as shown by minimal or no loss of its intended drug or biological activity) for a longer period of time (e.g., stable for at least about 1, 2, 3, 4, 5,6, 9, 12, 18, 24, 36 months or more upon storage at about 4 ℃). In other embodiments, the lyophilized lipid delivery vehicle can be stored non-refrigerated and remain stable for a longer period of time (e.g., stable for at least about 1, 2, 3, 4, 5,6, 9, 12, 18, 24, 36 months or more upon storage at about 25 ℃). In certain embodiments, upon reconstitution with a suitable rehydration medium (e.g., purified water, deionized water, 5% dextrose, and/or physiological saline), the reconstituted composition exhibits a pharmacological or biological activity comparable to that observed prior to lyophilization. For example, in certain embodiments, the pharmacological or biological activity of the encapsulated polynucleotide is equivalent to that observed prior to lyophilization of the composition, or alternatively exhibits a negligible reduction in pharmacological or biological activity (e.g., a reduction in biological or pharmacological activity of the encapsulated polynucleotide of less than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, or 50%).
Also disclosed herein are pharmaceutical compositions (e.g., lyophilized lipid nanoparticles) comprising a lyophilized lipid delivery vehicle further comprising or optionally prepared using one or more lyoprotectants (e.g., sugars and/or carbohydrates). In certain embodiments, inclusion of one or more lyoprotectants in the lipid nanoparticle may improve or otherwise enhance the stability of the lyophilized lipid delivery vehicle (e.g., under normal storage conditions) and/or facilitate reconstitution of the lyophilized lipid delivery vehicle using a rehydration medium, thereby preparing an aqueous formulation. For example, in certain embodiments, the lipid nanoparticles are prepared and the buffer present in the liposome formulation can be replaced (e.g., via centrifugation) with a lyoprotectant such as a sucrose solution or suspension (e.g., an aqueous solution comprising about 1-50% or 10-25% sucrose) prior to lyophilization. Other suitable lyoprotectants that may be used to prepare the lyophilized compositions described herein include, for example, trehalose, dextran (e.g., 1.5kDa, 5kDa, and/or 40kDa), and inulin (e.g., 1.8kDa and/or 4 kDa).
In some embodiments, the lyophilized compositions disclosed herein can also facilitate sustained release of the contents (e.g., polynucleotide) encapsulated in one or more lipid nanoparticles comprising such compositions. For example, contemplated are pharmaceutical compositions comprising a lyophilized lipid delivery vehicle, wherein the composition can be implanted into a subject without reconstitution (e.g., subcutaneously, e.g., as a membrane or disc). Such implanted lyophilized compositions may erode or otherwise disintegrate at a predetermined rate, for example, upon exposure to one or more biological fluids (e.g., serum, blood, cerebrospinal fluid, mucus, sweat, gastric secretions, urine, and/or saliva). In certain embodiments, such implanted pharmaceutical compositions comprising a lyophilized lipid delivery vehicle release, e.g., encapsulated, polynucleotides over at least 1, 2, 7, 10, 14, 21, 30, 45, 60, 90, 120 days or more. Optionally, such implanted compositions comprising a lyophilized lipid delivery vehicle release, e.g., encapsulated, polynucleotides over at least one, two, three, six, twelve, sixteen, twenty-four, thirty-six, or more.
In certain embodiments, a pharmaceutical composition provided herein that includes a lyophilized lipid delivery vehicle can be reconstituted prior to administration to a subject (e.g., a mammal). Upon reconstitution (e.g., using purified water or 5% dextrose as a rehydration medium), the reconstituted aqueous composition can be administered to a subject by one or more of the following routes of administration: intravenous, oral, rectal, transdermal, transmucosal, sublingual, subdural, nasal, intramuscular, subcutaneous, intramedullary injections, intrathecal, intraventricular, intraperitoneal, intranasal, ocular and/or intraocular.
The invention also provides methods of treating a disease (e.g., a disease associated with aberrant expression of a gene or nucleic acid) in a subject, wherein the method comprises administering to the subject one or more compounds and/or pharmaceutical compositions of the invention. Also contemplated is transfecting one or more target cells with one or more polynucleotides, wherein the method comprises contacting the one or more target cells with a compound or pharmaceutical composition described herein, such that the one or more target cells are transfected with the one or more encapsulated polynucleotides.
In certain embodiments, the treatment methods provided herein employ compositions comprising the lyophilized or reconstituted lipid delivery vehicles of the present invention that are capable of modulating the expression of aberrantly expressed nucleic acids and polynucleotides in one or more target cells and tissues. Accordingly, also provided herein are methods of treating a disease in a subject by administering to the subject an effective amount of a pharmaceutical composition provided herein that includes a lyophilized lipid delivery vehicle (e.g., when reconstituted with a rehydration medium such as sterile water for injection). In certain embodiments, such methods can enhance (e.g., increase) expression of the polynucleotide and/or increase production and secretion of the functional polypeptide product in one or more target cells and tissues (e.g., hepatocytes). In some embodiments, the targeted cell or tissue aberrantly expresses a polynucleotide encapsulated by one or more lyophilized lipid delivery vehicles (e.g., lipid nanoparticles) of the present invention.
The invention also provides methods of increasing the expression of one or more polynucleotides (e.g., mRNA) in one or more target cells, tissues, and organs. Typically, such methods comprise contacting the target cell with one or more compounds and/or a drug or liposome composition comprising one or more polynucleotides that are otherwise encapsulated. In some embodiments, the invention also relates to methods of transfecting one or more cells with a polynucleotide (e.g., comprising the steps of rehydrating a lyophilized composition and contacting such one or more cells with the rehydrated composition).
The above discussed and many other features and attendant advantages of the present invention will become better understood by reference to the following detailed description of the invention when taken in conjunction with the accompanying embodiments. The various embodiments described herein are complementary and can be combined or used together in a manner understood by the skilled person in view of the teachings contained herein.
Brief Description of Drawings
Figure 1 illustrates the luminosity output of firefly luciferase protein in mouse liver and spleen following intravenous administration of HGT 4003-based lipid nanoparticles filled with firefly luciferase (FFL) mRNA. The administered HGT 4003-based lipid nanoparticles provide enrichment of encapsulated mRNA in the liver over the spleen. Values are described as intermediate Relative Light Units (RLU)/mg total protein four hours after administration.
Figure 2 illustrates the luminosity output of firefly luciferase protein in mouse brain and spinal cord tissue following Intracerebroventricular (ICV) and Intrathecal (IT) administration of HGT 4003-based lipid nanoparticles filled with firefly luciferase (FFL) mRNA. HGT 4003-based lipid nanoparticles administered using the ICV administration route provide enrichment of encapsulated mRNA in brain compared to the IT administration route. Values are described as intermediate Relative Light Units (RLU)/mg total protein four hours after administration.
Description of the exemplary embodiments
The compounds of the invention are useful, for example, as liposomal delivery vehicles or as components of liposomal delivery vehicles. In certain embodiments, the compounds disclosed herein can be used as a liposome composition or alternatively as a component of a liposome composition (e.g., as a lipid nanoparticle). The compounds of the invention may also be employed in pharmaceutical compositions (e.g., lipid nanoparticles) and in methods of administering such pharmaceutical compositions to treat or prevent diseases, disorders or conditions or to deliver therapeutic molecules. In certain embodiments, such compounds and compositions facilitate delivery of, for example, an encapsulated substance (e.g., a polynucleotide) to one or more target cells, tissues, and organs.
The compounds disclosed herein typically comprise one or more cleavable groups, such as, for example, one or more disulfide (S-S) functional groups as described in formula I below. As used herein, the terms "cleave" and "cleavable" generally mean that one or more chemical bonds (e.g., one or more covalent bonds, hydrogen bonds, van der waals forces, and/or ionic interactions) between atoms in or adjacent to the subject functional group are broken (e.g., hydrolyzed) or are capable of being broken upon exposure to selected conditions (e.g., exposure to enzymatic conditions). In certain embodiments, the cleavable group is a disulfide functional group, and in particular embodiments is a disulfide group capable of cleaving upon exposure to a selected biological condition (e.g., intracellular conditions). In certain embodiments, the cleavable group is an ester functional group that is capable of cleaving upon exposure to a selected biological condition. For example, the disulfide group may be cleaved enzymatically or by hydrolysis, oxidation or reduction reactions. Upon cleavage of such disulfide functionality, one or more functional moieties or groups (e.g., one or more head groups and/or tail groups) bonded thereto may be released. Exemplary cleavable groups can include, but are not limited to: disulfide groups, ester groups, ether groups, and any derivatives thereof (e.g., alkyl and aryl esters). In certain embodiments, the cleavable group is not an ester group or an ether group.
The cleavable groups described herein are typically bonded (e.g., bonded by one or more hydrogen bonds, van der waals forces, ionic interactions, and covalent bonds) to one or more functional moieties or groups (e.g., at least one head group and at least one tail group). In certain embodiments, at least one functional moiety or group is hydrophilic (e.g., a hydrophilic head group comprising one or more of imidazole, guanidine, amino, imine, enamine, optionally substituted alkylamino, and pyridyl). As used herein, the term "hydrophilic" is used to indicate qualitatively that the functional groups are hydrophilic and typically such groups are water soluble. For example, disclosed herein are compounds comprising a cleavable disulfide (S-S) functional group bonded to one or more hydrophilic groups (e.g., hydrophilic head groups), wherein such hydrophilic groups include or are selected from the group consisting of: imidazole, guanidine, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl.
In certain embodiments, the selected hydrophilic functional groups or moieties may vary or otherwise impart properties to the compound or liposome composition having such a compound as a component (e.g., by improving the transfection efficiency of lipid nanoparticles having the compound as a component). For example, the incorporation of guanidine as a hydrophilic head group into the compounds described herein can promote the fusogenicity of such compounds (or liposome compositions having such compounds as a component) with the cell membrane of one or more target cells, thereby enhancing, for example, the transfection efficiency of such compounds. It is speculated that the nitrogen from the hydrophilic guanidine moiety forms a six-membered ring transition state that confers stability to the interaction and thus allows cellular uptake of the encapsulated substance. (Wender, et al, adv. drug del. rev. (2008) 60: 452-472.) similarly, the incorporation of one or more amino groups or moieties into the disclosed compounds (e.g., as head groups) can further facilitate endosomal/lysosomal membrane disruption of target cells by exploiting the fusogenicity of such amino groups. This is based not only on the pKa of the amino groups of the composition, but also on the ability of the amino groups to undergo hexagonal phase transformation and fusion with the target cell surface, i.e., the vesicle membrane. (Koltover, et al Science (1998) 281: 78-81.) this result is believed to promote the disruption of the vesicle membrane and the release of the lipid nanoparticle contents into the target cell.
Similarly, in certain embodiments, for example, incorporation of imidazole as a hydrophilic head group into the compounds described herein can serve to facilitate endosomal or lysosomal release, e.g., encapsulation of the contents in the liposomal compositions (e.g., lipid nanoparticles) of the invention. Such enhanced release may be obtained by one or two proton sponge-mediated disruption mechanisms and/or enhanced fusogenic mechanisms. The proton sponge mechanism is based on the compound and in particular on the ability of the functional moiety or group of the compound to buffer the acidification of endosomes. This can be manipulated or otherwise controlled by the pKa of the compound or the inclusion of one or more functional groups of such compounds (e.g., imidazole). Accordingly, in certain embodiments, the fusibility of imidazole-based compounds (e.g., HGT4001 and HGT4004) such as disclosed herein, which have a lower pKa relative to other traditional cationic lipids, is associated with endosome disruption properties facilitated by such imidazole groups. Such endosomal disruption properties in turn facilitate osmotic swelling and disruption of the liposome membrane, followed by transfection or intracellular release of the polynucleotide material packed or encapsulated therein into the target cell. This phenomenon can be applied to various compounds having desired pKa characteristics other than the imidazole moiety. Such embodiments also include polyaza-based functionalities such as polyamines, polypeptides (histidines), and nitrogen-based dendritic mechanisms.
The compounds described herein, particularly imidazole-based compounds (e.g., HGT4001 and HGT4004), are also characterized by their reduced toxicity, particularly relative to traditional lipids and cationic lipids. In some embodiments, the drug and liposome compositions described herein include one or more imidazole-based cationic lipid compounds such that the relative concentration of other more toxic cationic lipids in such drugs or liposome compositions can be reduced or otherwise removed. The imidazole-based compound or lipid (e.g., HGT4001 and/or HGT4004) may be used as the only cationic lipid in one or more of the pharmaceutical and liposomal compositions described herein (e.g., lipid nanoparticles), or alternatively may be combined with traditional cationic lipids (e.g., LIPOFECTIN or LIPOFECTAMINE), non-cationic lipids, helper lipids/cholesterol, and/or PEG-modified lipids. In certain embodiments, the total cationic lipid component of the compounds described herein, or alternatively of the drug and liposome compositions, can include the following molar ratios of total lipid present in such drugs or liposome compositions (e.g., lipid nanoparticles): about 1% to about 90%, about 2% to about 70%, about 5% to about 50%, about 10% to about 40%, or preferably about 20% to about 70% of the total lipid present in such a drug or liposome composition (e.g., lipid nanoparticle).
In certain embodimentsAt least one functional group of a moiety comprising a compound described herein is naturally hydrophobic (e.g., a hydrophobic tail group comprising a naturally occurring lipid such as cholesterol). As used herein, the term "hydrophobic" is used to indicate qualitatively that the functional group is water repellent and typically such groups are not water soluble. For example, disclosed herein are compounds comprising a cleavable functional group (e.g., a disulfide (S-S) group) bonded to one or more hydrophobic groups, wherein such hydrophobic groups include one or more naturally occurring lipids such as cholesterol and/or optionally substituted, variably saturated or unsaturated C6-C20Alkyl and/or optionally substituted, variably saturated or unsaturated C6-C20An acyl group.
In certain embodiments, the compounds described herein comprise, for example, at least one hydrophilic head group and at least one hydrophobic tail group, each bonded to at least one cleavable group, thereby rendering such compounds amphiphilic. The term "amphiphilic" as used herein to describe a compound or composition means the ability to be soluble in both polar (e.g., water) and non-polar (e.g., lipid) environments. For example, in certain embodiments, the compounds described herein comprise at least one lipophilic tail group (e.g., cholesterol or C)6-C20Alkyl) and at least one hydrophilic head group (e.g., imidazole), each bonded to a cleavable group (e.g., disulfide).
It should be noted that the terms "head group" and "tail group" as used to describe the compounds of the present invention, and in particular the functional groups comprising such compounds, are for ease of reference to describe the positioning of one or more functional groups relative to other functional groups. For example, in certain embodiments, a hydrophilic head group (e.g., a guanidine) is bonded (e.g., such as by one or more hydrogen bonds, van der waals forces, ionic interactions, and covalent bonds) to a cleavable functional group (e.g., a disulfide group), which in turn is bonded to a hydrophobic tail group (e.g., cholesterol).
Also disclosed herein are compounds having the structure of formula I,
wherein R is1Selected from the group consisting of: imidazole, guanidine, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl; wherein R is2Selected from the group consisting of formula II and formula III;
wherein R is3And R4Each independently selected from the group consisting of: optionally substituted, variably saturated or unsaturated C6-C20Alkyl and optionally substituted, variably saturated or unsaturated C6-C20An acyl group; and wherein n is zero or any positive integer (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or greater). In certain embodiments, each R is3And R4Containing optionally substituted, polyunsaturated C18Alkyl, and in other embodiments, R3And R4Each being unsubstituted, polyunsaturated C18An alkyl group. In certain embodiments, each R is3And R4Is (9Z, 12Z) -octadeca-9, 12-diene. In certain embodiments, n is 1 (such that the alkyl group is ethyl), 2 (such that the alkyl group is methyl), 3 (such that the alkyl group is, for example, propyl or isopropyl, 4 (such that the alkyl group is, for example, butyl, isobutyl, sec-butyl, or tert-butyl), 5 (such that the alkyl group is, for example, pentane), 6 (such that the alkyl group is, for example, hexane), 7 (such that the alkyl group is, for example, heptane), 8 (such that the alkyl group is, for example, octane), 9 (such that the alkyl group is, for example, nonane), or 10 (such that the alkyl group is, forAn alkane).
The term "alkyl" as used herein refers to straight and branched chain C1-C40Hydrocarbons (e.g. C)6-C20Hydrocarbons) and includes saturated or unsaturated hydrocarbons. In certain embodiments, alkyl groups may include one or more cycloalkyl groups and/or one or more heteroatoms such as oxygen, nitrogen, or sulfur, and may be optionally substituted with substituents (e.g., one or more alkyl groups, halogens, alkoxy groups, hydroxyl groups, amino groups, aryl groups, ethers, esters, or amides). In certain embodiments, contemplated alkyl groups include (9Z, 12Z) -octadeca-9, 12-diene. Such as "C6-C20The use of the name "means an alkyl group (e.g., straight or branched chain and including both olefin and alkyl) having the recited range of carbon atoms.
