COMPOSITIONS. CONSTRUCTS. CELLS AND METHODS FOR CELT, THERAPY
CROSS REFERENCE TO RELATED APPLICATIONS
[1] This application claims priority to U.S. Provisional Patent Application Serial Number 63/215,876 filed June 28, 2021 and entitled “COMPOSITIONS, CONSTRUCTS, CELLS AND METHODS FOR INCREASED RECOMBINANT PROTEIN EXPRESSION BY SITE SPECIFIC INTEGRATION”, and U.S. Provisional Patent Application Serial Number 63/254,997 filed October 12, 2021 and entitled “COMPOSITIONS, CONSTRUCTS, CELLS AND METHODS FOR CELL THERAPY”, the entire disclosure of each of which is hereby incorporated herein by reference.
FIELD OF THE DISCLOSURE
[2] The present disclosure relates to gene expression, and, in particular, to compositions, constructs, cells and methods for cell therapy.
BACKGROUND
[3] Mammalian artificial chromosomes (MACs) are chromosome-based vectors that can replicate in mammalian cells just as endogenous chromosomes do and are capable to carry very large amounts of recombinant genetic material. They are stable and typically segregate well in both mitosis and meiosis. MACs typically comprise a mammalian centromere, a region of specialized chromatin found within the chromosome that provides the foundation for the assembly of kinetochore, a protein structure where upon mitosis or meiosis the spindle fibers attach before segregation of sister chromatids. Thus, a centromere confers the ability to a chromosome to segregate to daughter cells. In higher eukaryotic cells, in particular in animal and plant cells, the boundary of a centromere is formed by highly repetitive DNA sequences which, via DNA binding proteins, create a genetically inactive zone around the centromere called pericentric heterochromatin. While no specific gene expression is observed in this region, the centromere could be detected by specific antibodies. Hadlaczky et al. [Proc. Natl. Acad. Sci. U.S. A 88, 8106-8110 (1991)] in 1991 described the isolation of a fragment of putative centromeric DNA by using such anti centromere antibodies. By co-transfecting this DNA with a selectable marker gene into a cell line, dicentric chromosomes, carrying an additional functional centromere, were obtained. The presence of two functional centromeres in the same chromosome caused specific breakages that resulted in the regular appearance of a minichromosome. Praznovszky et al. [De novo chromosome formation in rodent cells. Proc. Natl. Acad. Sci. U.S.A 88, 11042-11046 (1991)] observed that incorporation of a heterologous DNA fragment resulted in the generation of an excess centromere. Thus, they could achieve de novo chromosome formation in cells in which the marker centromere was separated from the dicentric chromosome and formed a stable, full-sized functional chromosome by the so- called amplification process. In EP0473253 Hadlaczky et al. teach a non-human mammalian cell line that carries a chromosome containing an excess centromere comprising a human DNA sequence associated with a dominant selectable marker gene. As mentioned above, regions of heterochromatin surrounding the centromere comprise highly repetitive satellite DNAs, which are transcriptionally inactive. This feature was utilized in WO1997040183, wherein Hadlaczky G. and Szalay A. disclosed satellite DNA based artificial chromosomes (SATACs) useful as gene expression vectors. SATACs are typically made up predominantly of repeating units of short satellite DNA, however, do not contain protein coding gene sequences unless heterologous or foreign DNA is inserted into them. These artificial chromosomes comprising foreign DNA, e.g. a selectable marker gene provided a promising platform to develop expression vectors. The potential use of SATACs in gene therapy at that time was reviewed by Hadlaczky G [Hadlaczky, G. Satellite DNA- based artificial chromosomes for use in gene therapy. Curr. Opin. Mol. Ther 3, 125-132 (2001)].
[4] Patents, including issued U.S. Pat. Nos. 6,077,697 and 6,025,155 describe the generation of ACes (also designated satellite artificial chromosomes (SATACs)) from any species.
[5] US2012064578 describes an artificial chromosome expression system (ACEs) developed from SATACs, which comprised multiple site-specific recombination sites. They disclose the introduction of multiple transgenes into an artificial chromosome with specific and different selection marker genes was applied for each transgene. The recombination site used is the lambda att site designed for recombination directed integration in the presence of lambda integrase. [see also Lindenbaum M. et al. "A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy" Nucleic Acids Research, 2004, Vol. 32, No. 21] [6] Kuroiwa Y et al. (Nature Biotechnol (2000) 18:1086-1090) disclose the use of the cre/lox homologous recombination system for the engineering of a human minichromosome. This system has the disadvantages of the possibility of excision and a lower recombination activity [Yamaguchi S et al. PLoS ONE 6(2) 17267 (2011)].
[7] While significant efforts have been made to create mammalian artificial chromosomes, there remains a need for safe, stable and reliable genetic vector systems to create them [Csonka, E. et al. J. Cell Sci 113 (Pt 18), 3207-3216 (2000); Hadlaczky, G Curr. 0pm. Mol. Ther 3, 125-132 (2001); Lindenbaum, M. etal. Nucleic Acids Res 32, el 72 (2004); Duncan, A. & Hadlaczky, G. Curr. Opin. Biotechnol 18, 420-424 (2007)]. The present invention in part provides such a vector system.
[8] Kennard M. L. reviewed the prospects of use of engineered mammalian chromosomes in cellular protein production and opined that "the most successful technique has been based on the artificial chromosome expression or ACE System, which consists of the targeted transfection of cells containing mammalian based artificial chromosomes with multiple recombination acceptor sites" and that this system eliminates the need for random integration into native host chromosomes [Kennard M. L. Methods in Molecular Biology 738 (2011) 217-238] The intervening decade has proven that these systems are also lacking.
[9] Gene and cell therapy are an extremely promising field for the applications of such MACs and vector systems. A novel gene therapy method was developed by a group including one of the inventors of the instant disclosure, called combined mammalian artificial chromosome-stem cell therapy and it has been shown that the ACE system is suitable for the treatment of an animal model for a devastating human disorder, Krabbe's disease, by delivering a therapeutic gene into mutant mice. Treated mutant mice lived more than four times longer. This gene therapy method used a novel therapeutic approach called combined mammalian artificial chromosome-stem cell therapy (CoMAC-Stem) [Katona, R. L. et al. “A combined artificial chromosome-stem cell therapy method in a model experiment aimed at the treatment of Krabbe's disease in the Twitcher mouse.” Cell. Mol. Life Sci 65, 3830- 3838 (2008)]. The art still needs, however, more efficient vector systems, e.g. for gene therapy applications, wherein multiple transgenes, e.g., therapeutic genes are required. The instant disclosure provides significant and novel methods that solve or ameliorate many of the serious issues that remain in spite of all the advances in prior cell and gene therapy methods.
[10] One of the major issues with cell therapy is low cell engraftment (see for example, Xiaofei et al (Stem Cells Int. 2016:7168797) caused by various factors, including low cell retention and poor cell survival. Engraftment is the process whereby the newly transplanted cells are accepted by the host and they begin to function, grow and replicate. Various approaches have been attempted, such as using biomaterials as cell carriers, controlled release of growth factors and oxygen, attempts to protect cells from inflammation and the immune system. All of these methods have disadvantages. Certain embodiments of the present disclosure solve or ameliorate these and other engraftment issues.
[11] Methods of the instant disclosure allow the loading of multiple (in principle, an unlimited number) genes onto chromosome vectors engineered with appropriate sites, referred to herein as genomic amplification drop site(s) (GADS).
[12] Certain embodiments of this disclosure are useful for gene therapy applications aimed at the treatment of simple or complex disorders and cancers, and cell therapy applications aimed at the treatment of simple and complex disorders and cancers. Certain embodiments are improved methods that are immediately useful in stem cell research, iPSC (induced pluripotent stem cell) production and research, production of differentiated cell lines from pluripotent and multipotent stem cells to study the differentiation process and also produce differentiated cell types for cell therapy, multiple protein production from one producer cell line for basic research and industry, production of immortalized and transiently immortalized somatic cell lines, which are difficult to expand by differentiation methods or direct ACE delivery, single and multiple gene overexpression research, single and multiple gene knock-down research.
[13] Approximately 20 to 30% of new drugs approved by the USFDA in recent years are biologies (in 2019 60 biologies were approved by CDER and CBER), up from 1 approval in 2000 (Batta et al., J Family Med Prim Care. 2020 Jan; 9(1): 105-114). There are currently more than 350 therapeutic biologies on the market and over 900 biologies in development. Development of expression systems for the efficient production of recombinant proteins is important for providing a source of a given protein for research or therapeutic use. The increased number of biologies in development has driven the need to develop simple and rapid high-output technologies for the development of recombinant protein expressing cell lines. The generation of commercial cell lines using conventional methods is a time-consuming, labor-intensive and repetitive process. The instant disclosure solves or ameliorates many of these problems.
[14] Expression systems have been developed for both prokaryotic cells and for eukaryotic cells, which include yeast, Pichia pastoris, insect and mammalian cells. Expression in mammalian cells, for example Chinese hamster ovary (or "CHO") cells, is often preferred for the manufacture of therapeutic proteins, since post-translational modifications in such expression systems are more likely to resemble those found in human cells expressing proteins than the type of post-translational modifications that occur in microbial (prokaryotic) expression systems. Human cell lines like HEK293, HT1080, Per.C6, and other well-known cell lines are even more preferred One skilled in the art can also develop means of using pluripotent, induced pluripotent, totipotent and adult stem cell lines using certain embodiments of the disclosure.
[15] Recombinant expression plasmids comprising a gene of interest that encodes all or a portion of a desired protein are routinely used to generate CHO cells expressing the desired recombinant protein. These recombinant plasmids randomly integrate into the genome of the host cells, which then can produce recombinant proteins, but the frequency of cell lines carrying stably integrated recombinant genes that are capable of expressing a desired recombinant protein at high levels is extremely low. A large number of transfected mammalian cell lines must be screened to identify clones which express the recombinant proteins at high levels. During the construction and selection of protein-producing cells lines, cell lines with a large range of expression, growth and stability profiles are obtained. These variations can arise due to the inherent plasticity of the mammalian genome. They can also originate from stochastic gene regulation networks or in variation in the amount of recombinant protein produced resulting from random genomic integration of a transgene principally due to the "position variegation effect" or merely from the plasmid copy number, especially in light of the large size of the mammalian genome and the fact that only a small percentage of the genomic DNA contains transcriptionally active sequences.
[16] As a consequence of these variations and the low (estimated at perhaps 1 in 10,000) frequency of genomic integration, resource-intensive and time-consuming efforts are required to screen many transfectants in the pool for these rare events, in order to isolate a commercially-compatible production cell line (e.g., cell lines exhibiting a combination of good growth, high productivity and stability of production, with desired product profile).
[17] Expression-augmenting sequences have been disclosed to increase expression of recombinant protein for eukaryotic expression systems (see for example WO 97/25420). An increase in the frequency of high-level recombinant gene expressing cell lines would provide a much greater pool of high protein-expressing cell lines to choose from. This task can be accomplished by generating homologous recombinant plasmids targeted to transcriptionally active sites as disclosed herein and by devising a means to select for such cell lines.
[18] There are a number of well-known and common amplification methods used to improve the yield of protein expression in mammalian cell systems. Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the cells that are generated are frequently unstable in culture. Further, the DHFR/MTX system has a long development time (taking up to 6 months), and the selection pressure is indirect.
[19] Another common amplification system is the GS/MSX system. The glutamine synthetase (GS) expression system has been used for decades but has numerous issue. L-Methionine sulfoximine (MSX) inhibits the activity of glutamine synthetase, an enzyme essential for the production of glutamine. MSX is used as a media supplement to aid selection and amplification processes in recombinant mammalian cell lines that use GS as a selective marker. However, this system also has a long development time, provides indirect selection pressure and the amplified gene to be expressed is even more unstable than the cell-lines developed by the DHFR/MTX system.
[20] The failings of existing amplification systems are discussed in Priola et al., 1, “High- throughput screening and selection of mammalian cells for enhanced protein production” Biotechnol. J. 2016, 11. pl-13. DOI 10.1002/biot.201500579.