As used herein, the term "aryl" refers to an aromatic group containing six to ten carbons in the ring portion (e.g., monocyclic, bicyclic, and tricyclic structures). The aryl group may be optionally substituted through available carbon atoms, and may include one or more heteroatoms such as oxygen, nitrogen, or sulfur in certain embodiments.
Also disclosed herein are pharmaceutical compositions comprising compounds of formula I, wherein R1Selected from the group consisting of: imidazole, guanidine, amino, imine, enamine, optionally substituted alkylamino (e.g., dimethylamino), and pyridyl; wherein R is2Is of formula II; and wherein n is zero or any positive integer (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or greater). Further disclosed herein are pharmaceutical compositions comprising compounds of formula I, wherein R1Selected from the group consisting of: imidazole, guanidine, imine, enamine, amino, optionally substituted alkylamino (e.g., dimethylamino), and pyridyl; wherein R is2Is of formula III; wherein R is3And R4Each independently selected from the group consisting of: optionally substituted, variably saturated or unsaturated C6-C20Alkyl and optionally substituted, variably saturated or unsaturated C6-C20An acyl group; and wherein n is zero or any positive integer. In certain embodimentsIn, R3And R4Each being optionally substituted, polyunsaturated C18Alkyl, and in other embodiments, R3And R4Each being unsubstituted, polyunsaturated C18An alkyl group. In certain embodiments, contemplated alkyl groups include (9Z, 12Z) -octadeca-9, 12-diene.
In certain embodiments, R1The group or head group is a polar or hydrophilic group (e.g., one or more imidazole, guanidine, and amino groups) and is bonded to R through a disulfide (S-S) cleavable linking group such as described in formula I2A lipid group. R1The group or head group may be through an alkyl group (e.g., C)1-C20Alkyl, where n is one to twenty) is covalently bonded to the cleavable linking group, or alternatively can be directly bonded to the cleavable linking group (e.g., where n is zero). The compounds and pharmaceutical compositions disclosed herein can be prepared such that upon exposure to selected conditions (e.g., suitable biological or enzymatic conditions), the cleavable linking group (e.g., disulfide group) cleaves and thereby causes dissociation of one or more functional groups or moieties (e.g., head and/or tail groups) bonded thereto. Functional groups or moieties (e.g., R)1Hydrophilic groups such as imidazole) may cause a phase transition in the liposome composition of which one or more compounds described herein are a component, which thereby destabilizes the liposome and facilitates fusion with one or more target cell membranes. Other contemplated cleavable linking groups can include those comprising a linker bonded to (e.g., covalently bonded to), for example, an alkyl group (e.g., C)1To C10Alkyl) groups, or one or more disulfide (S-S) linking groups.
In certain embodiments, the present invention provides a compound 5- (((10, 13-dimethyl-17- (6-methylhept-2-yl) -2, 3, 4,7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yl) disulfanyl) methyl) -1H-imidazole (referred to herein as "HGT 4001") having the structure of formula IV.
In certain embodiments, the present invention provides a compound 1- (2- (((3S, 10R, 13R) -10, 13-dimethyl-17- ((R) -6-methylhept-2-yl) -2, 3, 4,7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yl) disulfanyl) ethyl) guanidine (referred to herein as "HGT 4002") having the structure of formula V.
In certain embodiments, the present invention provides a compound 2- ((2, 3-bis ((9Z, 12Z) -octadec-9, 12-dien-1-yloxy) propyl) disulfanyl) -N, N-dimethylethylamine (referred to herein as "HGT 4003") having the structure of formula VI.
In other embodiments, the present invention provides a compound 5- (((2, 3-bis ((9Z, 12Z) -octadec-9, 12-dien-1-yloxy) propyl) disulfanyl) methyl) -1H-imidazole (referred to herein as "HGT 4004") having the structure of formula VII.
In still other embodiments, the present invention provides a compound 1- (((2, 3-bis ((9Z, 12Z) -octadec-9, 12-dien-1-yloxy) propyl) disulfanyl) methyl) guanidine (referred to herein as "HGT 4005") having the structure of formula VIII.
The compounds described herein can be used to construct liposome compositions that facilitate or enhance delivery and release of an encapsulating substance (e.g., one or more therapeutic polynucleotides) to one or more target cells (e.g., by penetrating or fusing with the lipid membrane of such target cells). In certain embodiments, the inclusion of one or more cleavable functional groups of such compounds allows, for example, the dissociation of a hydrophilic functional head group from a lipophilic functional tail group of the compound (e.g., upon exposure to acidic conditions), thereby promoting phase transition in the lipid bilayer of one or more target cells. For example, when the liposome composition (e.g., lipid nanoparticle) includes or is otherwise enriched for one or more compounds described herein, the phase transition in the lipid bilayer of one or more target cells facilitates delivery of an encapsulated substance (e.g., one or more therapeutic polynucleotides encapsulated in the lipid nanoparticle) into one or more target cells.
In certain embodiments, the compounds described herein are characterized by having one or more properties that provide advantages of such compounds over other similar classes of lipids. For example, in certain embodiments, the compounds described herein allow for control and tailoring of the properties of the liposome compositions (e.g., lipid nanoparticles) with which they are a component. In particular, the compounds described herein may be characterized by enhanced transfection efficiency and their ability to elicit specific biological outcomes. Such results can include, for example, enhanced cellular uptake, endosomal/lysosomal destruction capabilities, and/or enhanced intracellular release of encapsulated substances (e.g., polynucleotides).
In certain embodiments, the compounds described herein (and pharmaceutical and liposomal compositions comprising such compounds) employ a multi-functional strategy to facilitate delivery of an encapsulated substance (e.g., one or more polynucleotides) to and subsequent transfection of one or more target cells. For example, in certain embodiments, the compounds described herein (and pharmaceutical and liposomal compositions including such compounds) are characterized by having one or more of receptor-mediated endocytosis, clathrin-mediated and cytoplasmic membrane microcapsule-mediated endocytosis, phagocytosis and macroendocytosis, fusogenic, endosomal or lysosomal destruction, and/or exfoliatable properties that provide advantages of such compounds over other similar classes of lipids.
In certain embodiments, the compounds, and pharmaceutical and liposomal compositions (e.g., lipid nanoparticles) having such compounds as components, exhibit enhanced (e.g., increased) ability to transfect one or more target cells. Accordingly, also provided herein are methods of transfecting one or more target cells. Such methods generally comprise the step of contacting one or more target cells with a compound and/or pharmaceutical composition disclosed herein (e.g., an HGT 4003-based lipid nanoparticle encapsulated with one or more polynucleotides), such that one or more such target cells are transfected with the substance (e.g., one or more polynucleotides) encapsulated therein. As used herein, the term "transfection" or "transfection" refers to the intracellular introduction of one or more encapsulated substances (e.g., nucleic acids and/or polynucleotides) into a cell, or preferably into a target cell. The introduced polynucleotide may be stably or transiently maintained in the target cell. The term "transfection efficiency" refers to the relative amount of such encapsulated substances (e.g., polynucleotides) that are taken up, introduced, and/or expressed by the transfected target cells. In practice, transfection efficiency is assessed by the amount of reporter polynucleotide product produced by the transfected target cells. In certain embodiments, the compounds and pharmaceutical compositions described herein exhibit high transfection efficiency, thereby improving the likelihood that a suitable dose of the encapsulated substance (e.g., one or more polynucleotides) will be delivered to the site of pathology and subsequently expressed, while minimizing potential systemic adverse effects.
A wide range of substances that can exert a pharmacological or therapeutic effect can be delivered to target cells using the compounds, compositions and methods of the invention. Accordingly, the compounds and drugs and liposome compositions described herein can be used to encapsulate any substance suitable for intracellular delivery. In certain embodiments, such encapsulated substances are capable of conferring a therapeutic or diagnostic benefit to the cells in which such substances are delivered, and may include any drug, biological agent, and/or diagnostic agent. The substance may be organic or inorganic. The organic molecule can be a peptide, a protein, a carbohydrate, a lipid, a sterol, a nucleic acid (including peptide nucleic acids), or any combination thereof. In certain embodiments, the pharmaceutical and liposome compositions described herein can include or otherwise encapsulate more than one type of substance, e.g., two or more different polynucleotide sequences encoding proteins, enzymes, and/or steroids. In certain embodiments, the encapsulated substance is one or more of a polynucleotide and a nucleic acid.
As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to genetic material (e.g., DNA or RNA), and when referring to the compounds and compositions (e.g., lipid nanoparticles) described herein, such terms are generally used to refer to genetic material encapsulated by such compounds and compositions (e.g., lipid nanoparticles). In some embodiments, the polynucleotide is RNA. Suitable RNAs include mRNA, siRNA, miRNA, snRNA and snoRNA. Polynucleotides encompassed also include intergenic long-chain non-coding RNAs (lincrnas), which do not normally encode proteins but play a role in, for example, immune signaling, stem cell biology, and disease progression. (see, e.g., Guttman et al, 458: 223-. In a preferred embodiment, the polynucleotide is an mRNA. In certain embodiments, the polynucleotides encapsulated by the compounds or pharmaceutical and liposome compositions of the invention include RNA or stabilized RNA (e.g., mRNA encoding alpha galactosidase) encoding a protein or enzyme. The present invention encompasses the use of such polynucleotides (and in particular RNA or stabilized RNA) as therapeutics, for example as disclosed in international application No. pct/US 2010/058457 and US provisional application No. 61/494,881 filed 6/8/2011, which polynucleotides are capable of being expressed by target cells to facilitate production (and in some cases excretion) of a functional enzyme or protein by such target cells, the teachings all of which are incorporated herein in their entirety by reference. For example, in certain embodiments, upon expression of one or more polynucleotides by a target cell, production of a functional enzyme or protein (e.g., a uremic cycle enzyme or an enzyme associated with a lysosomal storage disorder) that is deficient in the subject can be observed. The term "functional" as used herein to limit a protein or enzyme means that the protein or enzyme has biological activity or alternatively is capable of performing the same or similar function as a native or normally functioning protein or enzyme.
The term "expression" in the context of the present invention, used in its broadest sense, refers to the transcription of a particular gene or polynucleotide into at least one mRNA transcript, or the translation of at least one mRNA or polynucleotide into a protein or enzyme. For example, in certain embodiments, the compounds and pharmaceutical or liposomal compositions described herein comprise a polynucleotide (e.g., mRNA) encoding a functional protein or enzyme. In the context of such mRNA polynucleotides, the term expression refers to the translation of such mRNA (e.g., by a target cell) to produce the polypeptide or protein encoded thereby.
In certain embodiments, the compounds and pharmaceutical compositions provided herein are capable of modulating the expression of aberrantly expressed nucleic acids and polynucleotides in one or more target cells and tissues. Accordingly, also provided herein are methods of treating a disease in a subject by administering to the subject an effective amount of a compound and/or a drug or a liposome composition described herein. In certain embodiments, such methods can enhance (e.g., increase) expression of the polynucleotide and/or increase expression and secretion of the functional polypeptide product in one or more target cells and tissues (e.g., hepatocytes). In some embodiments, the targeted cell or tissue aberrantly expresses a polynucleotide encapsulated by one or more compounds or drugs described herein and a liposome composition (e.g., a lipid nanoparticle). Also provided herein are methods of increasing the expression of one or more polynucleotides (e.g., mRNA) in one or more target cells, tissues, and organs. Typically, such methods comprise contacting the target cell with one or more compounds and/or drugs or liposome compositions comprising or otherwise encapsulating one or more polynucleotides.
In certain embodiments, the compounds disclosed herein can be used as liposomes or components of liposomes. In particular, the compounds described herein may be used in certain embodiments as the lipid (e.g., cationic lipid) component of a liposome composition (e.g., lipid nanoparticles). Such liposomes can be used to encapsulate substances and facilitate delivery of such substances to one or more target cells, tissues and organs. As used herein, the term "liposome" generally refers to a vesicle composed of lipids (e.g., amphipathic lipids) arranged in one or more spherical bilayers or bilayers. In certain embodiments, the liposome is a lipid nanoparticle (e.g., a lipid nanoparticle comprising one or more cationic lipid compounds disclosed herein). Such liposomes can be unilamellar or multilamellar vesicles having membranes formed from lipophilic materials and an aqueous interior containing encapsulated materials (e.g., polynucleotides) to be delivered to one or more target cells, tissues, and organs. In certain embodiments, the pharmaceutical and liposomal compositions described herein comprise one or more lipid nanoparticles. Contemplated liposomes include lipid nanoparticles. Examples of suitable lipids (e.g., cationic lipids) for forming liposomes and lipid nanoparticles encompassed thereby include one or more compounds disclosed herein (e.g., HGT4001, HGT4002, HGT4003, HGT4004, and/or HGT 4005). Such liposomes and lipid nanoparticles can also include additional cationic lipids such as C12-200, DLin-KC2-DMA, and/or HGT5001, non-cationic lipids, helper/cholesterol-based lipids, PEG-modified lipids, and phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides), and combinations or mixtures of the foregoing.
Several cationic lipids have been described in the literature, many of which are commercially available. In certain embodiments, in addition to one or more compounds or lipids disclosed herein (e.g., HGT4003), such cationic lipids are included in a medicament or liposome composition described herein. In some embodiments, a cationic lipid, N- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethylammonium chloride or "DOTMA" is used. Other suitable cationic lipids include, for example, ionizable cationic lipids such as (15Z, 18Z) -N, N-dimethyl-6- (9Z, 12Z) -octadeca-9, 12-dien-1-yl) dimyristyl-15, 18-dien-1-amine (HGT5000), (15Z, 18Z) -N, N-dimethyl-6- ((9Z, 12Z) -octadec-9, 12-dien-1-yl) ditetradecyl-4, 15, 18-triethylenetetramine-1-amine (HGT5001) and (15Z, 18Z) -N, N-dimethyl-6- ((9Z, 12Z) -octadec-9, 12-dien-1-yl) tetracosan-5, 15, 18-triethylenetetramine-1-amine (HGT 5002); c12-200(WO 2010/053572), 2- (2, 2-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) -1, 3-dioxolan-4-yl) -N, N-dimethylethylamine (DLinKC2-DMA)) (see, WO 2010/042877; semple et al, nature biotech.28: 172-, n-dimethyl-6- (9Z, 12Z) -octadec-9, 12-dien-1-yl) ditetradecyl-15, 18-dien-1-amine "HGT 5000", (15Z, 18Z) -N, N-dimethyl-6- ((9Z, 12Z) -octadec-9, 12-dien-1-yl) ditetradecyl-4, 15, 18-triethylenetetramine-1-amine "HGT 5001" and (15Z, 18Z) -N, N-dimethyl-6- ((9Z, 12Z) -octadec-9, 12-dien-1-yl) tetracos-5, 15, 18-triethylenetetramine-1-amine "HGT 5002", 5-carboxysperminylglycine-dioctadecylamide or "DOGS 2, 3-dioleyloxy-N- [2 (spermine-carboxamido) ethyl ] -N, N-dimethyl-1-propanaminium or "DOSPA" (Behr et al Proc. Nat.' l Acad. Sci.86, 6982 (1989); U.S. Pat. No.5,171,678; U.S. Pat. No.5,334,761), 1, 2-dioleoyl-3-dimethylammonium-propane or "DODAP", 1, 2-dioleoyl-3-trimethylammonium-propane or "DOTAP". Contemplated cationic lipids also include 1, 2-distearoyloxy-N, N-dimethyl-3-aminopropane or "DSDMA", 1, 2-dioleyloxy-N, N-dimethyl-3-aminopropane or "DODMA", 1, 2-dioleyloxy (dilinoleyloxy) -N, N-dimethyl-3-aminopropane or "DLinDMA", 1, 2-dilinoleyloxy (dilinoleyloxy) -N, N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyl-N, N-dimethylammonium chloride or "DODAC", N-distearoyl-N, N-dimethylammonium bromide or "DDAB", N- (1, 2-dimyridyloxyprop-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide or "DMRIE", 3-dimethylamino-2- (cholest-5-en-3-beta-oxybutyn-4-oxy) -1- (cis, cis-9, 12-octadecenyloxy) propane or "CLinDMA", 2- [5 ' - (cholest-5-en-3-beta-oxy) -3 ' -oxapentyloxy) -3-dimethyl-1- (cis, cis-9 ', 1-2 ' -octadecenyloxy) propane or "CpLinDMA", N-dimethyl-3, 4-dioleyloxybenzylamine or "DMOBA", 1, 2-N, N ' -dioleylcarbamoyl-3-dimethylaminopropane or "DOcarbDAP"), 2, 3-dioleyloxyl (dilinoloyloxy) -N, N-dimethylpropylamine or "DLInDAP", 1, 2-N, N' -dioleylcarbamoyl-3-dimethylaminopropane or "DLincarbDAP", 1, 2-dioleylcarbamoyl (dilinoylcarbamoyl) -3-dimethylaminopropane or "DLInCDAP", 2-dioleyl-4-dimethylaminomethyl- [1, 3] -dioxolane or "DLin-K-DMA", 2-dioleyl-4-dimethylaminoethyl- [1, 3] -dioxolane or "DLin-K-XTC 2-DMA" or mixtures thereof. (Heyes, J., et al, J Controlled Release 107: 276-. Also encompassed by the invention is the formulation of the composition (e.g., lipid nanoparticles) using cholesterol-based cationic lipids. Such cholesterol-based cationic lipids can be used alone or in combination with other cationic or non-cationic lipids. Suitable cholesterol-based cationic lipids include, for example, DC-Chol (N, N-dimethyl-N-ethylamidocholesterol, 1, 4-bis (3-N-oleylamino-propyl) piperazine (Gao, et al biochem. Biophys. Res. Comm.179, 280 (1991); Wolf et al BioTechniques23, 139 (1997); U.S. Pat. No.5,744,335).