[21] One of the most common methods of recombinant protein production involves transfection with one of a variety of viruses including lentivirus and retroviruses. See for example, Tandon, et al., Bio Protoc. 2018 November 5; 8(21): doi:10.21769/BioProtoc.3073. This approach also has many issues: 1) Safety issues: A very high titer of infectious viruses carrying the gene of interest is required in a packaging cell line, thus necessitating a high-level biosafety lab. Technicians must be highly trained to make and use these viruses, because the viruses can easily infect such technicians. The viruses may cause cancer or other problems because of virus integration triggered mutagenesis. Also, they have to wear special safety clothes, gloves, goggles etc. 17% of the human genome consists of long interspersed repeat element (“LINE”) sequences. These are hypothesized to be the remnants of previous virus infections. These viruses infected us and then some of them randomly integrated into the human genome. These virus sequences are a permanent part of the human genome and may even constitutively produce virus proteins such as reverse transcriptase. Reverse-transcriptase transcribes an RNA sequence and makes the DNA sequence from it in a reversal of the usual protein production process. This DNA sequence might then integrate into the human genome and cause mutations. Even if defected lentivirus vectors are used, such vectors might recombine with these pre existing LINE sequences and result in an active virus. The use of virus vectors even for making cell lines poses potentially dangerous risks.
2) Purification issues: Proteins purified from such viral systems require time consuming additional purification steps in order to ensure that the protein does not contain virus or viral toxin contaminants.
3) Cell line stability: During the process of creating cell lines with a virus, the lentivirus integrates into the target genome randomly at hundreds or even thousands of sites. This random integration causes mutations in the cell line which can be advantageous for protein production in the short term but in the long term these mutations are harmful for the cell line. These methods are most useful for short term protein production. Over a longer term these randomly integrated viruses cease to produce proteins due to silencing. There is an evolutionary mechanism to silence integrated viruses. That is thought to be a natural mechanism our cells use to keep the LINE sequences at bay.
[22] To achieve a long-term supply of a recombinant protein, a random integration system is not optimal. Embodiments of the present disclosure provide more stable protein production in a safer, faster, and easier system.
[23] The technical problem underlying the present disclosure is to overcome the above- identified disadvantages, in particular to provide, preferably in a safe, simple and efficient manner, high producing cell lines with a high stability and positive growth and productivity characteristics, in particular cell lines which provide a consistent productivity over a long cultivation and production period.
[24] In particular, the present disclosure solves or ameliorates many of these technical problems by the provision of a site-specific integration (SSI) host cell comprising an endogenous genomic amplification drop site(s) (GADS, which may also be referred to as genomic super expression drop site(s) or genomic overexpression drop site(s)), wherein an exogenous nucleotide sequence is integrated at said GADS. In some embodiments, the exogenous nucleotide sequence comprises at least one gene coding sequence of interest. In some embodiments, the nucleotide sequence comprises at least one selection marker gene, one promoter sequence and one enhancer sequence.
[25] This background information is provided to reveal information believed by the applicant to be of possible relevance. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art or forms part of the general common knowledge in the relevant art.
SUMMARY
[26] The following presents a simplified summary of the general inventive concept(s) described herein to provide a basic understanding of some aspects of the disclosure. This summary is not an extensive overview of the disclosure. It is not intended to restrict key or critical elements of embodiments of the disclosure or to delineate their scope beyond that which is explicitly or implicitly described by the following description and claims.
[27] In accordance with one broad aspect of the disclosure, a construct comprising a mammalian artificial chromosome or mammalian artificial chromosome arm that comprises a DNA fragment inserted into a region of open chromatin comprising a transgene wherein said transgene is amplified more than 2 times is disclosed.
[28] In one embodiment, the transgene is amplified more than 5 times. In another embodiment, the transgene is amplified more than 10 times. In yet another embodiment, the transgene is amplified more than 20 times.
[29] In one embodiment, the construct is created using a genomic amplification drop site (GADS) containing vector. In one particular embodiment, the GADS-containing vector comprises a sequence selected from any one of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or a functional fragment thereof
[30] In accordance with another broad aspect of the disclosure, a mammalian artificial chromosome or mammalian artificial chromosome arm is provided. The mammalian artificial chromosome or mammalian artificial chromosome arm comprises a sequence selected from any one of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, or a functional fragment thereof.
[31] In accordance with another broad aspect of the disclosure, a cell is provided, the cell comprising the construct described above, wherein the cell comprises a pluripotent cell or an adult stem cell modified by transfection with the GADS-containing vector.
[32] In one embodiment, the cell is a human embryonic stem cell, a human induced pluripotent stem cell, a gut stem cell, or a mesenchymal stem cell. In one particular embodiment, the cell is a human induced pluripotent stem cell. In another particular embodiment, the cell has been differentiated. In a more specific embodiment, the cell is differentiated into a blood cell. In some embodiments, the blood cell is a T-cell, a NK-cell, a human stem cell or the like.
[33] In one embodiment of the cell, the transgene expresses a protein selected from the group consisting of cystic fibrosis transmembrane conductance regulator (CFTR), Gamma-C, Factor VIII, Factor IX, adalimumab, atezolizumab, nivolumab, pembrolizumab, etanercept, trastuzumab, bevacizumab, rituximab, aflibercept, infliximab, ustekinumab, ranibizumab, proteins of tumor biology, proteins of food industry, proteins of animal health, proteins of ageing, proteins of genetic disorders, vaccines, virus-like particles (VLPs), single proteins, virus inhibitor proteins and the like.
[34] In accordance with another broad aspect of the disclosure, a method for treating a mammalian disease is provided. The method comprises transplanting a cell as described above into a mammal in need of such treatment.
[35] In some embodiments, the mammalian disease is hemophilia, cystic fibrosis, X chromosome-linked severe combined immunodeficiency syndrome (X-SCID), cancer, or the like.
[36] In accordance with another broad aspect of the disclosure, a method of performing gene therapy or cell therapy is provided. The method comprising preparing a mammalian artificial chromosome or mammalian artificial chromosome arm in accordance with the construct described above.
[37] In accordance with another broad aspect of the disclosure, a further method is disclosed, which comprises making one or more artificial chromosome arms or artificial chromosomes directly in stem cells and then differentiating the stem cells containing the one or more artificial chromosome arms or artificial chromosomes into therapeutic cell types for a specific mammalian disorder.
[38] In accordance with a further broad aspect of the disclosure, a use of a cell for treating a mammalian disease is disclosed, wherein the cell comprises any one of the cells described above.
[39] In some embodiments, the use comprises transplanting the cell into a mammal in need of such treatment. In some embodiments, the mammalian disease is hemophilia, cystic fibrosis, X chromosome-linked severe combined immunodeficiency syndrome (X-SCID), cancer, or the like.
[40] In accordance with another broad aspect of the disclosure, a use of the construct comprising the mammalian artificial chromosome or mammalian artificial chromosome arm described above is provided, the use comprising preparing the construct for use as a gene therapy agent or cell therapy agent.
[41] In accordance with another broad aspect of the disclosure, a use of the construct described above is provided, wherein the construct is used as a gene therapy agent or cell therapy agent.
[42] In accordance with a further broad aspect of the disclosure, a use of one or more artificial chromosome arms or artificial chromosomes in the preparation of therapeutic cell types for treating a specific mammalian disorder is provided, comprising producing the one or more artificial chromosome arms or artificial chromosomes directly in stem cells and differentiating the stem cells containing the one or more artificial chromosome arms or artificial chromosomes.
[43] In accordance with yet a further broad aspect of the disclosure, there is provided a mammalian cell comprising a mammalian artificial chromosome (MAC) or mammalian artificial chromosome arm (MACA), wherein the MAC or MACA comprises a DNA fragment inserted into a region of open chromatin, the DNA fragment comprises a transgene, and wherein the transgene is amplified more than 2 times, 5 times, 10 times, 20 times or 50 times.
[44] In one embodiment, the MAC or MACA is created using a genomic amplification drop site (GADS) containing vector.
[45] In various embodiments, the GADS containing vector comprises at least one sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, or a functional fragment thereof.
[46] In some embodiments, the mammalian cell is a pluripotent cell or an adult stem cell modified by transfection with said GADS containing vector.
[47] In accordance with yet a further aspect of the disclosure, there is provided a mammalian cell comprising the mammalian artificial chromosome or mammalian artificial chromosome arm described with reference to one of the embodiments above, wherein expression of the transgene by the mammalian cell is amplified by more than 2-fold, 5-fold, 10-fold, 20-fold, or 50-fold in comparison with an equivalent cell comprising the transgene in the absence of the GADS containing vector.
[48] In some embodiments, the mammalian cell is a pluripotent cell or an adult stem cell modified by transfection with the GADS containing vector.
[49] In some embodiments, the GADS containing vector comprises at least one sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, or a functional fragment thereof.
[50] Other aspects, features and/or advantages will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE FIGURES
[51] Several embodiments of the present disclosure will be provided, by way of examples only, with reference to the appended drawings, wherein:
[52] FIGURE 1 shows a plasmid map of one embodiment of a recombinant insertion and expression vector designed to create cell lines that express trastuzumab (Herceptin®, Genentech). [53] FIGURE 2 shows a plasmid map of another embodiment of a recombinant insertion and expression vector designed to create cell lines that express trastuzumab (Herceptin®, Genentech, i.e. another insertion vector for trastuzumab heavy and light chain production).
[54] FIGURE 3 shows a fluorescent in situ hybridization (FISH) image of a trastuzumab- producing CHO-K1 cell line, which cell line was produced in less than two weeks, wherein bright dots show the presence of trastuzumab expressing gene copies and wherein the arrows show that amplification is clearly visible on two chromosomes.
[55] FIGURE 4 shows an image of a Western blot of trastuzumab-producing CHO-K1 cell lines and also includes actin as a control, wherein the signal at 55 kDa is the trastuzumab heavy chain and the signal at 42 kDa is actin (internal control for quantitation of protein expression with Licor Odyssey gel documentation system).
[56] FIGURE 5 shows an image of a Western blot of Hek293 cells showing the comparison of trastuzumab heavy chain production (55kDa) and control actin production (42kDa). In addition to the controls, trastuzumab production is shown in lanes labelled 10/5, 10/6, 10/8, and 10/13. Each lane corresponds to particular clones.
[57] FIGURE 6 shows an image of a Western blot, showing purified trastuzumab production of both heavy (55 kDa) and light (25 kDa) chains from clone 10/13 of FIGURE 5, wherein purification was done by successive rounds of protein G (“SpinTrap”) purification.
[58] FIGURE 7 shows an image of an inverted Western blot of the results of subcloning of clone 10/13, wherein the signal at 55 kDa is the trastuzumab heavy chain and the signal at 42 kDa is actin (internal control for quantitation of protein expression with Licor Odyssey gel documentation system).
[59] FIGURE 8 shows a FISH analysis image demonstrating amplification of clone 10/13 across a variety of human chromosomes, wherein stars show targeted sites and amplification of trastuzumab genes.
[60] FIGURE 9 shows a plasmid map of a plasmid carrying GADS2 (SEQ ID NO:2) for transfecting, insertion and amplification.
[61] FIGURE 10 shows a FISH analysis image, wherein FISH stained MEF cells were transfected with a GADS2 carrying plasmid according to the disclosure and as shown in FIGURE 9, which in this embodiment carries the Influenza A virus Hemagglutinin MYMC_X-181 California strain sequence as a useful transgene, wherein targeting exclusively happened into a large acrocentric chromosome into the upper part of the long chromosomal arm in all 34 clones.
[62] FIGURE 11 shows a FISH analysis image, wherein FISH-stained cells Hamster chromosomes stained with DAPI (blue signal), and wherein the transgene was labeled with a green fluorescent dye. As shown, one hamster chromosome has an amplified chromosome arm with hundreds of transgene copies. As shown on the right side of the photo, a newly formed, autonomous mammalian artificial chromosome is visible. This chromosome is almost entirely consisting of transgene sequences in hundreds of copies (green signal).
[63] FIGURE 12 shows a plasmid map of a plasmid construct according to the disclosure carrying the GADS1 sequence (CDC27 pseudogene) and the Influenza A virus Hemagglutinin MYMC X-181 California strain sequence as a useful transgene.
[64] FIGURE 13 shows a FISH analysis image, wherein mouse chromosomes were stained with DAPI (blue) and transgenes were stained with a green fluorescent dye, the figure specifically showing an autonomous mammalian chromosome close to the middle of the photo with hundreds of copies from the transgene and more specifically showing two chromosomes with an amplified chromosome arm (30 copies and 60 copies from the transgenes are present). Cells were transfected with the construct shown in FIGURE 12.
[65] FIGURE 14 shows a plasmid map of a pIKRBBP7 plasmid for insertion and amplification of CIRBBP.
[66] FIGURE 15 shows various images of Western blots showing CIRBBP cell-lines generated using the pIKRBBP7 plasmid of FIGURE 14 showing expression of CIRBBP using an anti- AVI-tag antibody as a primary antibody and anti-mouse-HRP antibody as a secondary antibody in the Western blots. Western blots were developed by ECL and chemiluminescent signal was photographed.