Also contemplated are cationic lipids such as dialkylamino-based, imidazole-based, and guanidine-based lipids. For example, also encompassed is the use of cationic lipid ethyl (3S, 10R, 13R, 17R) -10, 13-dimethyl-17- ((R) -6-methylhept-2-yl) -2, 3, 4,7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yl 3- (1H-imidazol-4-yl) propionate or "ICE" as disclosed in international application No. pct/US 2010/058457 (the contents of which are incorporated herein by reference).
Also encompassed are polyethylene glycol (PEG) modified phospholipids and derivatized lipids such as derivatized ceramides (ceramides) (PEG-CER) (including N-octanoyl-sphingosine-1- [ succinyl (methoxypolyethylene glycol) -2000 ] in the liposomes and pharmaceutical compositions described herein](C8PEG-2000 ceramide)) preferably in combination with one or more compounds and lipids disclosed herein. Contemplated PEG-modified lipids include, but are not limited to: a polyethylene glycol chain up to 5kDa covalently linked to a linker having C6-C20Long alkyl chains on lipids. In some embodiments, the PEG-modified lipid employed in the compositions and methods of the invention is 1, 2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (2000MW PEG) "DMG-PEG 2000". The addition of PEG-modified lipids to lipid delivery vehicles can prevent complex aggregation and can also provide a means for increasing the circulating lifespan and delivery of lipid-polynucleotide compositions to target tissues, (Klibanov et al (1990) FEBS Letters, 268 (1): 235-237), or alternatively, they can be rapidly swapped out of formulation in vivo (see U.S. Pat. No.5,885,613). A particularly useful exchangeable lipid is PEG-ceramide having a shorter acyl chain (e.g., C14 or C18). The PEG-modified phospholipids and derivatized (derivitized) lipids of the invention may include the following molar ratios of total lipid present in the liposomal lipid nanoparticle: about 0% to about 20%, about 0.5% toAbout 20%, about 1% to about 15%, about 4% to about 10%, or about 2%.
The invention also encompasses the use of non-cationic lipids in one or more pharmaceutical or liposomal compositions (e.g., lipid nanoparticles). Preferably such non-cationic lipids are used in combination with one or more of the compounds and lipids disclosed herein. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic, or anionic lipid. As used herein, the phrase "anionic lipid" refers to any number of lipid species that carry a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include, but are not limited to: distearoylphosphatidylcholine (DSPC), Dioleoylphosphatidylcholine (DOPC), Dipalmitoylphosphatidylcholine (DPPC), Dioleoylphosphatidylglycerol (DOPG), Dipalmitoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylic acid methyl ester (DOPE-mal), Dipalmitoylphosphatidylethanolamine (DPPE), Dimyristoylphosphatidylethanolamine (DMPE), Distearoylphosphatidylethanolamine (DSPE), DLPE (1, 2-dilauroyl-sn-glycerol-3-phosphoethanolamine), DPPS (1, 2-dipalmitoyl-sn-glycero-3-phospho-L-serine), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), ceramides, sphingomyelin, cholesterol, or mixtures thereof. Such non-cationic lipids can be used alone, but are preferably used in combination with other excipients, such as one or more cationic lipid compounds disclosed herein (e.g., HGT4001, HGT4002, HGT4003, HGT4004, and/or HGT 4005). When used in combination with a cationic lipid, the non-cationic lipid may comprise the following molar ratio of total lipid present in the lipid nanoparticle, such as 5% to about 90%, or preferably about 10% to about 70%.
Also contemplated is the inclusion of a polymer into a lipid nanoparticle comprising a drug or a liposomal composition described herein. Suitable polymers may include, for example, polyacrylates, polyalkyl cyanoacrylates, polylactic acid-polyglycolide copolymers, polycaprolactone, dextran, albumin, gelatin, alginates, collagen, chitosan, cyclodextrins, and polyethylene imine. Such polymers may be used alone, but are preferably used in combination with other excipients, such as one or more cationic lipid compounds disclosed herein (e.g., HGT4001, HGT4002, HGT4003, HGT4004, and/or HGT 4005).
In certain embodiments, the drug and liposome compositions (e.g., lipid nanoparticles) are formulated based in part on their ability to facilitate transfection (e.g., of a polynucleotide) of a target cell. In another embodiment, the drug and liposome compositions (e.g., lipid nanoparticles) can be selected and/or prepared to maximize delivery of the polynucleotide to the target cells, tissues, and organs. For example, if the target cell is a hepatocyte, the properties (e.g., size, charge, and/or pH) of the drug and/or liposome composition can be maximized to effectively deliver such compositions (e.g., lipid nanoparticles) to the target cell or organ, reduce immune clearance, and/or promote retention in the target organ. Alternatively, if the target tissue is the central nervous system, the selection and preparation of the drug and liposome composition must take into account penetration and retention within the blood-brain barrier and/or use alternative methods of delivering such compositions (e.g., lipid nanoparticles) directly to such target tissue (e.g., via intracerebroventricular administration). In certain embodiments, the drug or liposome composition or component thereof lipid nanoparticle may be associated with an agent that facilitates the transfer of the encapsulated substance (e.g., disrupts or improves the permeability of the blood brain barrier and thereby enhances the transfer of such encapsulated polynucleotide to the target cell). While the drug and liposome compositions described herein (e.g., lipid nanoparticles) can facilitate the introduction of encapsulated substances such as one or more polynucleotides into a target cell, the addition of polycations (e.g., poly-L-lysine and protamine) to, for example, one or more lipid nanoparticles comprising the pharmaceutical composition as a copolymer can also facilitate and, in some cases, significantly enhance the transfection efficiency of several types of cationic liposomes by increasing the number of cell lines both in vitro and in vivo by a factor of 2-28. (see, N.J.Caplen, et al, Gene ther.1995; 2: 603; S.Li, et al, Gene ther.1997; 4,891.)
In certain embodiments of the invention, the drug and liposome compositions (e.g., lipid nanoparticles) are prepared to encapsulate one or more substances or therapeutic agents (e.g., polynucleotides). The method of incorporating a desired therapeutic agent (e.g., mRNA) into a liposome or lipid nanoparticle is referred to herein as "filling" or "encapsulation" (Lasic, et al, FEBS Lett., 312: 255-258, 1992). The substance (e.g., polynucleotide) filling or encapsulating the lipid nanoparticle may be located entirely or partially in the inner space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or in communication with the outer space of the lipid nanoparticle.
Packing or encapsulation, e.g., of polynucleotides into lipid nanoparticles, can serve to protect the polynucleotides from an environment that can contain enzymes or chemicals (e.g., serum) that degrade such polynucleotides and/or systems or receptors that cause rapid excretion of such polynucleotides. Accordingly, in some embodiments, the compositions described herein are capable of enhancing the stability of a polynucleotide encapsulated thereby, particularly with respect to the environment to which such polynucleotide will be exposed. Encapsulating substances such as polynucleotides into one or more of the drugs and liposome compositions described herein (e.g., lipid nanoparticles) also facilitates delivery of such polynucleotides into target cells and tissues. For example, a lipid nanoparticle comprising one or more lipid compounds described herein can allow the encapsulated polynucleotide to reach a target cell, or can preferentially allow the encapsulated polynucleotide to reach a target cell or organ based on the difference present (e.g., the lipid nanoparticle can be concentrated in the liver or spleen of a subject administered such lipid nanoparticles). Alternatively, the lipid nanoparticle may limit delivery of the encapsulated polynucleotide to other non-targeted cells or organs in which the presence of the encapsulated polynucleotide may be undesirable or of limited utility.
In certain embodiments, the pharmaceutical and liposomal compositions described herein (e.g., lipid nanoparticles) are prepared by combining a plurality of lipid components (e.g., one or more compounds described herein) with one or more polymer components. For example, HGT4003, DOPE, CHOL, and DMG-PEG2000 can be used to prepare lipid nanoparticles. Lipid nanoparticles can be composed of additional lipid combinations including, for example, HGT4001, DOPE, and DMG-PEG2000 in various ratios. The selection of the cationic lipid, non-cationic lipid, and/or PEG-modified lipid comprising the lipid nanoparticle and the relative molar ratio of such lipids to each other is based on the characteristics of the selected lipid, the desired properties of the target cell or tissue, and the characteristics of the substance or polynucleotide to be delivered by the lipid nanoparticle. Additional considerations include, for example, alkyl chain saturation as well as size, charge, pH, pKa, fusibility, and toxicity of the selected lipid.
The pharmaceutical and liposomal compositions (e.g., lipid nanoparticles) used in the present invention can be prepared by a variety of techniques now known to those skilled in the art. Multilamellar vesicles (MLVs) can be prepared by conventional techniques, for example by placing the selected lipid on the inner wall of a suitable container or vessel by dissolving the lipid in a suitable solvent and then evaporating the solvent to leave a thin film on the interior of the vessel, or by spray drying. The aqueous phase can then be added to the vessel and vortexed, which will result in the formation of MLVs. Unilamellar vesicles (ULV) can then be formed by homogenization, sonication, or extrusion of the multilamellar vesicles. Furthermore, unilamellar vesicles can be formed by detergent removal techniques.
In certain embodiments, the drug and liposome compositions of the present invention comprise lipid nanoparticles, wherein the encapsulated polynucleotide (e.g., mRNA) is associated with the surface of the lipid nanoparticle and the surface encapsulated within the same lipid nanoparticle. For example, during preparation of the compositions of the present invention, one or more cationic lipid compounds described herein and comprising lipid nanoparticles can associate with a polynucleotide (e.g., mRNA) by electrostatic interaction with such polynucleotide.
In certain embodiments, the pharmaceutical and liposomal compositions of the invention may be filled with diagnostic radionuclides, fluorescent substances, or other substances detectable in both in vitro and in vivo applications. For example, suitable diagnostic substances for use in the present invention may include rhodamine-dioleoylphosphatidylethanolamine (Rh-PE), green fluorescent protein mRNA (GFP mRNA), Renilla luciferase mRNA, and firefly luciferase mRNA (SEQ ID NO: 1).
During preparation of the liposome compositions described herein, they may also be encapsulated in an aqueous interior by including a water-soluble carrier agent in the hydration solution and may be incorporated into the lipid bilayer by including lipophilic molecules in the lipid formulation. For certain molecules (e.g., cationic or anionic lipophilic polynucleotides), packing of the polynucleotides into shaped lipid nanoparticles or liposomes can be accomplished by methods such as those described in U.S. patent No.4,946,683, the disclosure of which is incorporated herein by reference. Following encapsulation of the polynucleotide, the lipid nanoparticles may be treated by methods such as silica gel chromatography, diafiltration, or ultrafiltration to remove unencapsulated mRNA. For example, if it is desired to remove externally-bound polynucleotides from the surface of a liposome composition (e.g., lipid nanoparticles) described herein, such lipid nanoparticles can be passed through a diethylaminoethyl dextran cellulose column.
In addition to the encapsulated substance, a substance (e.g., a polynucleotide or one or more therapeutic or diagnostic agents) can be included or encapsulated in the lipid nanoparticle. For example, such additional therapeutic agents may be associated with the surface of the lipid nanoparticle, which may be incorporated into the lipid bilayer of the lipid nanoparticle by including such therapeutic agents in a lipid formulation or packing such therapeutic agents into a shaped lipid nanoparticle (see, U.S. patent nos. 5,194,654 and 5,223,263, the contents of which are incorporated herein by reference).
There are several methods of reducing the size or "styling" of the liposome compositions (e.g., lipid nanoparticles) disclosed herein, and any of these methods can generally be employed when styling is used as part of the present invention. Extrusion is one method of liposome styling. (Hope, M J et al Reduction of Liposome Size and Preparation of Unilamellar vehicles by Extrusion technologies. in: Liposome Technology (G. Gregoriadis, eds.) Vol.1, p.123 (1993)). The method consists of extruding liposomes through small pore polycarbonate membranes or asymmetric ceramic membranes to reduce the liposome size to a relatively well-defined size distribution. Typically, the suspension is circulated through the membrane one or more times until the desired liposome size distribution is achieved. Liposomes can be continuously extruded through smaller pore membranes to achieve a gradual reduction in liposome size.
Various alternative methods are available in the art for styling a set of lipid nanoparticles. One such sizing method is described in U.S. patent No.4,737,323, the contents of which are incorporated herein by reference. Sonication of the liposome or lipid nanoparticle suspension by bath or probe sonication results in small ULVs that gradually decrease in size down to diameters of less than about 0.05 microns. Homogenization is another method that relies on shear energy to fragment large liposomes into smaller liposomes. In a typical homogenization procedure, MLV is recirculated through a standard emulsion homogenizer until the selected liposome size is observed to be typically between about 0.1 and 0.5 microns. The size of the lipid nanoparticles can be determined by quasi-electro-optical scattering (QELS) as described in the following documents: bloomfield, ann.rev.biophysis.bioeng., 10: 421-450(1981), the contents of which are incorporated herein by reference. The diameter of the average lipid nanoparticle can be reduced by sonication of the formed lipid nanoparticles. Intermittent sonication cycles can be used alternating with QELS assessment to guide efficient liposome synthesis.
Selection of an appropriate size for the liposome compositions (e.g., lipid nanoparticles) described herein must take into account the location of the target cell or tissue, and to some extent, which application the lipid nanoparticles are used for. As used herein, the phrase "target cell" refers to a cell to which one or more of the drugs and liposome compositions described herein are localized or targeted. In some embodiments, the target cell comprises a specific tissue or organ. In some embodiments, the target cell lacks a protein or enzyme of interest. For example, when it is desired to deliver a polynucleotide to a hepatocyte, the hepatocyte represents the target cell. In some embodiments, the pharmaceutical or liposomal compositions of the invention (and, e.g., the polynucleotide species encapsulated therein) transfect target cells (i.e., do not transfect non-target cells) based on the differences that exist. The compositions and methods of the invention can be prepared to preferentially target a variety of target cells, including but not limited to: hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meningeal stellate cells, motor neurons, dorsal root ganglion cells, and anterior horn neurons), photoreceptor cells (e.g., rods and cones), retinal pigment epithelial cells, secretory cells, heart cells, adipocytes, vascular smooth muscle cells, cardiac muscle cells, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.
After transfection of one or more target cells with a polynucleotide, e.g., encapsulated in one or more lipid nanoparticles comprising a drug or liposome composition disclosed herein, it may be preferable to facilitate production of a product (e.g., a polypeptide or protein) encoded by such polynucleotides and enhance the ability of such target cells to express polynucleotides and produce a target, e.g., a polypeptide or protein. For example, transfection of a target cell with one or more compounds or pharmaceutical compositions that encapsulate mRNA will enhance (i.e., increase) the production of the protein or enzyme encoded by such mRNA.