[67] FIGURE 16 shows a FISH analysis image which shows BCHAT40 cell line with highest the trastuzumab expression (i.e. MAC produced in mouse cells with a trastuzumab expressing plasmid construct). The presence of the transgene carrying MAC is shown with the green signal. Mouse chromosomes were counterstained with DAPI (blue).
[68] FIGURE 17 shows an image of a Western blot of various clones with the chosen clone being clone (40) on the x-axis. [69] FIGURE 18 shows graphs of the results of fluorescence activated cell sorting (FACS) experiments, whereby trastuzumab produced by BCHAT40 cells recognizes cancer cells where Her2/Neu amplification is present. In particular, graphs 100 and 200 show curves 102 and 202, respectively, which are significantly shifted due to high level of trastuzumab binding in the breast cancer cell lines. Curves 104 and 204, in graphs 100 and 200, respectively, are control experiments where only the secondary antibody was added to the cells. In graphs 300 and 400, MDA-MB and MCF-7 human breast cancer cells, respectively, have normal level of Her2/Neu proteins in their cell membrane and so curves 302 and 402, respectively, are not significantly shifted due to low level of trastuzumab binding.
[70] FIGURE 19 shows microscopy images of SKBR3 human breast cancer cells that were immuno-stained with clinically approved trastuzumab and with purified trastuzumab that was produced by BCHAT40 cells containing MACs.
[71] FIGURE 20 shows a graph of the results of antibody-dependent cell-mediated cytotoxicity (ADCC) tests done to prove the functionality of clinically approved trastuzumab antibody produced by BCHAT40 cells, wherein the control is normal culture medium (negative control); the LMTK- CM is conditioned culture medium of LMTK- mouse cells (negative control); BCHAT40 CM is conditioned medium of BCHAT40 cells; and Herceptin is clinically approved trastuzumab which is included as a positive control. In all cases, SkB3 breast cancer cells and effector T-Cells were added together with these samples and the increase of apoptotic cell death was monitored. BCHAT40 CM gave the highest level of cell death compared to negative (LMTK-, LMTK- CM) and positive control (clinically approved Herceptin). Data shows that Herceptin produced by MACs are most effective in killing SkB3 breast cancer cells.
[72] FIGURE 21 shows an image of the results from when trastuzumab expressing MAC was transferred into mouse adipocyte stem cells (ADSCs) with microcell fusion, specifically showing the dot blotting experiment which indicates ADSC lines that are expressing trastuzumab after microcell fusion.
[73] FIGURE 22 shows images of the results of FISH analysis of the ADSC lines that were showing highest levels of trastuzumab production in dot blotting (as shown in FIGURE 21), which were then analyzed by FISH for the presence of delivered MAC, wherein green staining demonstrates the presence of trastuzumab producing MACs in these cell lines. [74] FIGURE 23 shows images of the FISH results of Gamma-C [yC] protein expressing MACs which were made in CHO-DG44 cells. Four cell line proved to be the best (3D11, 4F9, 5E4, 5G2). The FISH experiments demonstrate the presence of MACs in CHO cells: wherein red staining shows the presence of yC-MACs and wherein chromosomes were counterstained with DAPI (blue).
[75] FIGURE 24 shows an image of a Western blot that shows that 2B3, 3D11, 5E4, 4F9, 5G2 CHO cell express yC protein from MACs.
[76] FIGURE 25 shows images of the FISH results indicating that yC-MAC was delivered into mouse embryonic stem cells (mESCs, Rl) from CHO-DG44 cells by microcell fusion.
[77] FIGURE 26 shows images of the FISH results that demonstrate the presence of the yC gene carrying MACs in RGAMC3 subclones.
[78] FIGURE 27 shows an image of a Western blot showing that after delivery from CHO cells and subcloning, mESC cell lines still express yC from MACs.
[79] FIGURE 28 shows cell microscopy images showing that yC-MAC continued to express yC protein after differentiation into embryoid bodies (EBs), specifically showing images of identical-looking Rl ES (mESC), GMC27 ES (mESC), Rl EB and GMC27 EB cultures.
[80] FIGURE 29 shows an image of a Western blot showing that yC-MAC continued to express yC protein after differentiation into EBs, specifically reflecting continued protein expression at significantly higher levels as comparted to normalized b-actin expression.
[81] Elements in the several figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the figures may be emphasized relative to other elements for facilitating understanding of the various presently disclosed embodiments. Also, common, but well-understood elements that are useful or necessary in commercially feasible embodiments are often not depicted in order to facilitate a less obstructed view of these various embodiments of the present disclosure.
DETAILED DESCRIPTION
[82] Various implementations and aspects of the specification will be described with reference to details discussed below. The following description and drawings are illustrative of the specification and are not to be construed as limiting the specification. Numerous specific details are described to provide a thorough understanding of various implementations of the present specification. However, in certain instances, well-known or conventional details are not described in order to provide a concise discussion of implementations of the present specification.
[83] Various methods, processes and uses will be described below to provide examples of implementations of the disclosure disclosed herein. No implementation described below limits any claimed implementation and any claimed implementations may cover processes, methods or uses that differ from those described below. The claimed implementations are not limited to methods, processes or uses having all of the features of any one method or process described below or to features common to multiple or all of the methods, processes or uses described below. It is possible that a method or process described below is not an implementation of any claimed subject matter.
[84] Furthermore, numerous specific details are set forth in order to provide a thorough understanding of the implementations described herein. However, it will be understood by those skilled in the relevant arts that the implementations described herein may be practiced without these specific details. In other instances, well-known methods, procedures and components have not been described in detail so as not to obscure the implementations described herein.
[85] It is understood that for the purpose of this specification, language of “at least one of X, Y, and Z” and “one or more of X, Y and Z” may be construed as X only, Y only, Z only, or any combination of two or more items X, Y, and Z (e.g., XYZ, XY, YZ, ZZ, and the like). Similar logic may be applied for two or more items in any occurrence of “at least one ...” and “one or more...” language.
[86] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
[87] As used herein, the term “transgene” is given its broadest possible meaning to include any gene that one wants to express regardless of the species or source of that gene. For example, a wild-type human gene or a mutant mouse gene could both be a transgene when used in the constructs and methods herein regardless of the species of the host cell, for that matter.
[88] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in one of the embodiments” or “in at least one of the various embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” or “in some embodiments” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments may be readily combined, without departing from the scope or spirit of the innovations disclosed herein. The same logic may apply to examples.
[89] In addition, as used herein, the term “or” is an inclusive “or” operator, and is equivalent to the term “and/or,” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The meaning of "in" includes "in" and "on."
[90] The term “comprising” as used herein will be understood to mean that the list following is non-exhaustive and may or may not include any other additional suitable items, for example one or more further feature(s), component(s) and/or element(s) as appropriate.
[91] Novel insertion sequences, referred to herein as GADS (genomic amplification drop sites), that facilitate increased expression of recombinant proteins in mammalian host cells, are disclosed. A preferred embodiment of the disclosure is a GADS that was obtained from human cell genomic DNA. Vectors comprising such human derived GADS can be used for insertion into not only human but also mouse and hamster genomes as a result of the highly conserved nature of the GADS. Alternatively, one skilled in the art can easily obtain mouse or hamster GADS sequences. Another preferred embodiment of the disclosure is a GADS that was obtained from hamster cell genomic DNA. Another preferred embodiment of the disclosure is a GADS that was obtained from mouse cell genomic DNA.
[92] The present disclosure discloses a GADS sequence from a genomic locus in the human genome that is capable of high recombinant gene amplification and expression. In a most preferred embodiment of the disclosure, the GADS is selected from the group consisting of (a) DNAs comprising nucleotides of SEQ ID NO:l; (b) fragments of SEQ ID NO:l that are useful as insertion and expression sites; (c) nucleotide sequences complementary to (a) and/or (b); (d) nucleotide sequences that are at least about 80%, more preferably about 90%, and more preferably about 95% identical in nucleotide sequence to (a), (b) and/or (c) and that are useful for insertion and expression of exogenous proteins; and (e) combinations of the foregoing nucleic acid sequences that are useful for insertion and expression of exogenous proteins.
[93] Expression vectors comprising the novel GADS sequences are able to transform CHO, HEK293, or other mammalian cells to increase expression of recombinant proteins through genomic insertion into open chromatin and amplification. Thus, another embodiment of the disclosure is an expression vector comprising a GADS sequence. In a preferred embodiment, the expression vector further comprises a eukaryotic promoter/enhancer driving the expression of all or a portion of a protein of interest. Two or more different nucleic acids expressing exogenous proteins of interest can be present in an expression vector used to transfect a cell (e.g., CHO or HEK293 cells), wherein each nucleic acid sequence encodes a different polypeptide that assemble (when expressed) to form a desired protein. In an additional preferred embodiment, the expression vector comprises a plasmid that encodes a gene of interest and also encodes an amplifiable dominant selectable marker. A preferred marker is puromycin; other amplifiable markers known in the art are also suitable for use in certain embodiments of the expression vectors of the instant disclosure.
[94] Mammalian host cells can be transformed with an expression vector of the present disclosure to produce high levels of recombinant protein. Accordingly, another embodiment of the disclosure provides a mammalian host cell transformed with an expression vector of the present disclosure. Also within the scope of the present disclosure are mammalian host cells transformed with two expression vectors, wherein each of said two expression vectors encodes at least one polypeptide subunit that when co-expressed assembles into a desired protein with biological activity. In a most preferred embodiment, the host cells are CHO or HEK cells.
[95] The disclosure also provides a method for obtaining a recombinant protein, comprising transforming a host cell with an expression vector of the present disclosure, culturing the transformed host cell under conditions promoting amplification of the inserted exogenous vector and expression of the protein, and recovering the protein. In a preferred application of the disclosure, transformed host cells are selected with multiple selection steps with increasing concentrations of a selection antibiotic such as puromycin. Embodiments of this method are useful for creating an amplification and expression system which is tunable to the specific properties of different transgenes that one desires to produce. [96] In certain embodiments of the disclosure, the selectable antibiotic (preferably puromycin) concentration is increased in a series of steps to achieve the desired optimal amplification and expression level in each cell type (preferably human, mouse or hamster). The methods, constructs and systems disclosed herein also provide a large number and variety of individual clones with various levels of amplification providing different expression levels. The particular clones having optimal amplification levels can be selected to provide a stable cell line with a desired protein production level. One skilled in the art will understand that different proteins require different levels of production and the methods and constructs disclosed herein provide an adjustable combination of integration, amplification and protein production. One skilled in the art can use the methods and constructs disclosed herein to adjust the system for the best results for each individual desired protein product.
[97] Embodiments of the present disclosure therefore provide a novel approach to the existing problems of cell and gene therapy. The instant disclosure discloses vectors for insertion of one or more transgenic sequences into specific sequences, designated genomic amplification drop sites (GADS) which are novel non-coding regions that can be induced by methods disclosed herein to amplify the recombinant sequences many times even up to many hundreds of copies. These copies can be found on at least eleven mammalian chromosomes. In addition, once integrated, the transgenic sequences can be subjected to selective pressure that induces the formation of MACs or artificial chromosome arms.
[98] Certain embodiments of the disclosure disclose the creation of MACs expression in stem cells, including in certain embodiments, induced pluripotent stem cells (iPSCs), which can be differentiated into almost any desired cell type. Such MAC-containing cells can be introduced into a host, including human patients, to effect stable or transient cell therapy.
[99] In certain embodiments, the ubiquitous challenge of “engraftment” is solved (or at least, ameliorated) by the simple expediency of the induction of large scale, secreted transgenic, therapeutic proteins in a host. The numerous copies means that fewer stem cells are required to engraft to achieve an equivalent function.
[100] The instant disclosure comprises methods, compositions and constructs (together with respective uses) useful for production of large amounts of recombinant proteins in cell lines, including stem cells, that are stable for long periods of time. Embodiments of the disclosure include sequences and constructs for achieving non-random insertion of exogenous DNA sequences into the genome of mammalian cell lines followed by the amplification of the inserted DNA into multiple sites across numerous chromosomes. In one surprising aspect of the instant disclosure, the non-random insertion sites of the disclosure are substantially composed of euchromatin that are open and not silenced. Thus, the constructs of the instant disclosure are uniquely useful for the stable, long-term, large scale production of therapeutic proteins.
[101] Embodiments of the instant disclosure include methods for achieving rapid amplification of the inserted DNA across the genomes of the transformed cells.