In some embodiments, it may be desirable to limit the transfection of polynucleotides to certain cells or tissues. For example, due in part to its important role in metabolism and protein production, the liver represents an important target organ for the compositions of the invention, and accordingly diseases caused by defects in liver-specific gene products (e.g., urea cycle disorders) can benefit from specific targeting of cells (e.g., hepatocytes). Accordingly, in certain embodiments of the invention, the structural characteristics of the target tissue can be exploited to localize the distribution of the drug and liposome compositions of the invention (e.g., HGT 4001-based lipid nanoparticles) to such target tissue. For example, to target hepatocytes, one or more lipid nanoparticles comprising a drug or liposome composition described herein can be sized so that they are smaller in size than the perforations in the liver lining the antral endothelial layer; accordingly, one or more such lipid nanoparticles may readily penetrate such endothelial perforations to reach the target hepatocytes. Alternatively, the lipid nanoparticles may be sized such that the liposomes have a sufficient diameter to limit or specifically avoid their distribution into certain cells or tissues. For example, lipid nanoparticles comprising the drugs and liposome compositions described herein can be sized such that their size is larger than the perforations lining the lining of the antral lining, thereby limiting the distribution of the liposomal lipid nanoparticles to the hepatocytes. In this embodiment, large liposomal compositions (e.g., lipid nanoparticles) will not readily penetrate the endothelial perforation and instead be cleared by kupffer cells lining the antrum hepaticus. E.g., sizing of lipid nanoparticles comprising the pharmaceutical composition, thus may provide the opportunity to further regulate and precisely control the extent to which expression of the encapsulated polynucleotide may be enhanced in one or more target cells. Typically, the size of the at least one lipid nanoparticle comprising the drug and the liposome composition of the invention is in the range of about 25 to 250nm, preferably less than about 250nm, 175nm, 150nm, 125nm, 100nm, 75nm, 50nm, 25nm or 10 nm.
Similarly, the compositions of the invention can be prepared to preferentially distribute to other target tissues, cells or organs, such as the heart, lung, kidney, spleen. For example, the lipid nanoparticles of the present invention can be prepared to achieve enhanced delivery to target cells and tissues. Accordingly, the compositions of the present invention may be enriched with additional cationic, non-cationic, and PEG-modified lipids to further target tissues or cells.
In some embodiments, the compounds and pharmaceutical and liposomal compositions described herein (e.g., HGT 4002-based lipid nanoparticles) are distributed to hepatocytes and tissues to enhance delivery, transfection, and subsequent expression by, and corresponding production of polypeptides or proteins encoded by, the polynucleotides (e.g., mRNA) encapsulated therein. While such compositions may be preferentially distributed into hepatocytes and tissues, the therapeutic effect of the expressed polynucleotides and the proteins subsequently encoded thereby need not be limited to the target cells and tissues. For example, as disclosed in, e.g., international application No. pct/US 2010/058457 and U.S. provisional application No. 61/494,881, the teachings of which are incorporated by reference in their entirety, targeted hepatocytes may function as "reservoirs" or "reservoirs" capable of expressing or producing and excreting functional proteins or enzymes systemically or to the surroundings. Accordingly, in certain embodiments of the invention, one or more lipid nanoparticles (e.g., HGT 4005-based lipid nanoparticles) comprising the pharmaceutical and liposome compositions described herein may target hepatocytes (hepatocytes) and/or be preferentially distributed to hepatocytes and tissues by delivery. After transfection of a target hepatocyte by a polynucleotide encapsulated in one or more such lipid nanoparticles, such polynucleotides and excreted functional products (e.g., polypeptides or proteins) are expressed (e.g., translated) and systematically distributed where such functional products exert the desired therapeutic effect.
Polynucleotides encapsulated in one or more compounds or drugs described herein and liposome compositions can be delivered to and/or transfected into a target cell or tissue. In some embodiments, a functional polypeptide product that is capable of being expressed and produced (and in some cases excreted) by the target cell, thereby conferring a beneficial property, for example, to the target cell or tissue. Such encapsulated polynucleotides may encode, for example, hormones, enzymes, receptors, polypeptides, peptides, or other proteins of interest. In certain embodiments, such encapsulated polynucleotides may also encode small interfering RNA (sirna) or antisense RNA for the purpose of modulating or otherwise reducing or eliminating endogenous nucleic acid or gene expression. In certain embodiments, such encapsulated polynucleotides may be natural or recombinant in nature, and may exert their therapeutic activity using sense or antisense mechanisms of action (e.g., by modulating expression of a target gene or nucleic acid).
In some embodiments, the encapsulated polynucleotides (e.g., mRNA encoding the absent protein) may optionally include chemical or biological modifications, e.g., modifications that improve the stability and/or half-life of such polynucleotides or that improve or otherwise facilitate translation of such polynucleotides.
Also encompassed by the invention is the delivery of one or more single polynucleotides to a target cell by a compound or drug and liposome composition described herein, for example, by combining two single therapeutic agents or polynucleotides into a single lipid nanoparticle. Also contemplated is the delivery of one or more encapsulated polynucleotides to one or more target cells to treat a single disorder or defect, wherein each such polynucleotide acts through a different mechanism of action. For example, a drug or liposome composition of the invention may include a first polynucleotide, e.g., encapsulated in a lipid nanoparticle, intended to correct a defect in an endogenous protein or enzyme, and a second polynucleotide intended to inactivate or "knock down" the malfunctioning endogenous polynucleotide and its protein or enzyme product. Such encapsulated polynucleotides can encode, for example, mRNA and siRNA.
Although in vitro transcribed polynucleotides (e.g., mRNA) can be transfected into a target cell, such polynucleotides can be easily and efficiently degraded in vivo by the cell, thus rendering such polynucleotides ineffective. Furthermore, some polynucleotides are unstable in body fluids (in particular human serum) and may be degraded or digested even before reaching the target cells. Furthermore, native mRNA can decay within the cell, with a half-life of between 30 minutes and several days. Accordingly, in certain embodiments, the encapsulated polynucleotides provided herein, and in particular the mRNA polynucleotides provided herein, preferably retain at least some ability to be expressed or translated to produce a functional protein or enzyme within one or more target cells.
In certain embodiments, the pharmaceutical and liposomal compositions comprise one or more lipid compounds disclosed herein and one or more lipid nanoparticles comprising or encapsulating one or more stabilized polynucleotides (e.g., mRNA that has been stabilized against nuclease digestion or degradation in vivo) that modulate the expression of a gene or can be expressed or translated to produce a functional polypeptide or protein within one or more target cells. In certain embodiments, the activity of such encapsulated polynucleotides (e.g., mRNA encoding a functional protein or enzyme) is extended over an extended period of time. For example, the activity of the polynucleotide can be extended such that the pharmaceutical composition can be administered to the subject on a semi-weekly or bi-weekly basis, or more preferably monthly, bi-monthly, quarterly, or yearly basis. The prolonged or prolonged activity of the pharmaceutical compositions of the invention, and in particular the encapsulated mrnas, is directly related to the amount of functional protein or enzyme translated from such mrnas. Similarly, the activity of the compositions of the invention can be further lengthened or prolonged by making chemical modifications that further improve or enhance translation of the mRNA polynucleotide. For example, Kozac consensus sequences play a role in the initiation of protein translation and inclusion of such Kozac consensus sequences in an encapsulated mRNA polynucleotide may further lengthen or prolong the activity of the mRNA polynucleotide. In addition, the number of functional proteins or enzymes produced by a target cell is a function of the number of polynucleotides (e.g., mrnas) delivered to the target cell and the stability of such polynucleotides. Where the stability of a polynucleotide encapsulated by a compound or composition of the invention can be improved or enhanced, the half-life, activity, and frequency of administration of the composition of the translated protein or enzyme can be further increased.
In certain embodiments, the polynucleotide may be chemically modified, for example, to confer stability (e.g., stability relative to a wild-type or naturally occurring mRNA and/or an mRNA that is native to the target cell). Accordingly, in some embodiments, the encapsulated polynucleotides provided herein comprise at least one chemical modification that confers increased or enhanced stability to the polynucleotide, including, for example, improved tolerance to nuclease digestion in vivo. As used herein, the phrases "chemically modified" and "chemically modified," when such terms refer to a polynucleotide provided herein, include at least one change that preferably enhances stability and renders the polynucleotide more stable (e.g., resistant to nuclease digestion) than the wild-type or naturally-occurring type of such polynucleotide. When such terms "stable" and "stability" relate to polynucleotides encapsulated by the compounds or drugs of the invention and liposome compositions and particularly with respect to mRNA, such terms refer to increasing or enhancing resistance to degradation by, for example, nucleases (i.e., endonucleases or exonucleases) that are typically capable of degrading such RNAs. Increased stability can include, for example, less susceptibility to hydrolysis or other disruption caused by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within the target cell or tissue, thereby increasing or enhancing tolerance of such polynucleotides in the target cell, tissue, subject, and/or cytoplasm. The stabilized polynucleotide molecules provided herein exhibit a longer half-life relative to their naturally occurring, unmodified counterparts (e.g., wild-type versions of the polynucleotides).
The phrases "chemically modified" and "chemically modified" also encompass changes that improve or enhance translation of mRNA polynucleotides when such terms refer to polynucleotides encapsulated by the compounds or drugs of the invention and liposome compositions, including, for example, the inclusion of sequences that play a role in protein translation initiation (e.g., Kozac consensus sequences) (inclusions). (Kozak, M., nucleic acids Res15 (20): 8125-48 (1987)). The phrase "chemically modified" as used herein also includes modifications that introduce chemicals that are different from those found in naturally occurring polynucleotides, e.g., covalent modifications, such as the introduction of modified nucleotides (e.g., nucleotide analogs or the inclusion of non-naturally found pendant groups in such polynucleotide molecules). In some embodiments, the polynucleotides have been chemically or biologically modified to make them more stable prior to their encapsulation in the one or more lipid nanoparticles. Exemplary chemical modifications to a polynucleotide include deletion of a base (e.g., by deletion or substitution of a nucleotide) or chemical modification of a base.
In addition, suitable modifications include changes in one or more nucleotides of a codon such that the codon encodes the same amino acid but is more stable than the codon found in the wild-type form of the polynucleotide. For example, the inverse relationship between RNA stability and higher numbers of cytidine (C) and/or uridine (U) residues has been shown, and RNA without C and U residues has been found to be stable to most RNases (Heidenreich, et al J Biol Chem269, 2131-8 (1994)). In some embodiments, the number of C and/or U residues in the mRNA sequence is reduced. In another embodiment, the number of C and/or U residues is reduced by substituting one codon encoding a particular amino acid for another codon encoding the same or a related amino acid. Contemplated modifications to mRNA polynucleotides encapsulated by the compounds or drugs of the invention and liposome compositions also include the incorporation of pseudouridine. Incorporation of pseudouridine into mRNA polynucleotides encapsulated by the compounds or drug and liposome compositions of the present invention can enhance stability and translation and reduce immunogenicity in vivo (see, e.g., Karik Lou, K., et al, Molecular Therapy16 (11): 1833-1840 (2008)). Substitution and modification of the polynucleotides encapsulated by the compounds or drugs of the present invention and the liposome compositions can be carried out by methods readily known to one or ordinary skill in the art.
The constraints on reducing the number of C and U residues in the sequence will likely be greater in the mRNA coding region than in the untranslated region (i.e., it is unlikely that all C and U residues present in the message can be eliminated while still retaining the ability of the message to encode the desired amino acid sequence). However, the degeneracy of the genetic code represents an opportunity to allow a reduction in the number of C and/or U residues present in the sequence while maintaining the same coding capacity (i.e., there may be several different possibilities for modification of the RNA sequence based on the amino acids encoded by the codon). For example, the codon for Gly can be changed to GGA or GGG, but not GGU or GGC.
The term chemical modification also includes, for example, incorporation of non-nucleotide linked or modified nucleotides into a polynucleotide sequence of the invention (e.g., one or both end-capping modifications to the 3 'and 5' ends of an mRNA molecule encoding a functional protein or enzyme). Such modifications can include the addition of bases to a polynucleotide sequence (e.g., the inclusion of a poly a tail or longer poly a tail), alterations of the 3 'UTR or 5' UTR, complexing the polynucleotide with agents (e.g., proteins or complementary polynucleotide molecules), and the inclusion of elements that alter the structure of the polynucleotide molecule (e.g., form secondary structures).
The poly a tail is believed to stabilize natural messengers and synthetic antisense RNA. Thus, in certain embodiments, a long poly a tail may be added to an mRNA molecule thus making the RNA more stable. The poly a tail may be added using a variety of techniques recognized in the art. For example, long poly A tails can be added to synthetic or in vitro transcribed RNA using poly A polymerase (Yokoe, et al Nature Biotechnology.1996; 14: 1252-. The transcription vector also encodes a long poly-a tail. In addition, poly a tails can be added by transcription directly from the PCR product. The poly-A can also be ligated to the 3' end of the sense RNA using RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, second edition, Sambrook, Fritsch, and Maniatis eds (Cold Spring Harbor Laboratory Press: 1991). In certain embodiments, the poly a tail is at least about 90, 200, 300, 400, at least 500 nucleotides in length. In certain embodiments, the length of the poly a tail is modulated to control the stability of the modified sense mRMA molecules of the invention, and thus control the transcription of the protein. For example, since the length of the poly-A tail can affect the half-life of a sense mRNA molecule, the length of the poly-A tail can be adjusted to modify the level of nuclease resistance of the mRNA and thereby control the time course of polynucleotide expression and protein production in the target cell. In certain embodiments, the stabilized polynucleotide molecule is sufficiently resistant to degradation in vivo (e.g., by nucleases) such that it is delivered to the target cell in the absence of the lipid nanoparticle.
In certain embodiments, the chemical modification is a capping modification of one or more polynucleotides comprising the pharmaceutical composition of the invention. For example, such polynucleotides may be modified by incorporating 3 'and/or 5' Untranslated (UTR) sequences that are not naturally present in the wild-type polynucleotide. In certain embodiments, to modify the nucleotide sequence of an mRNA molecule, 3 'and/or 5' flanking sequences flanking the mRNA and encoding a second unrelated protein may be incorporated into the nucleotide sequence of the mRNA molecule encoding the functional protein. For example, 3 'or 5' sequences from a stabilized mRNA molecule (e.g., globin, actin, GAPDH, tubulin, histone, or citrate cycle enzyme) can be incorporated into the 3 'and/or 5' region of a sense mRNA polynucleotide molecule to increase the stability of the sense mRNA molecule.
Also encompassed by the invention are polynucleotide sequence modifications to one or both of the 3 'and 5' ends of the polynucleotide. For example, the invention encompasses modifications to the 3 'and/or 5' end of a polynucleotide (e.g., mRNA) comprising a partial sequence of the CMV immediate early 1(IE1) gene or a fragment thereof to improve nuclease resistance and/or to improve the half-life of the polynucleotide (such as, for example, as SEQ ID NO: 1). In addition to increasing the stability of the polynucleotide sequence, it has surprisingly been found that the inclusion of a CMV immediate early 1(IE1) gene partial sequence (e.g., to one or more 5 'untranslated regions and 3' untranslated regions of an mRNA) further enhances translation of the mRNA. Also contemplated is the inclusion of a sequence from the human growth hormone (hGH) gene or fragment thereof to one or both of the 3 'and 5' ends of a polynucleotide (e.g., mRNA) to further stabilize the polynucleotide (such as, e.g., as set forth in SEQ ID NO: 2). In general, contemplated chemical modifications improve the stability and/or pharmacokinetic properties (e.g., half-life) of polynucleotides relative to their unmodified counterparts, and include modifications made, for example, to improve the tolerance of such polynucleotides to nuclease digestion in vivo.
In some embodiments, the pharmaceutical composition, two or more lipid nanoparticles included therein, or a polynucleotide encapsulated by such lipid nanoparticles may include a stabilizing agent. The composition can include one or more agents that are directly or indirectly bonded to the polynucleotide and stabilize the polynucleotide, thereby enhancing the duration of tolerance in the cytoplasm of the target cell. Such agents preferably result in an improved half-life of the polynucleotide in the target cell. The stability and translation efficiency of an mRNA can be increased by incorporating "stabilizers" that form complexes with the polynucleotide (e.g., mRNA) naturally occurring within the cell (see, e.g., U.S. Pat. No.5,677,124). For example, incorporation of a stabilizer can be accomplished by combining poly a and protein with the mRNA to be stabilized in vitro prior to filling or encapsulating the mRNA within one or more lipid nanoparticles comprising the pharmaceutical composition. Exemplary stabilizing agents include one or more proteins, peptides, aptamers, translation accessory proteins, mRNA binding proteins, and/or translation initiation factors.