[102] Embodiments of the instant disclosure include methods and constructs for achieving rapid amplification of the inserted DNA of the transformed cells to produce artificial chromosomes.
[103] Embodiments of the instant disclosure include methods and constructs for achieving rapid amplification of the inserted DNA of the transformed cells to produce artificial chromosome arms.
[104] Certain embodiments of the recombinant expression vectors of the instant disclosure include novel sequences for achieving homologous recombination at specific sites in the genomes of mammalian cells.
[105] Recombinant expression vectors include synthetic or cDNA-derived DNA fragments encoding a protein, operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, viral, fungi, insect or bacterial genes. Such regulatory elements may include a transcriptional promoter, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Mammalian expression vectors may also comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5' or 3' flanking non-transcribed sequences, 5' or 3' non-translated sequences such as ribosome binding sites, a polyadenylation site, and transcriptional termination sequences. An origin of replication that confers the ability to replicate in a host, and a selectable gene to facilitate recognition of transformants, may also be incorporated. A preferred expression vector is shown in Figure 1.
[106] DNA regions are operatively linked when they are functionally related to each other. For example, a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. Transcriptional and translational control sequences in expression vectors used in transforming cells are known in the art.
[107] Transformed host cells are cells which have been transformed or transfected with expression vectors constructed using recombinant DNA techniques and which contain sequences encoding all or a portion of recombinant proteins. Expressed proteins may be secreted into the cell culture supernatant, depending on the DNA selected, but may also be deposited inside the cell and/or in the cell membrane. Various mammalian cell culture systems can be employed to express recombinant protein according to embodiments of the present disclosure, all well known in the art, for example COS lines of monkey kidney cells, CHO cells, HeLa cells, HEK293 cells, Per.C6 cells, LMTK- cells, pluripotent cells, induced pluripotent cells, totipotent cells, adult stem cells, primary cells and BHK cell lines.
[108] Several transformation protocols are known in the art, and are reviewed, for example, in Kaufman et. al., (1988) Meth. Enzymology 185:537. The transformation protocol chosen will depend on the host cell type and the nature of the gene of interest and can be chosen based upon routine experimentation. The basic requirements of any such protocol are first to introduce DNA encoding a protein of interest into a suitable host cell, and then to identify and isolate host cells which have incorporated the DNA in stable, expressible manner. Examples of methods useful for introducing DNA encoding a protein of interest can be found in Wigler et.al., (1980) Proc. Natl. Acad. Sci. USA 77:3567; Schaffner (1980) Proc. Natl. Acad. Sci. USA 77:2163; Potter et.al, (1988) Proc. Natl. Acad. Sci. USA 81:7161; and Shigekawa (1988) BioTechniques 6:742.
[109] A method of amplifying the gene of interest is also desirable for expression of the recombinant protein, and typically involves the use of a selection marker. The novel characteristics of the instant homologous recombination vectors are ideal for amplification using a selection marker. Resistance to cytotoxic drugs is the characteristic most frequently used as a selection marker and can be the result of either a dominant trait (i.e., can be used independent of host cell type) or a recessive trait (i.e., useful in particular host cell types that are deficient in whatever activity is being selected for). Many amplifiable markers are suitable for use in the present disclosure (for example, as described in Maniatis, Molecular Biology: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989)). Useful selectable markers for gene amplification in drug-resistant mammalian cells include DHFR-MTX (methotrexate) resistance (Alt et.al., (1978) J. Biol. Chem. 253:1357; Wigler et. al, (1980) Proc. Natl. Acad. Sci. USA 77:3567), and other markers known in the art (as reviewed, for example, in Kaufman et.al., (1988) Meth. Enzymology 185:537). The most widespread method for amplifying a target gene in cell culture is the use of methotrexate (Mtx) treatment to amplify dihydrofolate reductase (Dhfr), however, surprisingly, embodiments of the present disclosure provide a significantly better amplification method using increasing concentrations of puromycin.
[110] A preferred selection and amplification marker is the gene that encodes puromycin resistance. In certain embodiments of the present disclosure high levels of puromycin are used to apply selective pressure on the cells and the exogenous gene spreads along with the puromycin resistance gene throughout the GADS. In certain embodiments of the disclosure more than 5 copies of the exogenous gene are spread across different sites and chromosomes. In certain preferred embodiments, more than 10 copies of the exogenous gene are spread across different sites and chromosomes and in certain other preferred embodiments more than 25 copies of the exogenous gene are spread across different sites and chromosomes. In the most preferred embodiments, more than 50 copies of the exogenous gene are spread across different sites and chromosomes, leading to very high expression of the exogenous gene.
[111] In certain embodiments alternative selection and amplification markers other than the gene that encodes puromycin resistance may be used providing it can be obtained and applied in large doses. In industrial or therapeutic applications, the need for large amounts of antibiotic can easily become prohibitive and so a gene that codes for resistance to an inexpensive antibiotic is preferred.
[112] Previous methods of applying puromycin selection have been time consuming (see for example, Prieto et al., Prieto et al. BMC Proceedings 2011, 5(Suppl 8):P7 http://www.biomedcentral.com/ 1753-6561/5/S8/P7) and methods of the present disclosure found surprising and unexpected results using a novel protocol of multi-step increases in puromycin concentration. Preferred methods of the disclosure involve step wise increases in puromycin from 10 ug/ml up to 250 ug/ml with each increase taking place after less than 7 days. In more preferred embodiments puromycin concentration was increased after about 3 days.
[113] In certain embodiments of the present disclosure cell lines are created that express one recombinant protein or peptide of interest. In more preferred embodiments of the disclosure the cell lines of the disclosure produce two or more different proteins or peptides. In still further preferred embodiments, four different proteins or peptides may be produced in a single cell line. An example of the expression of two different peptides is provided herein as shown in Figure 6 which depicts the production of both the heavy and light chains of the trastuzumab antibody following with transformation of HEK293 cells by the vector depicted in Figure 2.
[114] Thus, preferred embodiments of the expression vectors may encode one recombinant protein or peptide of interest. In more preferred embodiments of the expression vectors two or more different proteins or peptides are encoded. In still further preferred embodiments, four different proteins or peptides may be encoded in a single vector. In other embodiments, multiple vectors may be used to express yet more exogenous proteins or peptides.
[115] In certain embodiments of the disclosure the recombinant proteins produced may be antibodies. In certain preferred embodiments, both light and heavy chains of antibodies may be produced by the same cell line.
[116] Embodiments of the disclosure can be used to produce almost any protein, even complex biologies containing multiple polypeptide chains. In certain embodiments of the disclosure the cell lines stably produce large amounts of a drug selected from adalimumab, atezolizumab, nivolumab, pembrolizumab, etanercept, trastuzumab, bevacizumab, rituximab, aflibercept, infliximab, ustekinumab, ranibizumab, proteins of tumor biology, proteins of food industry, proteins of animal health, proteins of ageing, proteins of genetic disorders, vaccines (VLPs, single proteins, virus inhibitor proteins).
[117] Applicants have isolated and identified novel sequence elements that can improve expression of recombinant proteins from at least two to ten-fold in stable cell lines when inserted in an expression vector. We refer to these novel sequence elements as GADS for genomic amplification drop sites.
[118] GADS according to the instant disclosure have been found in a variety of mammalian genomes and cell lines. Some are found in many species and some are found in only human cells.
[119] The novel insertion and amplification sites according to the disclosure comprise sequences that are found at sites that are adjacent to each other in wild type human cells, and some such sequences are found in the intergenic spacer regions of rDNA gene sequences. The useful GADS sequences were discovered in a research program to uncover the best possible insertion and amplification sites for recombinant protein production and mammalian artificial chromosome production.
[120] Methods according to the instant disclosure involve the novel use of sequences hypothesized to be used by mammalian cells to further chromosomal evolution. Sequences according to the instant disclosure are responsible for the amplification processes that occur in these vast rDNA regions in the continuously ongoing chromosomal evolution. These regions can amplify themselves and new chromosomes can be formed. These new chromosomes may be inherited through generations without any apparent side effects in humans. This process may seem slow, but in the evolutionary sense it is very fast. Extra chromosomes are formed naturally in the human population with a 0.043% frequency. These chromosomes are called sSMC chromosomes (small supernumerary marker chromosomes) and these are inherited in human families through many generations often without any apparent side effects (Fu S, Fu H et al. (1992). Yi Chuan Xue Bao 19(4):294- 7.; Gravholt CH, Friedrich U. (1995). Am J Med Genet 56(1): 106-11.; Csonka E. (2008). Hungarian Medical Journal 2(3):365-380.).
[121] An extensive research program was conducted to uncover the exact DNA sequences that are responsible for the amplification process described above and this research surprising lead to the development of the instant recombinant protein expression disclosure. During the examination of the vast amount of possible DNA it was found that no rDNA gene sequences are involved in the process and so the search began for sequences in the non coding intergenic spacers between the genes and upstream and downstream from the rDNA genes in the non-coding chromosomal regions. This massive search required the examination of hundreds of kilobases of DNA sequences.
[122] The extensive research described above uncovered two human GADS sequences: the CDC27 pseudogene sequence and a 2993 base pair (bp) sequence. Further examination and utilization of the 2993 bp sequence determined that a smaller portion of the sequence (904 bp) works well in certain embodiments of the methods for targeting and amplification in the HEK293 cell line according to the present disclosure. Embodiments of the disclosure utilizing this smaller sequence are advantageous when the exogenous gene to be expressed is large, or when several genes are to be expressed at the same time in the same cell line. In certain embodiments up to 4 genes can be expressed from one plasmid of the disclosure. In other embodiments multiple expression vectors may be used to transform the cells, increasing the number of polypeptides that can be produced simultaneously.
[123] The present disclosure relates to the identification of recombinant protein integration sites in a variety of host genomes, and the construction of homologous recombination vectors for achieving high, stable recombinant gene expression in mammalian cells.
[124] A number of GADS sequences have been identified and characterized. These sequences are disclosed as SEQ ID NO:l, SEQ ID NO:2 and SEQ ID NO:3, herein and also referred to as GADS1, GADS2 and GADS3, respectively.
GADS1: Human CDC27 pseudogene
[125] Certain embodiments of the instant disclosure include sequences known as the Homo sapiens cell division cycle 27 pseudogene (CDC27 pseudogene). This pseudogene has no previously known function. There are many versions of CDC27 pseudogenes which can be used in embodiments of the instant disclosure. CDC27 pseudogenes are found on at least 10 chromosomes in wild type human cells including chromosomes 2, 7, 13, 14, 15, 16, 20, 21, 22, and Y. CDC27 pseudogene 11 is found on chromosomes 7, 15, 16, 20, 21, 22, and Y in wild type human cells. However, in HEK293 cells CDC27 pseudogene 11 (hereinafter GADS1) sequences are found on 11 to 13 chromosomes by FISH analysis. The more widespread distribution in HEK293 is hypothesized to be the result of the altered genome found in this transformed cell line. The increased distribution of the GADS1 sequence in HEK293 cells is useful in certain embodiments of the disclosure because there are more target sites in these cultured cells. The same is true of CHO and mouse cell lines.
[126] A preferred sequence according to certain embodiments of the instant disclosure includes the 1978 base pair CDC27 pseudogene 11 sequence (GADS1). The CDC27 pseudogene sequences according to the disclosure are useful in embodiments of the instant disclosure where protein production in cell lines originating from a wide variety of different species are desired because the CDC27 pseudogene sequence is highly conserved in a variety of mammalian species. Nucleotide sequence identity to Cricetulus griseus (Chinese Hamster) CDC27 pseudogene DNA sequence is 85.87% compared to the whole human sequence with only 1% gaps were detected in a Blast search. Nucleotide sequence identity to Mus musculus (mouse) CDC27 pseudogene DNA sequence is 84.65% compared to the whole human sequence with only 1% gaps were detected in a Blast search. The CDC27 pseudogene is even more highly conserved across different primate species.
[127] GADS1 sequences according to the instant disclosure have been shown by FISH analysis to be present on at least 11 chromosomes in HEK293 cells, on the acrocentric arms in several hundred to thousands of copies. Thus, embodiments of the disclosure including constructs and methods that use these sequences are preferred for very high amplification and production of exogenous proteins.
[128] GADS1 sequences useful according to the present disclosure have been identified on chromosomes 2, 22 and Y. (See RefSeq, accessed May 2014: htps://www.alliancegenome.Org/gene/HGNC:1728T In addition, GADS1 sequences are found in Nucleolus organiser regions (NORs) which are chromosomal regions crucial for the formation of the nucleolus. In humans, the NORs are located on the short arms of the acrocentric chromosomes 13, 14, 15, 21 and 22, the genes RNRl, RNR2, RNR3, RNR4, and RNR5 respectively. Finally, wild type human cells carry GADS1 sequences on Chromosomes 7, 15, 16, 20, 21, 22, Y. These regions carry tens of copies of this sequence, each. So, the wild-type genome carries hundreds of copies of this sequence.
[129] GADS1 sequences were successfully used in methods according to the present disclosure utilizing CHO-K1 cells where insertion and amplification of exogenous DNA was demonstrated. Targeting and amplification was demonstrated in three different regions on large metacentric hamster chromosomes: at the very end of metacentric chromosomes; in the middle of one chromosomal arm of metacentric chromosomes; and in the middle (close to the centromere) of metacentric chromosomes.
[130] In other embodiments mouse GAD SI homologues may be used for exogenous gene expression in mouse cell lines.
[131] SEQ ID NO:l according to the present disclosure comprises the 5’-3’ DNA sequence disclosed in the Sequence Listing provided herewith, originating from Homo sapiens and having a length of 1978 bp ("SEQ ID NO:l” and “GADSl” used interchangeably herein).
[132] In preferred embodiments of the instant disclosure GADSl (SEQ ID NO:l) sequences are found in euchromatin regions, suitable for expression and are not silenced. This makes these sites ideal for recombinant protein production. There are many sites in the target genome where the gene of interest can be integrated with the help of CDC27 pseudogenes such as the preferred sequence of GADSl. [133] As discussed above and while not wanting to be bound by theory, the natural CDC27 pseudogenes are hypothesized to have an evolutionary function, because they are so extremely conserved. This CDC27 pseudogene sequence is a natural amplificator. In certain embodiments the application of antibiotic selection pressure to this sequence causes the GADS to be highly amplified together with the gene of interest.
[134] The chromatin around the CDC27 pseudogenes is always open for gene expression making the sites extremely useful in certain embodiments of the disclosure for recombinant gene expression. The inventors hypothesize that these and the other identified GADS sequences are involved in chromosomal evolution and have evolved to essentially be “untouchable” zones - remaining intact throughout evolutionary history. In addition, the natural amplification of these GADS, is accomplished without silencing, setting the systems of the present disclosure apart from all of the other amplification and expression systems previously developed.
[135] Further, a significant advantage of embodiments of the present disclosure over the previous known DHFR and GS based amplification processes that are used in the industry presently, is that the amplified chromosome arm created in embodiments of the present disclosure are stable and that is one reason why recombinant proteins are expressing stably at high levels in embodiments of the present disclosure.
[136] In addition to GADS1, the present disclosure encompasses fragments of SEQ ID NO:l that also exhibit GADS activity.
[137] Expression vectors comprising the isolated 1978 bp sequence (SEQ ID NO:l) and shorter fragments thereof are useful to transform CHO cells and result in high levels of stable protein expression. The present novel GADS1 is useful to improve expression of a recombinant protein driven by a promoter/enhancer region to which it is linked.
[138] Expression vectors comprising the isolated 1978 bp sequence (SEQ ID NO:l) and shorter fragments thereof are useful to transform HEK293 cells and result in high levels of stable protein expression. The present novel GADS1 is useful to improve expression of a recombinant protein driven by a promoter/enhancer region to which it is linked.
[139] Moreover, additional fragments of SEQ ID NO:l exhibiting GADS activity can be identified, as well as similar GADS motifs from other types of cells or from other integration sites in transformed cells. In addition, it is known in the art that subsequent processing of fragments of DNA prepared by restriction enzyme digestion can result in the removal of additional nucleotides from the ends of the fragments.
[140] A fragment (21 lbp - SEQ ID NO:4) of GADS1 (SEQ ID NO:l) is also a part of the 904 bp sequence of GADS3 (SEQ ID NO:3) and also the part of the 2993 bp sequence GADS2 (SEQ ID NO:2). One skilled in the art can thus devise various fragments of the sequences disclosed herein for use in additional embodiments of the present disclosure.
[141] Other combinations of fragments of SEQ ID NO:l can also be developed, for example, sequences that include multiple copies of all or a part of SEQ ID NO: 1. Such combinations can be contiguously linked or arranged to provide optimal spacing of the fragments. Additionally, within the scope of the present disclosure are expression vectors comprising the sequence of SEQ ID NO:l arranged with insertion sequences therein (e.g., insertion of a gene encoding a desired protein at a certain selected site in SEQ ID NO:l).
GADS2: 2993 bp long sequence
[142] In additional embodiments of the instant disclosure a 2993 bp sequence with no previously known function, hereinafter called “GADS2” is provided. GADS2 sequences are found in wild type human cells on chromosomes 7, 15, 16, 20, 21, 22 and Y. These regions each carry tens of copies of this sequence. Thus wild-type human genomes carry hundreds of copies from this sequence. A small part of this sequence overlaps with the sequence of GADS1.
[143] There is no known equivalent sequence in non-human species to GADS2 but this result might be merely because the non-coding portions of these genomes have been less well characterized.
[144] In certain embodiments the GADS2 sequence was used to make targeting and amplification of plasmids in mouse and hamster cell lines. These sequences were used to create independent mammalian artificial chromosomes as well as amplified chromosome arms. Thus, GADS2 sequences are useful for making artificial chromosome arms and independent artificial chromosomes. An embodiment of the instant application showing an example of such artificial chromosomes and chromosome arms is shown in Figures 16, 23 and 26.
[145] GADS2: SEQ ID NO:2 according to the present disclosure comprises the 5’-3’ DNA sequence disclosed in the Sequence Listing provided herewith, originating from Homo sapiens and having a length of 2993bp ("SEQ ID NO:2” and “GADS2” used interchangeably herein).
GADS3: 904 bp long sequence (a smaller fragment of the 2993 bp sequence)
[146] A smaller part of the GADS2 sequence is especially useful in developing human cell lines expressing recombinant proteins. The smaller sequence comprises 904 base pairs of the GADS2 sequence and is referred to as GADS3 or SEQ ID NO:3. Wild-type human cells carry these GADS3 sequences on chromosomes 7, 15, 16, 20, 21, 22, and Y. These regions carry tens of copies of this GADS3 sequence, each. The wild-type human genome carries hundreds of copies of GADS3 sequences.
[147] Cultured human cells (including but not limited to HEK293 cells) carry hundreds to thousands of copies from this GADS3 sequence based on FISH experiments. As discussed previously, cultured human cells have altered genomes, chromosome numbers and chromosome rearrangements and amplifications are very frequent events. Thus, GADS3 sequences are ideal for certain preferred embodiments of the methods and constructs of the instant disclosure.
[148] GADS3 has a number of desirable characteristics for use in preferred methods and constructs of the instant disclosure. 211 bp of the GADS3 sequence overlaps with GADSl at the 5’ end.
[149] No GADS3 nucleotide sequence identity was found to Cricetulus griseus Blast sequences.
[150] No GADS3 nucleotide sequence identity was found to Mus musculus Blast sequences. Thus, in certain embodiments of the disclosure, methods and constructs incorporating GADS3 sequences are useful for production and amplification in human cell lines.
[151] One significant advantage of embodiments of the instant disclosure comprising the GADS3 sequence is that GADS3 is small which is advantageous when you have large exogenous genes to express, or several genes to be expressed at the same time. Certain embodiments comprising expression vectors including GADS3 can include up to 4 exogenous genes to be expressed on a single plasmid. As disclosed in the examples, GADS3 vectors are useful for simultaneous expression of both the heavy and light chains of antibodies.
[152] GADS3: SEQ ID NO:3 according to the present disclosure comprises the 5’-3’ DNA sequence disclosed in the Sequence Listing provided herewith, originating from Homo sapiens and having a length of 904bp ("SEQ ID NO:3” and “GADS3” used interchangeably herein).
GADS4: SEQ ID NO:4 - A 211bp fragment of GADS2 and GADS3
[153] There is a 211 bp overlap between CDC27 pseudogene 11 (GADS1, SEQ ID NO:l), the 904 bp sequence (GADS3, SEQ ID NO. 3) and the 2993 bp sequence (GADS2, SEQ ID NO:2). This sequence, hereinafter referred to as “GADS4” or “SEQ ID NO:4”, can be used in certain embodiments of the disclosure.
[154] GADS4: SEQ ID NO:4 comprises the 5’-3’ DNA sequence disclosed in the Sequence Listing provided herewith, originating from Homo sapiens and having a length of 211 bp ("SEQ ID NO:4” and “GADS4” used interchangeably herein).
GADS5: SEQ ID NO:5
[155] A 293 bp fragment of the 2993 bp human sequence (GADS2, SEQ ID NO:2) has a 238 bp identity with 2% gaps with mouse genomic sequence. This sequence does not overlap with the 904 bp sequence (GADS3, SEQ ID NO:3) or with the CDC27 pseudogene 11 sequence
(GADSl, SEQ ID NO: 1).
[156] GADS5: SEQ ID NO:5 comprises the 5’-3’ DNA sequence disclosed in the Sequence Listing provided herewith, originating from Homo sapiens and having a length of 293 bp ("SEQ ID NO:5” and “GADS5” used interchangeably herein).
GADS6: SEQ ID NO:6
[157] A longer DNA sequence of 9456 bp which was used for mammalian artificial chromosome production in certain embodiments of the presented disclosure. In particular, GADS6: SEQ ID NO:6 comprises the 5’-3’ DNA sequence disclosed in the Sequence Listing provided herewith, originating from Homo sapiens and having a length of 9456bp ("SEQ ID NO:6” and “GADS6” used interchangeably herein).
[158] One skilled in the art will recognize that changes can be made in the nucleotide sequences set forth above including in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:6 by site directed or random mutagenesis techniques that are known in the art. The resulting GADS variants can then be tested for GADS homologous recombination activity as described herein. DNAs that are at least about 80%, more preferably about 85%, and more preferably about 90% identical in nucleotide sequence to SEQ ID NOs: 1, 2, or 3 or fragments thereof, having GADS homologous recombination activity are isolatable by experimentation and hypothesized to have GADS recombination. Accordingly, homologues of the disclosed GADS sequences and variants thereof are also encompassed by the present disclosure.
Cell Therapy with GADS Sequences
Gene and Cell Therapy for Hemophilia A
[159] Hemophilia A (HA) is a rare recessive X-linked bleeding disorder that is caused by mutations and/or deletions in the F8 gene, encoding the coagulation factor VIII (FVIII) (Bolton-Maggs and Pasi, 2003, Graw et al, 2005). On the basis of FVIII plasma activity, three forms of HA are recognized: severe (<1% of cases), moderate (l%-5% of cases), and mild (5%-40% of cases) (Bolton-Maggs and Pasi, 2003). Current therapy consists of repetitive infusions of recombinant or plasma-derived FVIII. This replacement therapy, however, does not represent a definitive cure and, moreover, 20%-40% of the treated patients develop anti-FVIII neutralizing antibodies (Den Uijl et al, 2011). Several alternatives have been proposed, such as the drug emicizumab, a prophylactic therapy for adult and pediatric patients (Lenting et al, 2017, Shima et al., 2016), and some gene and cell therapy approaches have been attempted. In HA patients the restoration of FVIII activity above 2%-5% could ameliorate the patients' quality of life. Gene transfer approaches for hemophilic patients have been attempted for almost 20 years (Nathwani et al, 2017). Presently, adeno-associated viruses (AAVs) are used for hemophilia gene therapy due to their relative safety, simplicity, and high liver tropism (Naso et al., 2017) but, despite positive results obtained with HB (Nathwani et al, 2011) and HA (Rangarajan et al., 2017), pre-existing immunity to AAVs and their long-term efficacy in young patients are still a concern. Several approaches for hemophilia gene therapy using lentiviral vectors (LVs) were developed and tested in preclinical models showing promising results (Cantore et al, 2015, Merlin et al, 2017). Nevertheless, there remains a need for novel approaches to treat hemophilia and embodiments of the instant disclosure that use high expression cell therapy solve or at least, ameliorate, many of the problems with existing approaches. Cell and gene therapy for cystic fibrosis
[160] Induced pluripotent stem cells (iPSCs) are a recently developed technology in which fully differentiated cells such as fibroblasts from individual cystic fibrosis (CF) patients can be repaired with the [wildtype] CFTR gene, and reprogrammed to differentiate into fully differentiated cells characteristic of the proximal and distal airways. The in vitro genetic roadmap which iPSCs follow as they are step-wise differentiated into either basal stem cells, for the proximal airway, or into Type II Alveolar cells for the distal airways were reviewed by Bette S Pollard, Harvey B Pollard Induced pluripotent stem cells for treating cystic fibrosis: State of the science Pediatr Pulmonol. 2018 Nov; 53(S3): S12-S29. doi: 10.1002/ppul.24118. iPSC-derived basal stem cells are penultimately dependent on NOTCH signaling for differentiation into club cells, goblet cells, ciliated cells, and neuroendocrine cells. Furthermore, given the proper matrix, these cellular progenies are also able to self-assemble into a fully functional pseudostratified squamous proximal airway epithelium. By contrast, club cells are reserve stem cells which are able to either differentiate into goblet or ciliated cells, but also to de-differentiate into basal stem cells. Variant club cells, located at the transition between airway and alveoli, may also be responsible for differentiation into Type II Alveolar cells, which then differentiate into Type I Alveolar cells for gas exchange in the distal airway. Using embodiments of the instant disclosure cystic fibrosis patients can receive cell therapy that provides stable expression of wild type CFTR protein. Certain embodiments of the method comprise introducing wildtype CFTR gene into iPSCs from CF patients at GADS sequences followed by the production of MACs expressing CFTR protein using pluromycin and differentiating the stem cells into fully functional epithelial cells through directed differentiation.