Stabilization of the drug and liposome compositions described herein (e.g., lipid nanoparticles) can also be improved by using opsonization-inhibiting moieties, which are typically large hydrophilic polymers, that are chemically or physically bonded or otherwise incorporated into the lipid nanoparticles (e.g., by intercalation of lipid-soluble anchors into the membrane itself, or by direct bonding to active groups of the membrane lipids). These opsonization-inhibiting hydrophilic polymers form a protective surface layer that significantly reduces uptake of liposomes by the macrophage monocyte system and reticuloendothelial system (e.g., as described in U.S. patent No.4,920,016, the entire disclosure of which is incorporated herein by reference). For example, delayed uptake of lipid nanoparticles by the reticuloendothelial system may be promoted by adding a hydrophilic polymer surface coating onto or into the lipid nanoparticles to mask recognition and uptake of the liposome-based lipid nanoparticles by the reticuloendothelial system. For example, in certain embodiments, one or more lipid nanoparticles comprising a pharmaceutical composition disclosed herein comprise a polyethylene glycol (PEG) polymer or PEG-modified lipid to further enhance delivery of such lipid nanoparticles to target cells and tissues.
RNA can avoid the enzyme when hybridized to a complementary polynucleotide molecule (e.g., DNA or RNA) (Krieg, et al, Melton. methods in enzymology.1987; 155, 397-. The stability of the hybridized mRNA may be attributed to the inherent single-stranded specificity of most RNases. In some embodiments, the stabilizing agent of the selected composite polynucleotide is a eukaryotic protein (e.g., a mammalian protein). In another embodiment, a polynucleotide (e.g., mRNA) for sense therapy can be modified by hybridization to a second polynucleotide molecule. Translation initiation may be diminished if the entire mRNA molecule is hybridized to a complementary polynucleotide molecule. In some embodiments, the 5' untranslated region and the AUG initiation region of an mRNA molecule can optionally be unhybridized. Following translation initiation, the unwinding activity of the ribosomal complex can even act on high affinity duplexes so that translation can proceed. (Liebhaber.J.mol.biol.1992; 226: 2-13; Monia, et al J Biol chem.1993; 268: 14514-22.) it is to be understood that any of the methods described above for enhancing the stability of a polynucleotide may be used alone or in combination with one or more of any of the other methods and/or compositions described above.
In certain embodiments, the pharmaceutical compositions of the present invention enhance delivery of the lipid nanoparticle encapsulated polynucleotide to one or more target cells, tissues or organs. In some embodiments, the enhanced delivery to the one or more target cells comprises increasing the amount of the polynucleotide that is contacted with or otherwise delivered to the target cells. In some embodiments, the enhanced delivery to the target cell comprises reducing the amount of polynucleotide in contact with non-target cells. In some embodiments, the enhanced delivery to the target cells comprises allowing transfection of at least some of the target cells with the encapsulated polynucleotide. In some embodiments, the level of expression of the polynucleotide encapsulated by the lipid nanoparticle comprising the subject pharmaceutical composition and the corresponding production of the functional protein or enzyme encoded by the polynucleotide is increased in the target cell.
The polynucleotide encapsulated by the compound or drug of the invention and the liposome composition may optionally be associated with a reporter gene (e.g., upstream or downstream of the polynucleotide coding region), e.g., which facilitates the determination of delivery of the polynucleotide to a target cell or tissue. Suitable reporter genes may include, for example, green fluorescent protein mRNA (GFP mRNA), Renilla luciferase mRNA (luciferase mRNA), firefly luciferase mRNA (SEQ ID NO: 1), or any combination thereof. For example, GFP mRNA can be fused to a polynucleotide encoding OTC mRNA to facilitate confirmation of mRNA localization in a target cell, tissue or organ.
In some embodiments, the pharmaceutical compositions of the invention comprise one or more additional molecules (e.g., proteins, peptides, aptamers, or oligonucleotides) that facilitate transfer of the polynucleotides (e.g., mRNA, miRNA, snRNA, and snoRNA) from the lipid nanoparticle into the intracellular compartment of the target cell. In some embodiments, the additional molecule facilitates delivery of the polynucleotide to a target cell, e.g., within the cytosol, lysosome, mitochondria, nucleus, nucleolus, or protease. Also included are agents that facilitate the transport of the translated target protein from the cytoplasm to its normal intracellular location (e.g., in the mitochondria) to treat deficiencies in this organelle. In some embodiments, the agent is selected from the group consisting of: proteins, peptides, aptamers, and oligonucleotides.
In some embodiments, the compositions of the invention promote the endogenous production of one or more functional proteins and/or enzymes in a subject, and in particular promote the production of proteins and/or enzymes that exhibit less immunogenicity relative to their recombinantly produced counterparts. In certain embodiments of the invention, the lipid nanoparticle comprises a polynucleotide encoding mRNA for a deficient protein or enzyme. When such compositions are distributed to a target tissue and subsequently transfected into such target cells, exogenous mRNA packed or encapsulated into the lipid nanoparticle comprising the composition can be translated in vivo to produce a functional protein or enzyme encoded by such encapsulated mRNA (e.g., a protein or enzyme deficient by the subject). Accordingly, in certain embodiments, the compositions of the present invention take advantage of the ability of a subject to: the ability to translate exogenous or recombinantly produced mRNA to produce endogenous translated protein or enzyme and thereby produce (and be suitable for excretion of) a functional protein or enzyme. The translated protein or enzyme is also characterized by the inclusion in vivo of natural post-transcriptional modifications, which may often be absent from recombinantly produced proteins or enzymes, thereby further reducing the immunogenicity of the translated protein or enzyme.
Encapsulating mRNA in lipid nanoparticles and administering pharmaceutical compositions comprising such lipid nanoparticles avoids the need to deliver mRNA to specific organelles (e.g., mitochondria) within target cells. Conversely, upon transfection of the target cell and delivery of the encapsulated mRNA to the target cell cytoplasm, the mRNA content of the lipid nanoparticle can be translated and a functional protein or enzyme produced.
The invention also encompasses differential targeting of one or more target cells and tissues by passive and active targeting methods. The phenomenon of passive targeting takes advantage of the natural distribution pattern of lipid nanoparticles in vivo, without relying on the use of additional excipients or methods of enhanced recognition of lipid nanoparticles by one or more target cells. For example, lipid nanoparticles that are phagocytosed by cells of the reticuloendothelial system may accumulate in the liver or spleen, and accordingly may provide a means for passively targeted delivery of compositions to such target cells.
Alternatively, the present invention encompasses active targeting involving the use of additional excipients, referred to herein as "targeting ligands" that can be bonded (covalently or non-covalently) to the lipid nanoparticles to aid in the localization of such lipid nanoparticles to certain target cells or target tissues. For example, targeting may be mediated by inclusion of one or more endogenous targeting ligands (e.g., apolipoprotein E) in or on the lipid nanoparticle to aid in distribution to the target cell or tissue. Active recognition of the targeting ligand by the target tissue facilitates tissue distribution to, and cellular uptake of the lipid nanoparticle and/or its contents by, the target cells and tissues. For example, in certain embodiments, one or more lipid nanoparticles comprising a pharmaceutical formulation may comprise a targeting ligand for apolipoprotein-E in or on such lipid nanoparticles to facilitate or assist such lipid nanoparticles in recognizing and binding to endogenous low density lipoprotein receptors expressed, for example, by hepatocytes. As provided herein, a composition can include a ligand that can enhance the affinity of the composition for one or more target cells. The targeting ligand may be attached to the outer bilayer of the lipid nanoparticle during or after formulation. These methods are well known in the art. In addition, some lipid nanoparticles may include fusogenic polymers such as PEAA, hemagglutinin, other lipopeptides (see U.S. patent application Ser. Nos. 08/835,281 and 60/083,294, the contents of which are incorporated herein by reference), and other features for in vivo and/or intracellular delivery (feature). In other embodiments, the compositions of the invention exhibit improved transfection efficiency, and/or exhibit enhanced selectivity for target cells or tissues of interest. Thus encompassed are compositions or lipid nanoparticles comprising one or more ligands (e.g., peptides, aptamers, oligonucleotides, vitamins, or other molecules) capable of enhancing the affinity of the composition or its component lipid nanoparticles and their polynucleotide content for one or more target cells or tissues. Suitable ligands may optionally be bonded or attached to the surface of the lipid nanoparticle. In some embodiments, the targeting ligand may span the surface of the lipid nanoparticle or be encapsulated within the lipid nanoparticle. The selection of suitable ligands can vary widely based on their physical, chemical, or biological properties (e.g., selective affinity and/or recognition of target cell surface markers or features) of the particular target of the cell and its corresponding targeting ligand. Suitable targeting ligands can be selected so as to take advantage of the unique characteristics of the target cell, thus allowing the composition to distinguish between target and non-target cells. For example, the compositions of the invention may have surface markers (e.g., apolipoprotein-B or apolipoprotein-E) that selectively enhance recognition or affinity for hepatocytes (e.g., by receptor-mediated recognition and binding of such surface markers). In addition, it may be desirable to localize the compositions of the present invention to a host hepatocyte using galactose as a targeting ligand, or alternatively to localize the compositions of the present invention to a hepatic endothelial cell using mannose containing a carbohydrate residue as a targeting ligand (e.g., mannose containing a carbohydrate residue that may preferentially bind to an asialoglycoprotein receptor is present in hepatocytes). (see Hillery AM, et al, "drug delivery and Targeting: For pharmaceuticals and Pharmaceutical Scientists" (2002) Taylor & Francis, Inc.) the presence of such Targeting ligands conjugated to moieties present in lipid nanoparticles has thus facilitated the recognition and uptake of the liposomal compositions of the invention by one or more target cells and tissues. Examples of suitable targeting ligands include one or more peptides, proteins, aptamers, vitamins, and oligonucleotides.
As used herein, the term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, rodents, and the like, to which the compounds, medicaments, or liposome compositions and methods of the invention can be administered. Generally, the terms "subject" and "patient" are used interchangeably herein when referring to a human subject.
The ability of the compounds and pharmaceutical or liposomal compositions (e.g., lipid nanoparticles) described herein to modulate or enhance the expression of encapsulated polynucleotides and production of polypeptides or proteins provides a novel and more efficient means for accomplishing the in vivo production of polypeptides and proteins for the treatment of host diseases or pathological conditions. Such lipid nanoparticle compositions are particularly useful in the treatment of diseases or pathological conditions associated with aberrant expression of nucleic acids encoding proteins or enzymes. For example, successful delivery of a polynucleotide (such as mRNA) to a target organ (such as the liver) and in particular to hepatocytes, can be used for the treatment and correction of innate errors of metabolism that localize to the liver. Accordingly, the compounds, pharmaceutical compositions, and related methods described herein can be employed to treat a wide range of diseases and pathological conditions, particularly those due to protein or enzyme deficiencies. Polynucleotides encapsulated by the compounds or drugs and liposome compositions described herein (e.g., HGT 4004-based lipid nanoparticles) can encode functional products (e.g., proteins, enzymes, polypeptides, peptides, functional RNAs and/or antisense molecules), and preferably encode products that are desired to be produced in vivo.
The compounds, pharmaceutical compositions, and related methods of the invention are broadly applicable to the delivery of therapeutic agents, such as polynucleotides (and in particular mRNA), to treat a variety of conditions. In particular, such compounds, compositions, and related methods of the invention are useful for the treatment of diseases or disorders associated with protein and/or enzyme deficiencies. In certain embodiments, the lipid nanoparticle encapsulated polynucleotide encodes a functional protein or enzyme that is excreted or secreted by one or more target cells into the surrounding extracellular fluid (e.g., mRNA encoding hormones and neurotransmitters). Alternatively, in another embodiment, the polynucleotide encapsulated by the compound or drug of the invention and the liposome composition encodes a functional protein or enzyme that is retained in the cytosol of one or more target cells (e.g., an mRNA encoding an enzyme associated with the urea cycle or lysosomal storage metabolic disorder). Other conditions for which the compounds, pharmaceutical compositions, and related methods of the invention are useful include, but are not limited to, the following conditions, such as: SMN 1-associated Spinal Muscular Atrophy (SMA); amyotrophic Lateral Sclerosis (ALS); GALT-related galactosemia; cystic Fibrosis (CF); SLC3a 1-associated disorders including cystinuria; COL4a 5-related disorders, including Alport syndrome (Alport syndrome); galactocerebrosidase deficiency; x-linked adrenoleukodystrophy and adrenomyeloneuropathy; huntington's disease; parkinson's disease; muscular dystrophy (such as, for example, duchenne and becker muscular dystrophy (Duchennc andBecker)); hemophilia, such as, e.g., hemophilia b (fix) and hemophilia a (fviii); friedreich's ataxia; peyre disease (Pelizaeus-Merzbacher disease); tuberous sclerosis associated with TSC1 and TSC 2; sanfilippo B syndrome (MPS IIIB); CTNS-associated cystinosis; FMR 1-related disorders including fragile X syndrome, fragile X-associated tremor/ataxia syndrome, and fragile X ovarian premature ovarian failure syndrome; par-widi syndrome (Prader-Willi syndrome); fabry disease (fabry disease); hereditary hemorrhagic telangiectasia (AT); Niemann-Pick disease Type C1 (Niemann-Pick disease Type C1); neuronal ceroid lipofuscinosis-related disorders including Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), Juvenile Barden disease (Juvenile battle disease), Morus-Ha disease (Santavuori-Haltia disease), Jansky-Bielschowsky disease, and PTT-1 and TPP1 deficiencies; EIF2B1, EIF2B2, EIF2B3, EIF2B4 and EIF2B5 associated childhood ataxia with central nervous system dysmyelination/white matter ablation; CACNA1A and CACNB4 associated periodic ataxia type 2; MECP 2-associated disorders including classical rett syndrome, MECP 2-associated severe neonatal encephalopathy, and PPM-X syndrome; CDKL 5-associated Atypical Rett Syndrome (advanced Rett Syndrome); kennedy's disease (SBMA); notch-3 associated autosomal dominant hereditary cerebral arteriopathy (CADASIL) with subcortical infarction and leukoencephalopathy; SCN1A and SCN 1B-related epilepsy; polymerase G-associated disorders including Alpers-huttenlocher syndrome, POLG-associated sensorimotor neuropathy, dysarthria and ophthalmoplegia, and progressive exo-ophthalmoplegia with mitochondrial DNA deletion in autosomal dominant and recessive inheritance; x-linked adrenal insufficiency; x-linked blood agammaglobulinemia; wilson's disease and Fabry's disease. In certain embodiments, the polynucleotides of the invention, and in particular the mrnas, may encode functional proteins or enzymes. For example, compositions of the invention can include mRNA encoded acarbosidase α, erythropoietin, α 1-antitrypsin, carboxypeptidase N, α -L-iduronidase, iduronate 2-sulfatase, N-acetylglucosamine-1-phosphotransferase, N-acetylglucosaminidase, α -glucosaminidase acetyltransferase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine-4-sulfatase, β -glucosidase, galactose-6-sulfatase, β -galactosidase, β -glucuronidase, glucocerebrosidase, heparan sulfamidase, hyaluronidase, galactocerebrosidase, or human growth hormone.
The compounds and pharmaceutical compositions described herein can be administered to a subject. In some embodiments, the composition is formulated in combination with one or more additional polynucleotides, vectors, targeting ligands or stabilizers or other suitable excipients. Techniques for formulating and administering drugs are found in "Remington's Pharmaceutical Sciences" Mack Publishing co., Easton, Pa., latest edition.
The compounds and drugs of the present invention and the liposome compositions (e.g., lipid nanoparticles) can be administered and dosed in accordance with current medical practice and in view of the subject's clinical condition, the nature of the encapsulating material, the site and method of administration, the time course of administration, the subject's age, sex, body weight, and other factors relevant to the clinician of ordinary skill in the art. For purposes herein, an "effective amount" can be determined by such relevant considerations known to those of ordinary skill in the experimental clinical studies, pharmacology, clinical, and medical arts. In some embodiments, the amount administered is effective to achieve at least some stabilization, amelioration, or elimination of the symptoms, and to obtain other indicators selected by those skilled in the art as suitable measures of disease progression, regression, or improvement. For example, suitable amounts and dosing regimens are those that result in at least transient expression of one or more polynucleotides in a target cell.
Suitable routes of administration for the compounds and pharmaceutical compositions disclosed herein include, for example, oral, rectal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, intracerebroventricular, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections or infusions. In certain embodiments, administration of a compound or composition described herein (e.g., a lipid nanoparticle) to a subject facilitates contact of such compound or composition with one or more target cells, tissues, or organs.
Alternatively, the compounds and compositions of the present invention may be administered locally rather than systemically, e.g., via direct injection or infusion of the pharmaceutical composition into the target tissue, preferably in a depot or sustained release formulation, such that contacting of the targeted cells with the component lipid nanoparticles may be further facilitated. Depending on the tissue targeted, local delivery can be affected in various ways. For example, an aerosol containing a composition of the invention may be inhaled (for nasal, tracheal or bronchial delivery); for example, the compositions of the present invention may be injected into the site of injury, disease manifestation, or pain; the composition may be provided in the form of a lozenge for oral, transtracheal, or transesophageal application; may be provided in the form of a liquid, tablet or capsule for administration to the stomach or intestine, may be provided in the form of a suppository for rectal or vaginal application; or even delivered to the eye by use of creams, drops or injections. Formulations containing the compounds of the invention complexed with therapeutic molecules or ligands may even be administered surgically, for example in combination with polymers or other structures or substances that may allow diffusion of the composition from the site of implantation to the surrounding cells. Alternatively, such compositions may be applied surgically without the use of a polymer or support.
In certain embodiments, the compositions of the present invention are formulated such that they are suitable for sustained release, e.g., encapsulation of a polynucleotide or nucleic acid therein. Such sustained release compositions may conveniently be administered to a subject at a delayed dosing interval. For example, in certain embodiments, the compositions of the present invention are administered to a subject twice daily, or every other day. In certain embodiments, the compositions of the invention are administered to a subject at the following intervals: twice weekly, once weekly, every ten days, every two weeks, every three weeks or more preferably every four weeks, monthly, every six weeks, every eight weeks, every other month, every three months, every four months, every six months, every eight months, every nine months or annually. Also contemplated are compositions and lipid nanoparticles formulated for depot administration (e.g., intramuscular, subcutaneous, intravitreal) to deliver or release a polynucleotide (e.g., mRNA) over a longer period of time. Preferably, the sustained release approach is used in combination with modifications (e.g., chemical modifications) introduced to the polynucleotide to enhance stability.
While certain compounds, compositions and methods of the invention have been described specifically according to certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the compounds of the invention. Each disclosure, reference material, etc. herein is incorporated by reference in its entirety to describe the background of the invention and to provide additional details regarding its implementation.
Lyophilized liquid delivery vehicle
The present invention provides pharmaceutical compositions comprising a lyophilized liposomal delivery vehicle and a liposomal formulation that enable complete delivery of encapsulated contents (e.g., polynucleotides) to one or more target cells, tissues or organs. For example, in delivering an encapsulated polynucleotide to one or more target cells, such polynucleotides are capable of modulating (e.g., increasing expression of) the expression of the polynucleotide or nucleic acid in the target cell. Also disclosed herein are related methods and processes for preparing such pharmaceutical compositions, and methods of treating one or more diseases or conditions by administering such pharmaceutical compositions to a subject in need thereof. It is also desirable that the lyophilized compositions described herein (e.g., lipid nanoparticles) have improved long-term stability (e.g., at least one, two, three, six, nine, twelve, eighteen, twenty-four, thirty months or more) when stored under refrigeration or at ambient temperatures (e.g., room temperature).
As used herein, the terms "lyophilization" and "lyophilized" refer to a process of preparing such liposome compositions in dry form by flash freezing, and in some cases to one or more drying steps (e.g., when exposed to vacuum conditions), whereby the water concentration in such liposome compositions is reduced to prevent or optionally limit further biological or chemical reactions.
Lyophilization of the liposome composition (e.g., lipid nanoparticles) can be performed by any suitable method, for example, a lyophilization cycle as provided according to the examples. After flash freezing the liposome composition (e.g., lipid nanoparticles), the liposome composition can be dried by one or more suitable methods, such as exposure to first and second vacuum drying conditions. In some embodiments, the liposome composition (e.g., lipid nanoparticles) can be dried at room temperature under vacuum conditions as provided in this example. After exposure to the lyophilization conditions described herein, the lyophilized lipid nanoparticle composition can be rehydrated and administered to a subject using, for example, a suitable aqueous rehydration medium (e.g., sterile water, physiological saline, and/or 5% dextrose).
In certain embodiments, a lyophilized pharmaceutical composition described herein is characterized in that it is stable (e.g., relative to being an un-lyophilized pharmaceutical composition). As used to describe the lyophilized liposomal compositions described herein, the term "stable" refers to preventing such liposomal compositions (e.g., lipid nanoparticles) from aggregating or flocculating (e.g., after reconstitution). The stability of such lyophilized pharmaceutical compositions can be determined with reference to a number of physical properties. For example, stability may be determined with reference to the particle size of the lipid nanoparticles comprising such compositions. Preferably, after rehydration of the lyophilized composition disclosed herein, the size distribution and physical characteristics of the reconstituted composition are the same as or alternatively equivalent to the composition prior to lyophilization. Accordingly, in certain embodiments, lyophilization of the lipid nanoparticles does not significantly alter or change the particle size of the lipid nanoparticles after lyophilization and/or reconstitution. For example, lipid nanoparticles comprising a lyophilized pharmaceutical composition do not flocculate or aggregate when reconstituted (e.g., with purified water), or alternatively exhibit limited or negligible flocculation or aggregation (e.g., as determined by the particle size of the reconstituted lipid nanoparticles).
In certain embodiments, reconstituted liposomal compositions (e.g., lipid nanoparticles) of the invention exhibit enhanced (e.g., increased) ability to transfect one or more target cells. Accordingly, also provided herein are methods of transfecting one or more target cells. Such methods generally include the step of contacting one or more target cells with, for example, a reconstituted lyophilized pharmaceutical composition of the invention (e.g., a lyophilized HGT 4003-based lipid nanoparticle encapsulated with one or more polynucleotides), such that the one or more target cells are transfected with the substance (e.g., one or more polynucleotides) encapsulated therein.
In certain embodiments, one or more lipids (e.g., cationic lipids) may be used as liposomes or alternatively as a component of a lipid delivery vehicle (e.g., lipid nanoparticles) used in the compositions of the present invention. As noted above, suitable lipid delivery vehicles are lipid nanoparticles comprising a nucleic acid, a cationic lipid (such as a cleavable cationic lipid), such as: the above-mentioned HGT4001, HGT4002, HGT4003, HGT4004 and HGT4005 or are selected from the group consisting of: c12-200, ICE, DOTMA, DOGS, DOSPA, DODAP, DOTAP, DSDMA, DODMA DLInDMA DLDMADDAB DMRIECLinDMA CpLinDMA DMOBA DOcarbDADLINPDLincarbDAP DLinCDAP DLin-K-DMA DLin-K-XTC2-DMA, DLinKC2-DMA, HGT5000, HGT5001, HGT5002 or mixtures thereof.
Other suitable components of the lipid delivery vehicle include non-cationic lipids, helper lipids, such as cholesterol and PEG-modified lipids as described above. For example, HGT4003, DOPE, CHO and DMG-PEG2000 can be used to prepare lipid nanoparticles. Lipid nanoparticles can be composed of additional lipid combinations in various ratios, including, for example, HGT4001, DOPE, and DMG-PEG 2000. The selection of the cationic lipid, non-cationic lipid, and/or PEG-modified lipid comprising the lipid nanoparticle and the relative molar ratio of such lipids to each other is based on the characteristics of the selected lipid, the nature and grade of the intended target cell or tissue, and the characteristics of the substance or polynucleotide to be delivered by the lipid nanoparticle. Additional considerations include, for example, the degree of saturation of the alkyl chain and the size, charge, pH, pKa, fusibility, and toxicity of the selected lipid.
In one embodiment, the lyophilized lipid delivery vehicle further comprises at least one lyoprotectant. The term "lyoprotectant" as used herein refers to one or more compounds that, when combined with or included in the preparation of one or more liposomal compounds described herein, enhance (e.g., increase) the chemical and/or physical stability of the liposomal compound (e.g., lipid nanoparticle) during lyophilization, storage, or reconstitution of such liposomal compounds. For example, in certain embodiments, inclusion of one or more lyoprotectants in the lipid nanoparticle may improve or otherwise enhance the stability of the lyophilized composition (e.g., under normal storage conditions) and/or facilitate reconstitution of the lyophilized composition using a rehydration medium, thereby preparing an aqueous formulation. In some embodiments, the buffer present in the liposome formulation can be replaced (e.g., via centrifugation) with a suitable lyoprotectant (e.g., an aqueous sucrose solution comprising about 1-50% or 10-25% sucrose) prior to preparing the lipid nanoparticles and lyophilizing. In some embodiments, a lyoprotectant is included (e.g., during hydration, diafiltration, and/or dilution) as a buffer or part of the medium in which the lipid formulation is prepared or lyophilized. Examples of suitable lyoprotectants that can be used to prepare the lyophilized compositions described herein include, for example, trehalose, dextran (e.g., 1.5kDa, 5kDa, and/or 40kDa), inulin (e.g., 1.8kDa and/or 4kDa), and any combination thereof.
It is believed that the inclusion of a sugar lyoprotectant may serve to stabilize the lyophilized composition during lyophilization. (see, Anchordoquy, et al, J.Pharm.Sci. (2000) 89: 289-296.) one possible explanation for the observed stabilization may include the particle segregation hypothesis, which refers to the formation of a sugar matrix as a physical barrier between liposome particles.
Lyophilized drugs and component liposomes (e.g., lipid nanoparticles) for use in the present invention can be prepared by various techniques currently known in the art. Multilamellar vesicles (MLVs) can be prepared by conventional techniques, for example by depositing the selected lipid onto the inner wall of a suitable container or vessel by dissolving the lipid in a suitable solvent via a slurry, and then evaporating the solvent to leave a thin film inside the vessel, or by spray drying. The aqueous phase can then be added to the vessel and vortexed, which will result in the formation of MLVs. Unilamellar vesicles (ULV) can then be formed by homogenization, sonication, or extrusion of the multilamellar vesicles. Furthermore, unilamellar vesicles can be formed by detergent removal techniques.
As used herein, the articles "a" and "an" are to be understood as including a plurality of referents in the specification and claims, unless expressly stated to the contrary. Claims or descriptions that include an "or" between one or more members of a group are deemed to satisfy the requirement that one or more or all of the group members be present in, employed in, or otherwise relevant to a given product or process, unless the contrary is indicated or otherwise evident from the context. The invention includes embodiments in which a member of a group is specifically present in, employed in, or otherwise associated with a given product or process. The invention also includes embodiments in which more than one or all of the members of the group are present in, employed in, or otherwise associated with a given product or process. Moreover, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the listed claims is introduced into another claim that depends on the same base claim (or, as related any other claim), unless the contrary is indicated, or unless one of ordinary skill in the art would appreciate that a contradiction or inconsistency would arise. When elements are presented as a list (e.g., in a Markush group or similar format), it is to be understood that each subgroup of elements is also disclosed and that any element can be removed from the group. It will be understood that, generally, when the invention or aspects of the invention are referred to as including a particular element, feature, etc., certain embodiments or aspects of the invention consist or consist essentially of such element, feature, etc. For the sake of simplicity, those embodiments have not been shown in each case with so much text. It should also be understood that any embodiment or aspect of the invention is explicitly excluded from the claims, regardless of whether a particular exclusion is recited in the specification. The disclosures and other references cited herein to describe the background of the invention and to provide additional details regarding its practice are incorporated herein by reference.
Examples
Example 1 preparation of HGT4001
The compound 5- (((10, 13-dimethyl-17- (6-methylhept-2-yl) -2, 3, 4,7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yl) disulfanyl) methyl) -1H-imidazole (imidazole-cholesterol disulfide) (referred to herein as "HGT 4001") was prepared according to the general synthetic scheme shown below in reaction 1.
Reaction 1
The intermediate compound identified as compound (3), 2- (((3S, 10R, 13R,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2, 3, 4,7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta [ a ] a]Phenanthren-3-yl) disulfanyl) pyridine (pyridylcholesterol disulfide). A solution containing 3.0g (7.45mmol) of compound (1) and 1.8g (8.17mmol) of compound (2) in chloroform (35ml) was prepared and stirred at room temperature for four days. The solvent was evaporated, methanol (50ml) was added to the residue and evaporated. The resulting solid was suspended in methanol (50ml) and stirred at room temperature overnight. The pyridyl cholesterol disulfide product (3) was collected by filtration, washed with methanol and dried under high vacuum. Yield: 3.6g (95%).1H NMR(300MHz，CDCl3)δ8.43(m，1H)，7.76(m，1H)，7.62(m，1H)，7.05(m，1H)，5.32(bd，J=4Hz，1H)，2.75(m，1H)，2.35(d，J=8Hz，2H)，2.05-1.7(m，5H)，1.7-1.2(m，8H)，1.2-0.8(m，25H)，0.65(s，3H)。MS(APCI，Pos)：512(M+1)。
The intermediate compound, 4- ((benzylthio) methyl) -1H-imidazole, identified as compound (6) in reaction 1 was prepared as follows. A solution containing 12.15g (123.9mmol) of compound (4) and 15.5ml (132mmol) (5) in glacial acetic acid (200ml) was prepared and heated to reflux temperature for 24 h. The reaction mixture was allowed to cool overnight. The solvent was evaporated and the residue was dissolved in chloroform (800 ml). The resulting solution was washed with diluted ammonia (4: 1 water: concentrated ammonia, 200ml) and brine (200 ml). The organic phase was dried (Na)2SO4) Filtered and the solvent evaporated. Flash chromatography (silica gel, 500 g; 5-7% methanol in chloroform) afforded 23g of the desired product 4- ((benzylthio) methyl) -1H-imidazole (compound (6)), which represented a 91% yield. NMR showed the presence of a small impurity (4 wt%) identified as acetate and identified as compound (8) below. Compound 6 material was used to produce HGT4001 without further purification.1H NMR(300MHz，CDCl3)δ7.60(d，J=1Hz，1H)，7.35-7.2(m，5H)，6.90(d，J=1Hz，1H)，3.67(s，2H)，3.62(s，2H)。MS(APCI，Pos)：205(M+1)。
The intermediate compound (1H-imidazol-4-yl) methanethiol identified as compound (7) in scheme 1 was prepared as follows. The liquid ammonia (200ml) solution was concentrated by suspension containing 15g of compound (6) (70.5mmol) in ether (30 ml). To this resulting yellow solution 5g of solution (217mmol) were added portionwise until the mixture remained dark blue. It was then stirred for 40 minutes. Adding about 10-15g of solid NH4Cl until the color disappeared and the solvent was evaporated using a nitrogen stream to afford crude compound (7), which was used without purification.
HGT4001 was prepared by the following steps: 3.6g of the compound (3) (7mmol) and 10ml of triethylamine (71.8mmol) were added to chloroform (200ml), and the resulting solution was degassed using vacuum and nitrogen, and the resulting solution was rapidly added to the compound (7), and the resulting mixture was stirred at room temperature under nitrogen. After 3 days, 200ml of water were added and the mixture was extracted with chloroform (2 × 500 ml). The organic extracts were washed with brine (200ml) and dried (Na)2SO4) Filtered and the solvent evaporated. Flash chromatography (silica gel, 200g, neutralized with 1% triethylamine in chloroform; 2-5% ethanol in chloroform) afforded 1.25g HGT4001 (35% yield over two steps).1H NMR(300MHz，CDCl3)δ7.61(s，1H)，7.00(s，1H)，5.33(d，1H)，3.93(s，2H)，2.58-2.46(m，1H)，2.29(d，2H)，1.91(m，5H)，1.61-0.84(m，33H)，0.66(s，3H).13C NMR(300MHz，CDCl3) δ 141.6, 135.3, 134.3, 121.4, 118.1, 56.8, 56.2, 50.3, 50.2, 42.4, 39.8, 39.6, 39.1, 36.8, 36.2, 35.8, 31.9, 29.1, 28.3, 28.1, 24.4, 23.9, 22.9, 22.6, 21.0, 19.4, 18.8, 11.9. MS (APCI, Pos)515(M + 1). Elemental analysis: c31H50N2S2C (calculated 72.32), actual 72.04; h (calculated 9.79), actual 9.84; n (5.44, calculated), actual value 5.41.
Example 2 preparation of HGT4002
The compound 1- (2- (((3S, 10R, 13R) -10, 13-dimethyl-17- ((R) -6-methylhept-2-yl) -2, 3, 4,7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yl) disulfanyl) ethyl) guanidine (referred to herein as "HGT 4002") was prepared according to the following general synthetic scheme shown in reaction 2.