XSCID cell and gene therapy project
[161] X chromosome-linked severe combined immunodeficiency syndrome (X-SCID) is a rare inherited disorder, which affects boys. The X-SCID syndrome is caused by faulty expression of the gamma chain (g) of the interleukin-2 receptor (IL2RG), which results in the complete absence of mature T and natural killer (NK) cells, whereas B cells are frequently present in increased numbers. Disruption of IL-2 mediated signaling, however, does not account for the X-SCID phenotype, because IL-2 deficiency does not stop T-cell development. DiSanto JP, Keever CA, Small TN, Nicols GL, O'Reilly RJ, Flomenberg N: Absence of interleukin 2 production in a severe combined immunodeficiency disease syndrome with T cells. J.Exp.Med 1990, 171:1697-1704.
[162] Further studies established that yc also participated in the receptors for IL-4, IL-7, IL-9, and IL-15 (reviewed by Sugamura K, Asao H, Kondo M, Tanaka N, Ishii N, Nakamura M, Takeshita T: The common yC chain for multiple cytokine receptors. Adv.Immunol 1995, 59:225-277.) The X-SCID phenotype, therefore, results from combined defects in these 5 cytokine systems. Children born with X-SCID lack a working immune system, so their bodies are unable to fight off any infections. As a result, they suffer from severe infections since several months after birth, and without treatment these babies with X-SCID rarely survive beyond their first birthday.
[163] Until recently, the only cure for X-SCID was a bone marrow transplant. About 30% of children have the option of an HLA-identical bone-marrow transplant (BMT), which provide them a >90% chance for survival. BMT with a partially matched donor the survival rates are much lower and the complication rates higher. For children where no suitable donor is available at all, gene or cell therapy is their only real hope.
[164] During its 40-year history, the only unequivocal success story in gene therapy is the successful treatment of children with X-SCID. Alain Fischer’ s team at the Necker Hospital for Sick Children (Paris, France) pioneered the X-SCID gene therapy treatment. First, bone-marrow cells were extracted from the patient and immature hematopoietic cells were selectively isolated. Then, the correct gene (gamma c or yC) was introduced to these cells by repeated infection of a retrovirus containing the normal version. The final step is to transplant these cells back into the patient (Cavazzana-Calvo M, Hacein-Bey S, de Saint BG, Gross F, Yvon E, Nusbaum P, Selz F, Hue C, Certain S, Casanova JL, Bousso P, Deist FL, Fischer A: Gene therapy of human severe combined immunodeficiency (SCID)-Xl disease. Science 2000, 288:669-672. Hacein-Bey- Abina S, Le Deist F, Carlier F, Bouneaud C, Hue C, De Villartay JP, Thrasher AJ, Wulffraat N, Sorensen R, Dupuis-Girod S, Fischer A, Davies EG, Kuis W, Leiva L, Cavazzana-Calvo M: Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N.Engl.J.Med 2002, 346: 1185-1193). [165] Of 11 children who have received this treatment in France, nine have developed functional immune systems. Using basically the same protocol similar successes have been seen in the USA, UK, Germany, and Italy.
[166] A serious setback for gene therapy occurred in 2002 when it was reported that a child being treated in France for X-SCID showed signs of having developed leukemia after undergoing treatment. To date, T-cell acute lymphoblastic leukemia (T-ALL)-like disease occurred in 4 out of a total of 20 patients with X-SCID, treated in two separate gene-therapy trials (Pike- Overzet K, van der Burg M, Wagemaker G, van Dongen JJM, Staal FJT: New insights and unresolved issues regarding insertional mutagenesis in X-linked SCID gene therapy. Mol Ther 2007, 15:1910-1916).
[167] Occurrences of serious adverse events (SAEs) in otherwise successful gene therapy trials for X-linked severe combined immunodeficiency were somewhat unexpected as it was not predicted from prior preclinical studies in mice. The development of leukemia after this gene therapy has been attributed to the upregulated expression of the oncogene LM02 because of vector integration. (7Thrasher AJ, Gaspar HB, Baum C, Modlich U, Schambach A, Candotti F, Otsu M, Sorrentino B, Scobie L, Cameron E, Blyth K, Neil J, Abina SH, Cavazzana-Calvo M, Fischer A: Gene therapy: X-SCID transgene leukaemogenicity. Nature 2006, 443: E5-E6. Howe SJ, Mansour MR, Schwarzwaelder K, Bartholomae C, Hubank M, Kempski H, Brugman MH, Pike-Overzet K, Chatters S J, de Ridder D, Gilmour KC, Adams S, Thornhill SI, Parsley KL, Staal FJT, Gale RE, Linch DC, Bayford J, Brown L, Quaye M, Kinnon C, Ancliff P, Webb DK, Schmidt M, von Kalle C, Gaspar HB, Thrasher AJ: Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J Clin Invest 2008, 118:3143- 3150. Hacein-Bey- Abina S, von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, Lim A, Osborne CS, Pawliuk R, Morillon E, Sorensen R, Forster A, Fraser P, Cohen JI, de Saint BG, Alexander I, Wintergerst U, Frebourg T, Aurias A, Stoppa-Lyonnet D, Romana S, Radford-Weiss I, Gross F, Valensi F, Delabesse E, Macintyre E, Sigaux F, Soulier J, Leiva LE, Wissler M, Prinz C, Rabbitts TH, Le Deist F, Fischer A, Cavazzana- Calvo M: LM02-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003, 302:415-419).
[168] To investigate the origin of these adverse events, IL2RG was expressed from a lentiviral vector in a murine model of X-SCID, and the fates of mice were followed for up to 1.5 years post-transplantation. Unexpectedly, 33% of these mice (n=15) developed T-cell lymphomas that were associated with a gross thymic mass. Worldwide, some 88 mice have been treated with IL2RG in retroviral vectors but these studies were limited in their duration, which usually did not exceed 6 months post-transplant. Woods et al. reported that their first IL2RG induced lymphoma appeared 6 months post-transplant. Longer-term analysis did not reveal leukemogenesis in dogs, rhesus macaques or sheep carrying human chimeric genes for up to one-year post-transplant, but this may have been attributable to features inherent in large-animal models. However, more recently the death of a monkey 5 years after administration of CD34+ cells transduced by a retroviral vector containing marker genes was reported. Autopsy revealed that the monkey developed a fatal myeloid sarcoma, a type of acute myeloid leukemia. Tumor cells contained 2 clonal vector insertions.
[169] One of the insertions was found in BCL2-A1, in an antiapoptotic gene. This event suggests that currently available retroviral vectors may have long-term side effects, particularly in hematopoietic stem and progenitor cells. In the human gene-therapy trials, leukemias did not appear until 2-3 years after treatment, therefore SAE can be regarded as a long-term risk. Woods NB, Bottero V, Schmidt M, von Kalle C, Verma IM: Gene therapy: therapeutic gene causing lymphoma. Nature 2006, 440:1123. Soudais C, Shiho T, Sharara LI, Guy- Grand D, Taniguchi T, Fischer A, Di Santo JP: Stable and functional lymphoid reconstitution of common cytokine receptor g chain deficient mice by retroviral-mediated gene transfer. Blood 2000, 95:3071-3077. Otsu M, Sugamura K, Candotti F: Lack of dominant-negative effects of a truncated gamma(c) on retroviral-mediated gene correction of immunodeficient mice. Blood 2001, 97: 1618-1624. Aviles Mendoza GJ, Seidel NE, Otsu M, Anderson SM, Simon-Stoos K, Herrera A, Hoogstraten-Miller S, Malech HL, Candotti F, Puck JM, Bodine DM: Comparison of five retrovirus vectors containing the human IL- 2 receptor g chain gene for their ability to restore T and B lymphocytes in the X-linked severe combined immunodeficiency mouse model. Mol.Ther 2001, 3:565-573. Lo M, Bloom ML, Imada K, Berg M, Bollenbacher JM, Bloom ET, Kelsall BL, Leonard WJ: Restoration of lymphoid populations in a murine model of X-linked severe combined immunodeficiency by a gene-therapy approach. Blood 1999, 94:3027-3036. Otsu M, Anderson SM, Bodine DM, Puck JM, O'Shea JJ, Candotti F: Lymphoid development and function in X-linked severe combined immunodeficiency mice after stem cell gene therapy. Mol.Ther 2000, 1:145-153. Otsu M, Sugamura K, Candotti F: In vivo competitive studies between normal and common g chain-defective bone marrow cells: implications for gene therapy. Hum.Gene Ther 2000, 11:2051-2056. Whitwam T, Haskins ME, Henthorn PS, Kraszewski JN, Kleiman SE, Seidel NE, Bodine DM, Puck JM: Retroviral marking of canine bone marrow: long-term, high-level expression of human interleukin-2 receptor common g chain in canine lymphocytes. Blood 1998, 92:1565-1575. An DS, Kung SK, Bonifacino A, Wersto RP, Metzger ME, Agricola BA, Mao SH, Chen IS, Donahue RE: Lentivirus vector-mediated hematopoietic stem cell gene transfer of common g-chain cytokine receptor in rhesus macaques. J.Virol 2001, 75:3547-3555. Seggewiss R, Pittaluga S, Adler RL, Guenaga FJ, Ferguson C, Pilz IH, Ryu B, Sorrentino BP, Young, W S, III, Donahue RE, von Kalle C, Nienhuis AW, Dunbar CE: Acute myeloid leukemia is associated with retroviral gene transfer to hematopoietic progenitor cells in a rhesus macaque. Blood 2006, 107:3865-3867. Saffery R, Choo KH: Strategies for engineering human chromosomes with therapeutic potential. J.Gene Med 2002, 4:5-13.
[170] At present, taking all the options into account, regulatory authorities allow retrovirus vector-mediated yc clinical trials to continue if there were no acceptable alternative therapies for trial participants who would have died without this intervention. X-SCID gene therapy is limited to patients who have failed identical or haploidentical stem cell transplantation or for whom no suitable stem cell donor can be identified. The procedure is still being reviewed and used on a case-by-case basis including appropriate risk-benefit analysis accompanied by implementation of informed consent and monitoring plans. At the same time, regulatory authorities urged for the development of safer vectors to reduce the risk of insertional mutagenesis and vectors capable of regulated therapeutic gene expression.
[171] Mammalian artificial chromosomes are safe, stable, non- integrating vectors with large transgene carrying capacity. Irvine DV, Shaw ML, Choo KHA, Saffery R: Engineering chromosomes for delivery of therapeutic genes. Trends Biotechnol 2005, 23:575-583.
[172] Grimes BR, Monaco ZL: Artificial and engineered chromosomes: developments and prospects for gene therapy. Chromosoma 2005, 114:230-241. Basu J, Willard HF: Artificial and engineered chromosomes: nonintegrating vectors for gene therapy. Trends Mol.Med 2005, 11:251-258. Basu J, Willard HF: Human artificial chromosomes: potential applications and clinical considerations. Pediatr.Clin.North Am 2006, 53:843-853. Ren X, Katoh M, Hoshiya H, Kurimasa A, Inoue T, Ayabe F, Shibata K, Toguchida J, Oshimura M: A novel human artificial chromosome vector provides effective cell lineage-specific transgene expression in human mesenchymal stem cells. Stem Cells 2005, 23:1608-1616. Wilson A, Trumpp A: Bone-marrow haematopoietic-stem-cell niches. Nat Rev Immunol
2006, 6:93-106. Shoichi Iriguchi et al. A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy. NATURE COMMUNICATION S (2021) 12: 430-https://doi.org/10.1038/s41467-020-20658-3. All incorporated herein by reference in their entirety. Embodiments of the present disclosure can include expression of not only gamma c but also IL-4, IL-7, IL-9, and IL-15.
[173] We have developed a method to target gene constructs into open chromatin sites in mammalian cells and at the same time achieve amplification of the delivered transgenes. The amplification may range from 15 copies to hundreds of copies. Therefore, in this protocol we can make artificial chromosome arm (MACArm) or even individual MACs, depending on the expected protein production levels. These transgenes then express the therapeutic proteins in the right levels. Depending on the desired protein amount for therapeutic applications, we have the chance to choose the cell lines with the optimal amplification level and protein production level for the desired therapy.
[174] The following examples are meant to be illustrative of embodiments of the present disclosure and do not limit the scope of the disclosure in any way. All references cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
EXAMPLES
EXAMPLE 1: GADS 3 CHO-K1 cell line production of trastuzumab
[175] The optimal DNA sequence for expression of trastuzumab was determined and the DNA was synthesized in a pUC plasmid and transferred into an appropriate insertion vector according to the disclosure for insertion in CHO-K1 cells. A typical plasmid is shown in
Figure 1. [176] This plasmid was used to establish stable CHO-K1 cell lines with amplified “trastuzumab” as follows: a. CHO-K1 cells were seeded into one well of a 24- well plate (approximately 100000 cells). The next day cells were transfected with 1 pg trastuzumab containing plasmid. For this cell line we use the Turbofect reagent (see https : // www. thermofisher com/ order/ catalo g/ product/R0532#/R0532. accessed
May 31, 2021). b. The insertion plasmid was diluted with 100 mΐ serum-free DMEM medium. c. The Turbofect reagent was mixed thoroughly by vortexing. d. 2 mΐ Turbofect reagent was added to the insertion plasmid solution. e. This reaction was mixed by pipetting and incubated at room temperature for 20 minutes. f. The transfection mix was added evenly and drop by drop to the 24 cells in the wells with each well containing serum containing 1 ml DMEM medium. g. After 24 hours, the cells were collected using TrypLE Select reagent and then distributed into the wells of 2x96-well plates. About 40 ml of DMEM was used in order to achieve volume of about 200 mΐ x 96 x 2 ml altogether. h. After 24 hours antibiotic selection is begun by the addition of 10 pg/ml puromycin. i. After 3 days the selection medium is exchanged for fresh medium. j. After an additional 3 days the selection medium is again exchanged. k. After an additional 3 days the growing cell clones are collected from the wells of the 96-well plate by TrypLE Select reagent, and individually transferred into one well of a 24-well plate. l. 24 hours later antibiotic selection with 50 pg/ml Puromycin is started. m. 3 days later the selection medium is exchanged for medium containing 100 pg/ml Puromycin containing medium. n. The growing cell lines were examined by lysing the cells and purifying the total protein. Western blot analysis (see Figure 4) was used to determine the production of trastuzumab. o. The highest producing cell lines are checked with FISH experiments for the presence of the insertion plasmid. See for example Figure 3. p. The best clones are further selected with increased antibiotic selection. The cells that are growing at 100 pg/ml puromycin are split into 3 wells of a 24-well plate and after 24 hours the cells are subjected to 150 pg/ml, 175 pg/ml and 200 pg/ml puromycin. q. Surviving clones are examined with western blotting for protein production and with FISH for the presence of amplification. r. The best producing clones are grown in serum-free medium. s. Trastuzumab is purified from the medium. t. The concentration of trastuzumab is determined by spectrophotometer, HPLC or similar procedures. u. Highest yielding cell lines will be selected and trastuzumab can be purified.
EXAMPLE 2: GADS 3 HEK293 cell line production of trastuzumab
[177] Plasmids containing the GADS3 sequence (SEQ ID NO:3) were used to transfect HEK293 cell line. The insertion vector is shown in Figure 2. The production of trastuzumab was confirmed by Western blotting as shown in Figure 5 using the 42kDa actin protein expression for quantitation.
[178] Trastuzumab is purified using protein G columns. The Protein GHP SpinTrap column was used for small scale purification. Figure 6 shows a blot of the purified trastuzumab both heavy and light chains (for SpinTrap see https://www.sigmaaldrich.com/catalog/product/ sigma/ge28903134?lang=hu®ion=HU&gclid=CiwKCAiwtdeFBhBAEiwAKOIv5914n 5Xlr4X4sllmGD Z46KOJtAlk-rlomuiFYESnStEUWOXp-aaThoCi-4QAvD BwE. accessed May 31, 2021).
[179] Based on the Bradford protein concentration measurement, we found that trastuzumab production was at least 111 pg/cell.
[180] The highest producing cell line (10/13) was subjected to subcloning (see Figure 7) and the highest producing clone (#11) was subjected to FISH analysis (see Figure 8).
EXAMPLE 3: GADS2 cell line production in mouse cell lines
[181] Mouse cell lines: Mouse embryonic fibroblast (MEF) cells from 3.5 days old individual mouse embryos were isolated and used to establish stable cell lines. The cells were importalized with well documented methods: basically, cells were passaged every 3 days until immortalized. These cells show several markers of mesenchymal stem cells by FACS experiments. This process and these cell lines are useful for adult mesenchymal stem cells from adult tissues (preferably from adipose tissue after liposuction), modify those cells with mammalian artificial chromosomes created using the instant disclosure and using them directly or after differentiation into certain cell types (fat, cartilage, bone etc.)) for gene therapy and/or cell therapy experiments. The first step, shown here, demonstrates a vector according to the disclosure that carried the 2993 bp sequence (GADS2: SEQ ID NO:2) into which was inserted a useful gene (Influenza A virus Hemagglutinin MYMC X-181 California strain) as shown in Figure 9. This embodiment of the disclosure was transfected into the immortalized MEFs, producing 34 stable cell lines with small-scale amplification that resulted in 30-50 copies of the exogenous sequence, as shown in Figure 10. Surprisingly, targeting exclusively happened into a large acrocentric chromosome in the upper part of the long chromosomal arm in all 34 clones.
EXAMPLE 4: GADS2 cell line production in CHO cell lines
[182] A plasmid according to the disclosure (shown in Figure 9) was used to transfect CHO- DG44 cells producing 79 clones. Autonomous mammalian artificial chromosomes were formed in 16 cell lines out of the 79. Figure 11 shows an example of an autonomous mammalian artificial chromosome and an amplified chromosome arm together. One hamster chromosome has an amplified chromosome arm with hundreds of transgene copies. The autonomous mammalian artificial chromosome consists almost entirely of transgene sequences, representing hundreds of transgene copies.
EXAMPLE 5: GADS1 cell line production in mouse cell lines
[183] LMTK- mouse cells were transfected with a plasmid construct according to the disclosure carrying GADS1 sequence (CDC27 pseudogene: SEQ ID NO:l) and the Influenza A virus Hemagglutinin MYMC X-181 California strain sequence was inserted as a useful exogenous gene (Figure 12). We produced 32 cell lines. Eight cell lines carried autonomous mammalian artificial chromosomes (Figure 13).
EXAMPLE 6: GADS1 cell line production in mouse cell lines [184] CHO-K1 cell lines were made with a plasmid according to the disclosure carrying the GADS1 sequence (CDC27 pseudogene: SEQ ID NO:l) and expressing RBBP7 protein (pIKRBBP7, see Figure 14). Two types of cell lines were constructed in this embodiment. One type of cell line expresses and secretes the RBBP7 protein into the culture medium (CIRBBP cell lines). In this embodiment, the RBBP7 protein is expressed with a hamster IgK secretion signal on the N-terminal. After the secretion signal, there are two tags for labeling and purification (an AVI tag and a 6xHis tag). 53 cell lines were produced with this construct and 24 of them stably produce the RBBP7 protein (see Figure 15). This was shown by Western blotting experiments.
EXAMPLE 7: GADS1 cell line production in mouse cell lines
[185] Additional RBBP7 protein producing cell lines were produced without the hamster IgK secretion signal to cause the protein to remain inside the cells rather than be excreted. Proteins produced in this embodiment are purified from cell lysates.
EXAMPLE 8: Methods of generating mammalian artificial chromosomes and transgenic stem cells
[186] The following method was used to generate mammalian artificial chromosomes and transgenic stem cells in certain embodiments of the disclosure. Other suitable methods according to the disclosure are described below. The gene of interest /transgene was cloned into the pPur plasmid (from Clontech) together with CMV promoter and SV40 polyA signal to create plasmid pPUR-TG. a. BAC JH10 was digested with Pad and Xcml restriction enzymes. The 9456 bp fragment was isolated (GADS6). b. pPUR-TG and the GADS6 fragment was co-transfected into CHO cell lines (CHO- Kl, CHO-DG44) or mouse cells (LMTK-). A 1 : 1 ratio of DNA molecules was used for transfection experiments. Transfection was done with Turbofect (Thermo- Fisher, R0531) reagent as the manufacturer suggested. Transfections were done in 1x60 mm Petri dishes for each experiment. Cells were seeded into the Petri dish in a 2.0-6.3x105 concentration a day before transfection. c. 48 hours after transfection, the cells were split into 4x100 mm Petri dishes. d. 24 hours later antibiotic selection was started with 5 pg/ml Puromycin. e. Selection medium was replaced with fresh selection medium every other day for 2 weeks. At this time, colonies originated from single cells were grown up to a size of 100-300 cell masses. f. We started to pick the largest colonies individually by using the cylinder method. g. Individual colonies were seeded into individual wells of 24-well plates. Hundreds of clones were picked in the next 7 to 14 days. h. Individual clones were cultured up to 6 wells of a 24-well plate. Cells from 5 wells were collected into 5xfreezing vials in freezing medium (culture medium with 10% FBS and 10% DMSO (Sigma, D2650)) and stored at -80°C. i. Cells were cultured from 1 well of a 24-well plate until they grow into confluency in 1x60 mm Petri dish. Then cells were lysed in lxLaemli buffer with -mercapto- ethanol (Thermo-Fisher, 21985023) as follows: 200 mΐ lxLaemli was added to the cell pellet, mixed, and stored on ice until it became highly viscous. At this point lysed cells were boiled for 3 minutes. After boiling cells were vortexed for 1 minute. Boiling and vortexing were repeated two more times. The resulted solution was aliquoted (20 mΐ each) and stored at -80°C. One aliquot was used for Western blotting experiments without freezing. j. Cell lines were screened by Western blotting for protein production. Cell lines with highest protein production rates were examined by FISH experiments. k. One vial of frozen cells was thawed into one well of a 24-well plate. These cells were cultured until they reached 100 mm Petri dishes with 50-60% confluency. Next day the cells were blocked in metaphase by using Colchicine (5 pg/ml, BDH, 27805) for 6 hours. Cells in metaphase stage were collected, cell membrane was disrupted with low salt conditions (75 mM kalium-chloride) and fixed in methanol: acetic acid (3:1) solution. About 30-50 mΐ of this solution was dropped onto surface treated glass slides and the chromosomes and interphase nuclei were fixed onto the slides with methanol: acetic acid (3:1) solution and drying. FISH experiments were performed with fluorescently labeled transgenes on these slides with standard protocol. Artificial chromosome arms were identified and individual artificial chromosomes by FISH experiments. l. Further experiments were done with cell lines, which showed high-yield protein production by Western blotting and carried individual artificial chromosomes. m. Total chromosome set was isolated from these cell lines and transfected into mouse R1 ESCs (embryonic stem cells) as described in Robert Katona Dendrimer Mediated Transfer of Engineered Chromosomes in: Hadlaczky, G (szerk.) Mammalian Chromosome Engineering: Methods and Protocols Totowa (NJ), USA: Humana Press (2011) 265 p. pp. 151-160., pi 0. n. Mammalian artificial chromosome carrying R1 ESCs were established and the best clones were selected by Western blotting and FISH experiments. o. R1 ESCs were differentiated into various types of adult cells depending on the therapeutic need and used for cell and gene therapy experiments as it was described in Examples below and as shown in the Figures.
EXAMPLE 9: Cell Therapy for hemophilia A
[187] Wild-type Factor VIII are cloned into an INPAMAC GADS vector (pINPF8). iPSCs are extracted from from PB CD34+ cells. The pINPF8 vector is transfected into the iPSC cells and iPSC cell lines carrying targeted and amplified pINPF8 plasmid are created. Cell lines that produce the highest level of F8 protein are selected and differentiated into ECs (endothelial cells). ECs are transplanted into the patients (animal for pre-clinical, human for clinical protocols).