Reaction 2
Tert-butyl (2- (pyridin-2-yldisulfanyl) ethyl) carbamate, an intermediate compound identified as compound (10) in reaction 2 above, can be prepared by adding 5.0g of compound (9) (28.2mmol) and 6.82g of compound (2) (31mmol) to 100ml of chloroform (100ml) and stirring at room temperature for four days to form a solution. The solvent was evaporated and the resulting yellow solid was purified by flash chromatography (SiO)250-100% ethyl acetate in hexane) to afford 9.0g of the impure compound (10). NMR showed the presence of the desired material (56 wt%), along with the starting compound (2) (24%) and the disulfide compound (11) (20%) identified below. The resulting mixture was used in the following step without further purification.1H NMR(300MHz，DMSO-d6)δ8.55-8.45(m，1H)，7.9-7.8(m，2H)，7.3-7.2(m，1H)，7.07(bt，J=5Hz，1H)，3.25-3.15(m，2H)，2.87(t，J=7Hz，2H)，1.37(s，9H)。MS(APCI，Pos)287(M+1)，231(M+1-C4H8)。
The intermediate compound bis N, N' -tert-butyl-1- (2- (pyridin-2-yldisulfanyl) ethyl) guanidine carbamate (14) was prepared by adding 2.0g of compound (10) (56% pure, 3.9mmol) to anhydrous dichloromethane (12ml), and then adding TFA (6ml) thereto, and the resulting solution was stirred at room temperature for 5 hours. The solvent was evaporated and the residue was dried under high vacuum to afford crude compound (13) (TFA salt).The compound (13) salt was dissolved in 25ml of anhydrous dichloromethane, excess triethylamine (7ml) was added, then 2.7g of compound (12) (7.0mmol) was added and the reaction mixture was stirred overnight at room temperature, then diluted with chloroform (175ml) and washed with water (2 × 50ml) and brine (50 ml). The organic solution was dried (Na)2SO4) Filtered and the solvent evaporated. By flash chromatography (SiO)20-10% methanol in chloroform) to provide 1.9g of the impure compound (14). NMR showed the presence of the desired compound (14) (73 wt%), along with the disulfide compound (15) (27 wt%) identified below. The mixture was used in the following step without further purification.1H NMR(300MHz，CDCl3) δ 11.48(bs, 1H), 8.86(bt, 1H), 8.55-8.5(m, 1H), 7.65-7.6(m, 2H), 7.25-7.15(m, 1H), 3.8-3.65(m, 2H), 2.99(t, J =6Hz, 2H), 1.51(s, 9H), 1.49(s, 9H). MS (APCI, Pos): complex, not detected (M + 1).
1- (2- (pyridin-2-yldisulfanyl) ethyl) guanidine trifluoroacetate, an intermediate compound identified as compound (16) in reaction 2 above, was prepared by adding 1.6g of compound (14) (73% pure, 2.8mmol) to anhydrous dichloromethane (33ml), adding TFA (11ml) thereto, and the resulting solution was stirred at room temperature overnight. The solvent was evaporated and the residue was dried under high vacuum to afford crude compound (16) (TFA salt), which was then used in the following step without purification.
HGT4002 was prepared by dissolving the TFA salt of compound (16) in anhydrous dichloromethane (50ml) followed by addition of excess triethylamine (5 ml). 1.13g of thiocholesterol (1) (2.8mmol) was added and the reaction mixture was stirred at room temperature overnight, then diluted with chloroform (200ml) and washed with water (2X50ml) and brine (100 ml). The resulting organic solution was dried (Na)2SO4) Filtered and the solvent evaporated. By flash chromatography (SiO)20-30% ethanol in chloroform) and trituration in acetoneTo purify the residue to provide 80mg of HGT 4002.1H NMR(300MHz，DMSO-d6) δ 7.60-6.90 (width s, 4H), 5.35(d, 1H), 3.39(t, 2H), 2.84(t, 2H), 2.72(m, 1H), 2.28(m, 2H), 1.91(m, 5H), 1.58-1.28(m, 10H), 1.20-0.82(m, 23H), 0.65(s, 3H).13C NMR(300MHz，DMSO-d6) δ 157.5, 141.5, 121.5, 56.7, 56.1, 50.1, 49.6, 42.4, 38.3, 36.7, 36.2, 35.7, 31.9, 29.0, 28.3, 27.9, 24.4, 23.7, 23.2, 22.9, 21.0, 19.5, 19.1, 12.2. MS (APCI, Pos): 520(M + 1). Elemental analysis: c30H53N3S2-SiO2C (calculated 62.13), actual 62.33; h (calculated 9.21), actual 9.08; n (7.25, calculated), actual 7.07; s (11.06, calculated), actual 10.83.
Example 3 preparation of HGT4003
The compound 2- ((2, 3-bis ((9Z, 12Z) -octadec-9, 12-dien-1-yloxy) propyl) disulfanyl) -N, N-dimethylethylamine (referred to herein as "HGT 4003") was prepared according to the following general synthetic scheme shown in reaction 3.
Reaction 3
By adding 11.37g of compound (18) (90.3mmol) dropwise to 9.73g of compound (17) (90.3mmol) and 18.64g of K2CO3(135.1mmol) in 60mL of ACN to prepare 3- (benzylthio) propane-1, 2-diol, an intermediate identified as compound (19) in reaction 3 above. The resulting mixture was heated at reflux for 2 hours, and after cooling the reaction mixture to room temperature, the reaction mixture was filtered and the solids were washed with 20mL ACN. The filtrate was evaporated and the grey liquid residue was purified by column chromatography (eluent: 10-100% EtOAc in hexane) to give 17.03g of compound (19) (95%) as a clear liquid.
By passingNaH (60% in mineral oil, 0.82g, 20.5mmol) to 1.56g of compound (19) (7.88mmol) and 6.91g of compound (20) (21.00mmol) in THF (200mL) in N was added2The intermediate compound benzyl (2, 3-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) propyl) sulfane, identified as compound (21) in reaction 3 above, was prepared from the stirred mixture. The resulting mixture was heated at reflux for 44 hours. After the reaction mixture was cooled to room temperature, the mixture was taken up in Et2O (400mL) was diluted and washed with water (300mL) and brine (300 mL). The organic layer was washed with anhydrous Na2SO4Dried and evaporated, and the yellow liquid residue purified by column chromatography (eluent: 0-20% EtOAc in hexanes) to give compound (21) (2.04g, 37.3%) as a yellow liquid.
By adding Et of Compound (21) (0.7g, 1.01mmol)2O (30mL) solution to liquid NH3(30mL) to prepare the intermediate compound 2, 3-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) propane-1-thiol, identified as compound (22) in reaction 3 above, and the intermediate compound was N-78 ℃ in a 2-neck RBF2Then concentrated, and then a small piece of Na (90mg, 3.91mmol) was added. When TLC showed complete disappearance of Compound (21), the resulting mixture was stirred at-78 deg.C for 30min and 340mg NH was added4Cl (6.34 mmol). The dark blue color of the reaction mixture faded to light yellow within 10min and the dry ice acetone bath was removed. With N2The reaction mixture was purged while gradually warming to room temperature. In the passage of N2(reducing the volume of the reaction mixture to about 20mL) already reduced most of the NH3After stripping, aqueous HCl (3N, 30mL) was added. The mixture was extracted with DCM (60 mL). Extract DCM with anhydrous Na2SO4Dried and evaporated. The yellow liquid residue was purified by column chromatography (eluent: 0-20% EtOAc in hexane) to give 490mg of compound (22) (80%) as a pale yellow liquid.
The intermediate compound, N-dimethyl-2- (pyridin-2-yldisulfanyl) ethylamine, identified as compound (24) in reaction 3 above, 2.8g of compound (2) (12.7mmol) and 1.41g of compound (23) (10mmol) were combined in DCM (30 mL). With N2The mixture was purged for 10 minutes while stirring the mixture and 1.5mL Et was added3N (11.2 mmol). The resulting solution was stirred at room temperature for 16 hours and added to a 230g silica gel column. The column was eluted with 40-100% EtOAc/hexanes and then 8-10% MeOH/DCM to give 0.72g of compound (24) as a yellow liquid (34%).
By combining 487mg of compound (22) (0.81mmol) and 180mg of compound (24) (0.84mmol) in 2mL DCM, then in N2Stirring was carried out at room temperature for 16 hours to prepare HGT 4003. The reaction solution was purified by column chromatography three times (eluent: 20-100% EtOAc in hexane) to give 252mg of HGT4003 as a yellow liquid (44%). 213mg of compound (25) (37%) identified in reaction 4 below was also obtained from column chromatography purification.1H NMR(300MHz，CDCl3)δ5.36-5.33(m，8H)，3.65(m，1H)，3.56-3.50(m，4H)，3.43(td，2H)，2.96-2.74(m，8H)，2.60(t，2H)，2.25(s，6H)，2.04(m，8H)，1.62-1.50(m，5H)，1.39-1.22(m，32H)，0.88(t，6H).13C NMR(300MHz，CDCl3) δ 130.3, 128.0, 71.8, 71.6, 70.6, 58.8, 45.5, 41.4, 36.9, 31.6, 30.1, 29.7, 29.5, 29.4, 27.3, 26.2, 25.7, 22.6, 14.2. MS (APCI, Pos): 709(M + 1). Elemental analysis: c43H81NO2S2C (calculated 72.92), actual 72.75; h (calculated 11.53), actual 11.50; n (1.98, calculated), actual 2.08; s (9.05, calculated), actual 8.95.
Reaction 4
An alternative HGT4003 synthetic pathway is described in reaction 4 above, which employs a pyridyl disulfide bis (alkyl) intermediate. By dissolving 1.35g of Compound (22) (2.24mmol) and 0.54g of Compound (2) (2.45mmol) in 10mL of CHCl3In combination with N2Stirring at room temperature for 16 hours was carried out to prepare intermediate compound 2- ((2,3-bis ((9Z, 12Z) -octadeca-9, 12-dien-1-yloxy) propyl) disulfanyl) pyridine. The reaction solution was purified three times by column chromatography (eluent: 0-20% EtOAc in hexane) to obtain 1.1g of compound (25) (67%) as a pale yellow liquid. 1.09g of Compound (23) (7.71mmol) was added to Compound (25) (1.1g, 1.54mmol) and Et3N (2.6mL, 18.5mmol) in CHCl3(20mL) in solution and in N2Stirring the mixture. TLC after 16 hours showed complete disappearance of compound (25). The reaction solution was then washed with aqueous NaOH (1N, 20mL), anhydrous Na2SO4Dried and evaporated. The yellow liquid residue was purified by column chromatography (eluent: 5-100% EtOAc in hexane) to give 0.37g HGT4003 as a pale yellow liquid (34%).
Example 4
Lipid nanoparticles of firefly luciferase (FFL) mRNA (SEQ ID NO: 1) comprising HGT4001, DOPE and DMG-PEG2000 and encapsulating optimized codons were formed via standard ethanol injection methods. (Ponsa et al, int.J.pharm. (1993) 95: 51-56.) A stock ethanol solution of lipid at a concentration of 50 mg/mL was prepared in advance and stored at-20 ℃.
Synthesizing a codon optimized firefly luciferase (FFL) mRNA by: plasmid DNA template from the encoding gene was transcribed in vitro, followed by the addition of a5 'Cap construct (Cap1) (Fechter, P. et al, J.Gen.virology (2005) 86: 1239-1249) and a 3' poly (A) tail (approximately 200 nucleotides in length as determined by gel electrophoresis). As shown below, the 5 'and 3' untranslated regions present in each FFL mRNA product are represented by SEQ id nos: x and Y in 4.
Codon-optimized firefly luciferase (SEQ ID NO: 3):
XAUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGCCAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGAGCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGUGUAAY
X=GGGAUCCUACC(SEQ ID NO：5)
Y＝UUUGAAUU(SEQ ID NO：6)
the FFL mRNA was stored in water at-80 ℃ at a final concentration of 1 mg/mL until use. The concentration of all mrnas was determined via a Ribogreen assay (Invitrogen). mRNA encapsulation was calculated by Ribogreen assay in the presence and absence of 0.1% Triton-X100. Particle size (dynamic light scattering (DLS)) and zeta potential were determined using a malvern zetasizer instrument in 1x PBS and 1mM KCl solutions, respectively.
Aliquots of 50 mg/mL of the imidazole-based cationic lipid HGT4001. The ethanolic solutions of DOPE and DMG-PEG2000 were mixed and diluted with ethanol to a final volume of 3 mL. Separately, an aqueous buffered solution of FFL mRNA (10mM citrate/150 mM NaCl, pH4.5) was prepared from the stock solution at 1 mg/mL. The lipid solution was injected rapidly into the aqueous mRNA solution and shaken to produce the final suspension in 20% ethanol. The resulting nanosuspension was filtered, diafiltered with 1x PBS (ph7.4), concentrated and stored at 2-8 ℃. Final concentration =0.69 mg/mL CO-FF mRNA (encapsulated). Zave=70.3nm(Dv(50)=43.2nm；Dv(90)=80.3nm)。
Example 5
This example illustrates that HGT 4003-based lipid nanoparticles provide a highly efficient method of delivering polynucleotide constructs to one or more target cells, tissues and organs. HGT4003 based lipid nanoparticles were formed via standard ethanol injection methods. (Ponsa et al, int.J.pharm. (1993) 95: 51-56.) A stock ethanol solution of lipid at a concentration of 50 mg/mL was prepared in advance and stored at-20 ℃.
Synthesizing a codon optimized firefly luciferase (FFL) mRNA by: plasmid DNA template from the encoding gene was transcribed in vitro, followed by the addition of a5 'Cap construct (Cap1) (Fechter, P. et al, J.Gen.virology (2005) 86: 1239-1249) and a 3' poly (A) tail (approximately 200 nucleotides in length as determined by gel electrophoresis). The 5 'and 3' untranslated regions present in each mRNA product consist of SEQ ID NO: x and Y in 4 represent. The FFL mRNA was stored in water at-80 ℃ at a final concentration of 1 mg/mL until use. The concentration of all mrnas was determined via a Ribogreen assay (Invitrogen). mRNA encapsulation was calculated by Ribogreen assay in the presence and absence of 0.1% Triton-X100. Particle size (dynamic light scattering (DLS)) and zeta potential were determined using a malvern zetasizer instrument in 1x PBS and 1mM KCl solutions, respectively.
Aliquots of 50 mg/mL ethanolic solutions of HGT4003, DOPE, cholesterol and DMG-PEG2000 were mixed and diluted with ethanol to a final volume of 3 mL. Separately, an aqueous buffered solution of FFL mRNA (10mM citric acid) was prepared from a1 mg/mL stock solutionSalt/150 mM NaCl, pH 4.5). The lipid solution was injected rapidly into the aqueous mRNA solution and shaken to produce the final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with 1x PBS (ph7.4), concentrated and stored at 2-8 ℃. Final concentration =1.27 mg/mLCO-FF mRNA (encapsulated). Zave=60.9nm(Dv(50)=47.9nm；Dv(90)=75.3nm)。
To determine whether HGT 4003-based lipid nanoparticles are capable of delivering the encapsulated polynucleotide construct to one or more target cells, CD-1 mice were injected with a single dose of HGT 4003-based lipid nanoparticles encapsulated with FFL mRNA and sacrificed four hours later. As discussed below, animals were administered a single dose of HGT 4003-based lipid nanoparticles encapsulated with FFL mRNA via one of Intravenous (IV), Intracerebroventricular (ICV), or Intrathecal (IT) routes of administration. The activity of firefly luciferase protein produced in the liver, spleen, brain and spinal cord of animals following FFL mRNA expression was determined in a bioluminescent assay.
Briefly, bioluminescence assays were performed using the Promega luciferase assay System (article # E1500/E4500 Promega). Tissue preparation was performed as follows: portions of the desired (flash frozen) tissue samples were thawed, washed with RO/DI water and placed in ceramic bead homogeneous tubes. The tissue was treated with lysis buffer and homogenized. Upon five freeze/thaw cycles, followed by centrifugation at 4 ℃, the supernatant was transferred to a fresh centrifuge tube. The tissue extracts were repeated and stored at-80 ℃.
Luciferase assay reagents were prepared by adding 10mL of luciferase assay buffer to the luciferase assay substrate and mixing via vortexing. mu.L of the homogenate samples were loaded into 96-well plates, and then 20. mu.L of the plate control for each sample was loaded. Separately, 120 μ L of luciferase assay reagent was loaded into each well of a 96-well flat-bottom plate and each plate was inserted into an appropriate chamber using a Biotek Synergy2 instrument and the luminosity was measured in Relative Light Units (RLU).
By administering a single bolus Intravenous (IV) injection to the study animalsLipid nanoparticle formulations based on HGT4003 encapsulated with FFL mRNA described herein were evaluated. After four hours, animals were sacrificed and liver and spleen were harvested from each animal. The luminosity of FFL proteins produced via exogenous FFL messengers delivered is detected and analyzed. Figure 1 illustrates an example of using an intravenously administered HGT4003 based lipid nanoparticle system and shows that more than an order of magnitude of FFL protein (2.34 x10, respectively) is produced enriched in the liver compared to the spleen6RLU/protein mg vs 1.71x105RLU/protein mg), which demonstrates that the use of HGT4003 based nanoparticles provides enrichment of encapsulated substances in the liver over the spleen.