[188] This same protocol is applicable to treatment of hemophilia B by cloning Factor IX cDNA into the INPAMAC GADS plasmid (pINPF9).
[189] Procedures outlined in Olgasi et al. "Patient- Specific iPSC-Derived Endothelial Cells Provide Long-Term Phenotypic Correction of Hemophilia A.” Stem Cell Reports. (2018); 11:1391-1406. doi: 10.1016/j.stemcr.2018.10.012 (the disclosure of which is hereby incorporated herein by reference in its entirety) can be adapted for embodiments of the present disclosure.
EXAMPLE 10: Cell Therapy for Cystic Fibrosis
[190] Below is provided an exemplary method for producing cell lines for cell therapy of conditions such as cystic fibrosis: 1. The appropriate transgene (CFTR cDNA) is inserted into an INPAMAC GADS vector. The CFTR transgene is synthesized and then cloned into an INPAMAC GADS vector (pINPCFTR plasmid).
2. The previously mentioned iPSCs are used for these experiments. pINPCFTR plasmids are transfected into these stem cells.
3. Puromycin antibiotic selection is utilized as described herein to create cell lines with amplified transgenes.
4. Antibiotic resistant cell lines are identified and checked for CFTR protein production by Western blotting.
5. The best cell lines based on Western blotting results are evaluated by FISH experiments, to identify cell lines with the best amplifications of the transgenes for CFTR protein production.
6. Based on FISH and Western blotting experiments, the best cell lines (one or two) are selected and differentiated these to cell types as described in Pollard and Pollard “Induced pluripotent stem cells for treating cystic fibrosis: State of the science” in Pediatr Pulmonol. 2018 Nov; 53(S3): S12-S29. doi: 10.1002/ppul.24118 -the disclosure of which is hereby incorporated by reference herein in its entirety.
8. The differentiated cell types are checked to confirm that they express CFTR protein by Western blotting and the best cell lines are used for transplantation into host animals and/or patients.
EXAMPLE 11: Cell and Gene Therapy for Therapy for X-SCID
[191] In the X-SCID clinical protocols, multiple transfections of bone marrow hematopoietic stem cells (HSCs) with retroviral vectors are necessary to produce enough HSCs for the therapy. Attempts at in vitro expansion of hematopoietic stem cells have so far has been unsuccessful. To establish an HSC clone carrying transgenes delivered and amplified with the INPAMAC technology, 50-100 cell divisions or more in the undifferentiated state will be carried out in order to obtain amounts sufficient for therapeutic applications.
[192] Gamma-C [yC] protein expressing MACs were made in CHO-DG44 cells. Four cell line proved to be the best (3D11, 4F9, 5E4, 5G2). In Figure 23, FISH experiments demonstrating the presence of MACs in CHO cells are shown: Red staining shows the presence of yC-MACs. Chromosomes were counterstained with DAPI (blue).
[193] Figure 24 shows Western blots of CHO cells expressing yC protein from MACs. The yC- MAC was delivered into mouse embryonic stem cells (mESCs, Rl) from CHO-DG44 cells by microcell fusion (Figure 25 - wherein mESC clones carrying yC-MAC were named RGAMC. Four RGAMC clones were isolated and RGAMC3 was the highest expressor. FISH experiments demonstrate the presence of MACs in mESCs, wherein in RGAMCl, MAC is stained green; and in RGAMC2 to RGAMC4, MAC is stained red; and wherein DAPI was used as a counterstain (blue) to show all chromosomes and nuclei) and the presence of the yC gene [yC] carrying MACs in RGAMC3 subclones were demonstrated by FISH experiments (Figure 26 - RGMAC3 was the best, but not a single cell clone. Thus, this mESC cell line was subcloned. Five subclones were obtained: GM14, 19, 20, 22, 27. FISH experiments demonstrated the presence of the yC gene carrying MACs (Green signal: yC gene on MACs, Blue signal: mouse chromosomes with DAPI staining)). Figure 27 shows that even after delivery from CHO cells and subcloning, mESC cell lines still express yC from MACs (in Figure 27, M is the Protein size marker; 1 is GMC14; 2 is GMC19; 3 is GMC20; 4 is GMC22; 5 is GMC27; the negative control (-) is mESC; and the positive control (+) is 3D 11 (CHO)); and Figures 28 and 29 show that yC-MAC continued to express yC protein even after differentiation into embryoid bodies (EBs). With reference to Figure 29, note that wild type Rl mESCs were differentiated to EBs. Human yC is not present in these cells. When yC-MAC carrying mESCs were differentiated into EBs, both expressed human yC, and EBs expressed it in a significantly higher level when normalized to b-actin expression.
[194] In order to overcome shortcomings of existing technologies, pluripotent and adult stem cells (human ESCs, human iPSCs, gut stem cells, or mesenchymal stem cells for example) will be modified by transfection with the GADS vectors described herein and the amplified cells will be differentiated into blood cell types (T-cells, NK-cells or HSC cells). These blood cells can be delivered into the patient’s blood stream to produce the missing protein, specifically: yC 1. The human yC transgene is inserted into an INPAMAC GADS vector. The original yc transgene will be synthesized and then cloned into an INPAMAC GADS vector (pINPAXSCID plasmid).
2. pINPAXSCID plasmids will be transfected into appropriate stem cells. Most preferably, induced PSCs (iPSCs) derived from an antigen-specific cytotoxic T-cell clone, or from T-cell receptor (TCR)-transduced iPSCs, will be used, since these cell lines have the highest promise to differentiate into T-cells.
3. Puromycin antibiotic selection, as described herein, will be used to create cell lines with amplified transgenes.
4. The antibiotic resistant cell lines will be checked for yC protein production by Western blotting.
5. Best cell lines will be selected based on Western blotting results, and will then be evaluated by FISH experiments, in order to identify the cell lines having optimal amplifications of the transgenes for yC protein production.
6. The best-performing cell lines will be selected based on FISH and Western blotting experiments, and will be differentiated to T-cells using the protocol described herein.
7. The differentiated T-cells will be checked to confirm continued expression of yC protein by Western blotting.
8. T-Cells that are confirmed to be expressing yC protein, will be suitable for therapeutic experiments if converted into GMP conditions.
[195] This procedure can be done in mice, too, as a pre-clinical experiment. In this case, mouse
ESCs are stabilized and stable cell lines are created as described by Iriguchi et al. “A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy” in NATURE COMMUNICATIONS (2021) 12: 430- https://doi.org/10.1038/s41467-020-20658-3, the disclosure of which is hereby incorporated by reference herein in its entirety. T-cells can then be differentiated from the mESCs and transplanted into XSCID mice for treatment.
[196] Using analogous techniques, human iPSCs and mESC cell lines can be created in embodiments of the present disclosure using the yC transgene. EXAMPLE 12: Creation of Artificial Chromosomes with Trastuzumab
[197] In order to demonstrate tumor therapy with combined artificial chromosomes and the use of stem cells, the heavy chain and light chain sequences of trastuzumab with the IgK secretion signal were cloned into a plasmid with a puromycin resistance gene and co transfected with a mouse rDNA targeting DNA sequence. This can also be done efficiently with any of the GADS sequences disclosed in this application.
[198] A MAC was constructed that expresses trastuzumab protein in a mouse cell line (LMTK-) and separately in CHO cells. The LMTK line expressed a higher level of trastuzumab production and so was chosen to move forward. The cell line which produced the highest level of trastuzumab protein was BCHAT40 (1.8 g/L). Trastuzumab was purified from the culture medium of BCHAT40 cells and the antibody’s functionality was assayed with FACS, immunostaining and ADCC experiments.
[199] FISH experiments (see Figure 16) show the BCHAT40 cell line with the highest level of trastuzumab expression. The presence of the transgene carrying MAC is shown with the green signal. Mouse chromosomes were counterstained with DAPI (blue). Figure 17) shows the Western blots of various clones with the chosen clone (40) highlighted in red.
[200] FACS experiments are shown in Figure 18. Cells were incubated with trastuzumab that was purified from BCHAT40 cells. Trastuzumab binds to Her2/Neu receptor on the surface of the tumor cells and after washing out unbound trastuzumab antibodies the cells were incubated with anti-human-FITC secondary antibody which recognizes Her2/Neu bound trastuzumab. SK-BR3 and BT474 are human breast cancer cells with high level of Her2/Neu proteins in the cell membrane. In graphs 100 and 200, curves 102 and 202, respectively, are significantly shifted due to high levels of trastuzumab binding in the breast cancer cell lines. Curves 104 and 204 are control experiments where only the secondary antibody was added to the cells. In graphs 300 and 400, MDA-MB and MCF-7 human breast cancer cells, respectively, have normal level of Her2/Neu proteins in their cell membrane and so curves 302 and 402 are not significantly shifted due to low level of trastuzumab binding.
[201] SKBR3 human breast cancer cells were immunostained with clinically approved trastuzumab and with purified trastuzumab that was produced by BCHAT40 cells containing MACs. Results (Figure 19) show that trastuzumab from BCHAT40 cells are as good as the clinically approved trastuzumab in recognizing Her2/Neu protein in the cell membrane of SK-BR3 tumor cells. In Figure 19, 502 shows a negative control: anti- human-RHO 1:500; 504 shows a positive control: therapeutic trastuzumab (16mg/ml) anti- human-RHO (1:500); 506 shows BCHAT40 trastuzumab (7 mg/ml) anti-human-RHO (1:500); and 508 shows BCHAT40 trastuzumab (13.2 mg/ml) anti-human-RHO (1:500).
[202] ADCC tests were conducted (Figure 20) to prove the functionality of the trastuzumab antibody produced by BCHAT40 cells. Trastuzumab binds to Her2/Neu receptor and if trastuzumab antibody has the right binding and configuration (folding, secondary modifications etc.), then CD16.NK-92 effector cells can recognize it and kill the tumor cells which are tagged by the trastuzumab antibody. Results in the graph show: Control: SKBR3 human breast cancer cells were incubated with BCHAT40 produced trastuzumab, but no effector cells were added. There are only a few cells are dead in this control experiment. B: Effector cells are added and there are significant dead cells present. Effector cells killed the trastuzumab tagged SKBR3 breast cancer cells. C: BCHAT40 produced trastuzumab induces high levels of cell death in SKBR3 cells when effector cells are added.
[203] These experiments showed that the MAC-produced trastuzumab is functional. To carry out cell therapy with this trastuzumab producing MAC, mouse adipocyte stem cells (ADSCs) were isolated and immortalized. These cells are mesenchymal stem cells (MSCs) from fat tissue. MSCs show tropism to tumor sites and so can be used to deliver the MAC produced trastuzumab to the tumor sites in a mouse model of human breast cancer. Figures 21 and 22 show the successful creation of these cells.
[204] The MACs were delivered into ADSCs and many trastuzumab producing therapeutic ADSC lines were recovered (see Figure 1). These cell lines are ready for transplantation into animals.
[205] This procedure can be done using the GADS-containing vectors and iPSC and mESC cells followed by differentiation into to MSCs for tumor targeting.
[206] While the present disclosure describes various embodiments for illustrative purposes, such description is not intended to be limited to such embodiments. On the contrary, the applicant's teachings described and illustrated herein encompass various alternatives, modifications, and equivalents, without departing from the embodiments, the general scope of which is defined in the appended claims. Except to the extent necessary or inherent in the methods or processes themselves, no particular order to steps or stages of methods or processes described in this disclosure is intended or implied. In many cases the order of method or process steps may be varied without changing the purpose, effect, or import of the methods described.
[207] Information as herein shown and described in detail is fully capable of attaining the above- described object of the present disclosure, the presently preferred embodiment of the present disclosure, and is, thus, representative of the subject matter which is broadly contemplated by the present disclosure. The scope of the present disclosure fully encompasses other embodiments which may become apparent to those skilled in the art, and is to be limited, accordingly, by nothing other than the appended claims, wherein any reference to an element being made in the singular is not intended to mean "one and only one" unless explicitly so stated, but rather "one or more." All structural and functional equivalents to the elements of the above-described preferred embodiment and additional embodiments as regarded by those of ordinary skill in the art are intended to be encompassed by the present claims. Moreover, no requirement exists for a system or method to address each and every problem sought to be resolved or ameliorated by the present disclosure, for such to be encompassed by the present claims. Furthermore, no element, component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. However, that various changes and modifications in form, material, work-piece, and fabrication material detail may be made, without departing from the spirit and scope of the present disclosure, as set forth in the appended claims, as may be apparent to those of ordinary skill in the art, are also encompassed by the disclosure.