In addition, lipid nanoparticle formulations based on HGT4003, encapsulated with FFL mRNA, were evaluated by administering a single bolus injection to the central nervous system of study animals via the Intracerebroventricular (ICV) or epidural (IT) administration routes. Four hours later, animals were sacrificed and brain and spinal cord were collected from each animal. The luminosity of FFL proteins produced via exogenous FFL messengers delivered is detected and analyzed. As illustrated in figure 2, after administration of HGT 4003-based lipid nanoparticles, the production of FFL protein was enriched in the brain after the ICV administration route compared to the IT administration route.
A detectable luminescent signal above baseline was observed in each animal administered (independent of the chosen route of administration) HGT 4003-based lipid nanoparticle formulation encapsulating FFL-mRNA. The expression of exogenously administered FFL mRNA and the production of firefly luciferase protein from such FFL mRNA is inferred from the presence of luminescent signals above background. The increase in luminosity observed in the liver of the animals exceeded similar signals observed in the spleen, indicating an enrichment of lipid nanoparticles in hepatocytes and tissues. Similarly, when nanoparticles based on HGT4003, encapsulating FFL mRNA, were administered via the ICV administration route, the FFL protein IT produced was enriched in the brain relative to after IT route administration. Accordingly, this example illustrates that HGT 4003-based lipid nanoparticles provide a highly efficient method of delivering polynucleotide constructs to one or more target cells, tissues and organs.
Example 6 lyophilized Liposome preparation
Lipid nanoparticles were formed via standard ethanol injection methods (Ponsa, et al, int.J.pharm. (1993) 95: 51-56.) an ethanol stock solution of lipid at a concentration of 50 mg/mL was prepared in advance and stored at-20 ℃. Codon optimized firefly luciferase (FFL) mRNA (SEQ ID NO: 3) was stored at a final concentration of 1 mg/mL in water at-80 ℃ until use.
The concentration of all FFL mRNA was determined via a Ribogreen assay (Invitrogen). mRNA encapsulation was calculated by Ribogreen assay in the presence and absence of 0.1% Triton-X100. Particle size (dynamic light scattering (DLS)) and particle size were determined using a malvern zetasizer instrument in 1x PBS and 1mM KCl solutions, respectively. 293T cells were used to assess the in vitro activity of the encapsulated mRNA preparations and an equal amount of 10. mu.g mRNA from the selected preparation was incubated with 293T cells for 8 hours at 37 ℃. Luciferase production was measured using the Perkin elmer britelite Plus kit.
Typically, lyophilization of lipid nanoparticles is performed by freezing the prepared liposomes in a solution containing a lyoprotectant (sucrose) and then removing any water or moisture by vacuum sublimation. In particular, prior to lyophilization, the buffer present in the liposome formulation was replaced by 10% sucrose via centrifugation. The resulting lipid nanoparticle solution was then subjected to a lyophilization process characterized by the specific parameters of the freezing, primary drying, and secondary drying steps, as identified in table 1 below. The lyophilized cake was reconstituted with an appropriate amount of purified water prior to physical characterization and biochemical analysis as described below.
TABLE 1
Example 7
Formulations of lipid nanoparticles were prepared to include a lipid encapsulated in a mixture of C12-200: DOPE: CHOL: firefly luciferase mRNA (FFL) in DMG-PEG2000 (40: 30: 20: 10, N/P2) lipid nanoparticles. A portion of the batch of the prepared lipid nanoparticle formulation was then lyophilized according to the protocol shown in table 1.
The observed physical properties of the fresh (non-lyophilized) and lyophilized lipid nanoparticle formulations were compared according to the protocol described above and were found to be consistent. As illustrated in table 2 below, the average particle size (Z) of the fresh and lyophilized lipid nanoparticlesave) 103.8nm and 117.0nm, respectively. The polydispersity index (PDI) of the fresh lipid nanoparticles was 0.236 compared to 0.247 for the lyophilized lipid nanoparticles. Dv of fresh lipid nanoparticles50And Dv9060.2nm and 156nm, respectively, compared to the Dv of lyophilized lipid nanoparticles50And Dv9049.0nm and 176nm, respectively. Accordingly, the observed physical characteristics also indicate that both fresh and lyophilized lipid nanoparticles are stable and, furthermore, the particle size remains relatively comparable.
TABLE 2
| Batches 5926-48 | Zave(nm) | PDI | Dv50(nm) | Dv90(nm) |
| Before freeze-drying | 103.8 | 0.236 | 60.2 | 156 |
| After freeze-drying | 117.0 | 0.247 | 49.0 | 176 |
Example 8
The formulation of lipid nanoparticles was prepared to comprise a lipid encapsulated in DLinKC 2-DMA: DOPE: CHOL: firefly luciferase mRNA (FFL) in DMG-PEG2000 (50: 25: 20: 5, N/P5) lipid nanoparticles. One batch of the prepared lipid nanoparticle formulation was frozen according to the protocol shown in table 5 below.
The lyophilization process is performed by freezing the prepared liposomes in a solution containing a lyoprotectant (sucrose) and then removing any water or moisture by vacuum sublimation. In particular, prior to lyophilization, the buffer in the liposome formulation was replaced with 10% sucrose via centrifugation. The resulting liposome solution is then subjected to a lyophilization process characterized by the specific parameters of the freezing, primary drying and secondary drying steps, as identified in table 3 below. The lyophilized cake was reconstituted with an appropriate amount of purified water prior to physical characterization and biochemical analysis as described below.
TABLE 3
Prepared fresh (not lyophilized) and lyophilized formulations were used to deliver encapsulated FFL mRNA to 293T cells and luminosity was determined according to the protocol described above. As illustrated in table 4 belowThe luminescence value of fresh lipid nanoparticles before lyophilization was observed to be 4.21x106In contrast, the value of the luminescence observed after reconstitution of the lyophilized formulation was 2.65x106。
Average particle size (Z) of fresh and lyophilized lipid nanoparticlesave) 89.11nm and 96.41nm, respectively. The polydispersity index (PDI) of the fresh lipid nanoparticles was 0.205 compared to 0.204 for the lyophilized lipid nanoparticles. Dv of fresh lipid nanoparticles50And Dv9063.8nm and 117nm, respectively, compared to the Dv of lyophilized lipid nanoparticles50And Dv9065.1nm and 135nm, respectively. As table 6 shows, particle size and encapsulation efficiency were better maintained during lyophilization. The encapsulation efficiency of FFL mRNA of fresh and lyophilized lipid nanoparticles was 93% and 87%, respectively. Furthermore, the observed physical characteristics indicate that both fresh and lyophilized lipid nanoparticles are stable and furthermore the particle size remains relatively comparable.
TABLE 4
Example 9
The preparation of lipid nanoparticles was prepared to include Erythropoietin (EPO) mRNA (SEQ ID NO: 4) located at the 5 'and 3' ends of SEQ ID NO: 1 and SEQ ID NO: 2 and is enclosed by dlinck 2-DMA: DOPE: CHOL: DMG-PEG2000 (50: 25: 20: 5, N/P5) lipid nanoparticles. One batch of the prepared lipid nanoparticle formulation was lyophilized according to the protocol shown in table 3.
Human Erythropoietin (EPO) mRNA (SEQ ID NO: 4)
AUGGGGGUGCACGAAUGUCCUGCCUGGCUGUGGCUUCUCCUGUCCCUGCUGUCGCUCCCUCUGGGCCUCCCAGUCCUGGGCGCCCCACCACGCCUCAUCUGUGACAGCCGAGUCCUGGAGAGGUACCUCUUGGAGGCCAAGGAGGCCGAGAAUAUCACGACGGGCUGUGCUGAACACUGCAGCUUGAAUGAGAAUAUCACUGUCCCAGACACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUGGAGGUCGGGCAGCAGGCCGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAGCUGUCCUGCGGGGCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCCCCUGCAGCUGCAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACCACUCUGCUUCGGGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAGAUGCGGCCUCAGCUGCUCCACUCCGAACAAUCACUGCUGACACUUUCCGCAAACUCUUCCGAGUCUACUCCAAUUUCCUCCGGGGAAAGCUGAAGCUGUACACAGGGGAGGCCUGCAGGACAGGGGACAGAUGA
Physical properties of the lipid nanoparticle formulation observed before and after lyophilization were compared according to the above protocol and were found to be consistent. The average particle size (Z) of fresh (not lyophilized) and lyophilized lipid nanoparticles is illustrated in Table 5 belowave) 85.9nm and 95.4nm, respectively, indicating that both fresh and lyophilized lipid nanoparticles are stable. The polydispersity index (PDI) of the fresh lipid nanoparticles was 0.188 compared to 0.231 for the lyophilized lipid nanoparticles. Dv of fresh lipid nanoparticles50And Dv9061.0nm and 112nm, respectively, compared to the Dv of lyophilized lipid nanoparticles50And Dv9067.2nm and 134nm, respectively. The encapsulation efficiency of EPO mRNA of fresh and lyophilized lipid nanoparticles was 94% and 86%, respectively. As also shown in table 7, particle size and encapsulation efficiency were better maintained during lyophilization.
Finally, the R & D Systems Human EPO Quantikine IVD ELISA kit was used to measure the erythropoietin protein produced by 293T cells. As described in table 5, the erythropoietin protein produced after delivering EPO mRNA to 293T was comparable for the formulations before and after lyophilization, and no significant difference was present in the production of erythropoietin protein when comparing the lipid nanoparticle formulations before and after lyophilization.
TABLE 5
Example 10
A six month stability study was performed on lipid nanoparticles encapsulated with lyophilized EPO mRNA. The particle size distribution, the efficiency of mRNA encapsulation and the expression of EPO in CD-1 mice were determined.
The lipid formulation comprised KC2 as described in example 9: DOPE: CHOL: EPO mRNA encapsulated in DMGPEG2K (50: 25: 20: 5). The N/P ratio (defined as the ratio of the number of nitrogens in the cationic lipid to the number of phosphates in the nucleic acid) is 5.
One vial was stored at 2-8 ℃. One vial was stored at room temperature. The humidity of both storage conditions was not controlled.
The lyophilized cake was reconstituted with an appropriate amount of water for injection prior to physical characterization and animal studies.
Particle size was obtained with a Malvern Zetasizer Nano-ZS. The Invitrogen RiboGreen assay kit was used to determine the efficiency of mRNA encapsulation in lipid particles. Unencapsulated mRNA was detected directly. Total mRNA was measured after lysis of lipid nanoparticles in the presence of 0.45% w/v Triton X-100. Encapsulation efficiency was calculated as (total mRNA-unencapsulated mRNA)/total mRNAx 100%.
Wild-type CD-1 mice were used to assess the relative expression of EPO following a single IV administration of two formulations of lipid nanoparticles encapsulating hEPO mRNA. The EPO levels in serum were measured 6 hours after administration of the drug. Four 7-week-old CD-1 mice (2 males, 2 females) were used in this study. At arrival, animals were randomized to 2 treatment groups, each containing 2 animals (1 male, 1 female per group). On the first day, animals were weighed and body weights recorded. Each mouse received a single IV dose of 99. mu.g mRNA/animal, with a dose volume of 300. mu.L/animal. At 6 hours after dosing administration, mice were CO dosed2Asphyxiation was performed to euthanasia, which was then thoracically opened and the maximum available amount of blood was collected and processed to obtain serum. All treatments administered were well tolerated in CD-1 mice after a single IV administration. Serum levels of hEPO can be measured by ELISA. In all study animals from which either formulation was receivedEPO was observed in the serum of (a).
The test results are summarized in table 6. No significant change in particle size distribution was observed after 6 months of storage of the lyophilized lipid nanoparticles at refrigeration temperature and room temperature. Furthermore, the efficiency of mRNA encapsulation in lipid nanoparticles during storage remained essentially unchanged. These results indicate that the integrity of the lipid particles is better maintained in the lyophilized configuration during storage. Stability at accelerated room temperature conditions for 6 months supports a potential 2-year shelf life under refrigerated conditions. In addition, serum hEPO was detected in wild-type CD-1 mice at 6h after intravenous injection of the reconstituted lyophilized lipid nanoparticle (after storage at refrigeration temperature or room temperature) suspension. These results show that the integrity of the lipid particles is effectively protected during storage in the lyophilized configuration.
TABLE 6
Abbreviations: 1) zave (Zave average) is the average of the intensity distribution; 2) PDI (polydispersity index) describes the distribution width; 3) dv50 is the median value of the volume distribution; 4) dv90 means that 90% of the volume distribution lies below this value.
Example 11
Lyophilization studies of lipid nanoparticles encapsulated with mRNA were performed using 2-hydroxypropyl- β -cyclodextrin as a lyoprotectant. As a comparison, sucrose lyoprotectants were also evaluated.
mRNA was encapsulated by ethanol dilution method in C12-200: DOPE: CHOL: DMGPEG2K (40: 30: 25: 5) lipid particles. The N/P ratio was 20. The buffer in the formulation was replaced by an aqueous solution containing the appropriate amount of sucrose or 2-hydroxypropyl- β -cyclodextrin via centrifugation prior to lyophilization. The resulting solution is subjected to a lyophilization process characterized by the specific parameters of the freezing, primary drying and secondary drying steps. Table 7 describes the lyophilization cycle of the sucrose-containing formulation. Table 8 describes the lyophilization cycle of the formulation containing 2-hydroxypropyl- β -cyclodextrin. The lyophilized cake was reconstituted with an appropriate amount of purified water prior to physical characterization and biochemical analysis. Particle size was obtained with a Malvern Zetasizer Nano-ZS. The encapsulation efficiency of mRNA in lipid particles was determined using the Invitrogen RiboGreen assay kit. Unencapsulated mRNA was detected directly. Total mRNA was measured after lysis of lipid nanoparticles in the presence of 0.45% w/v Triton X-100. Encapsulation efficiency was calculated as (total mRNA-unencapsulated mRNA)/total mRNAx 100%.
Wild-type CD-1 mice were used to assess the relative expression of EPO in mice following a single IV administration of two formulations of lipid nanoparticles encapsulated with EPOmRNA. The EPO levels in serum were measured at 6h after administration. Three 7-week-old male CD-1 mice were used per group. At arrival, animals were randomized to treatment groups containing 3 animals per group. On day-one, animals were weighed and body weights recorded. Each mouse received a single IV dose of 15. mu.g mRNA/animal, with a dose volume of 50. mu.L/animal. At 6h after the administration of the drug, the mice were euthanized by asphyxiation with CO2, then they were thoracically opened and the maximum available amount of blood was collected and processed to give serum. All treatments administered were well tolerated in CD-1 mice after a single IV administration. Serum levels of EPO can be measured by ELISA. EPO was observed in sera from all study animals that received either formulation.
TABLE 7
TABLE 8
All test results are summarized in table 9. When sucrose was used as a lyoprotectant in a weight ratio of 6: 1 to total lipid, particle size growth was observed during lyophilization. However, particle size is better maintained when 2-hydroxypropyl- β -cyclodextrin is used instead even at a relatively low weight ratio (5: 1). The N/P ratio is 20. Furthermore, the encapsulation efficiency of mRNA in lipid nanoparticles is better maintained during lyophilization. These results indicate that the integrity of the lipid particles is effectively protected during lyophilization. Furthermore, serum hEPO levels in wild-type CD-1 mice 6 hours after administration were comparable before and after lyophilization. In summary, 2-hydroxypropyl- β -cyclodextrin is an effective lyoprotectant for mrna-encapsulated lipid nanoparticles prepared with C12-200 lipids.
TABLE 9
Abbreviations: 1) zave (Zave average) is the average of the intensity distribution; 2) PDI (polydispersity index) describes the distribution width; 3) dv50 is the median value of the volume distribution; 4) dv90 means that 90% of the volume distribution lies below this value
The above examples illustrate that the lyophilized lipid nanoparticle formulation exhibits physical characteristics, including relative stability, particle size of the lipid nanoparticles, and encapsulation efficiency, that are comparable or equal relative to the non-lyophilized lipid nanoparticles prepared. Lyophilized lipid nanoparticles also showed comparable protein production with respect to the encapsulated mRNA polynucleotide. For example, several lyophilized lipid nanoparticle compositions evaluated showed comparable firefly fluorescent protein production as determined by the presence of a luminescent signal, and thus inferred expression and/or production of exogenously administered encapsulated mRNA. The above results indicate that the lyophilized lipid nanoparticle compositions and formulations described herein are stable and capable of minimizing degradation of the encapsulated compound (e.g., polynucleotide). Such lyophilized lipid nanoparticle compositions are expected to have an increased shelf life under refrigerated or ambient temperature storage conditions, presenting an attractive approach to improving the availability and potential costs associated with such pharmaceutical compositions.