Compositions and methods for using non-human primate personalized genome assemblies and induced pluripotent stem cell lines for preclinical evaluation

Abstract

Described herein are methods of preclinical drug evaluation, including using a personalized genome assembly of a subject and induced pluripotent stem cells (iPSCs) generated from the subject. Additionally, a method of preclinical testing of a human therapeutic includes obtaining tissue or blood from at least one individual of a population that includes a single species of non-human primate, isolating PBMCs from the at least one individual, generating iPSCs from the PBMCs from the at least one individual, and performing one or more of in vitro, in vivo, or genetic tests on the at least one individual. Described herein are compositions for generating iPSCs from non-human primates.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 257,997, filed October 20, 2021, the contents of which are incorporated herein by reference in their entirety. Literature citations

[0002] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0003] The present disclosure relates to bioinformatics, efficacy, functionality, and toxicity of drugs and related chemical entities for preclinical testing. Additionally, the present disclosure relates to compositions and methods for using non-human primate personalized genome assemblies and induced pluripotent stem cell lines for preclinical evaluation. [Background technology]

[0004] In the pharmaceutical industry, preclinical studies help manufacturers determine whether a drug candidate can be safely tested in humans. Traditional preclinical testing relies heavily on animal testing. Typically, FDA policy requires that drug candidates be tested in rodents and non-rodents. Although there is no requirement that the non-rodents be primates, non-human primates (NHPs) are often used for preclinical testing because they are anatomically and physiologically similar to humans. Exposure of these primate subjects to the drug candidate helps determine the candidate's safety in humans. The use of non-human primates is extremely costly. In some cases, it can cost tens of thousands of dollars for a single animal just to sacrifice it in an experiment. Furthermore, there is a growing aversion to animal testing in general and NHP testing in particular.

[0005] Thus, there is a great need for testing methods that minimize the number of animals that must be sacrificed in preclinical trials. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been made to solve the problems in the conventional techniques described above. [Means for solving the problem]

[0007] In some aspects, the technology described herein relates to a method of generating induced pluripotent stem cells (iPSCs) from individuals within a population of non-human primates (NHPs), the method comprising obtaining peripheral blood mononuclear cells (PBMCs) from at least one individual of the population comprising a single species of non-human primate; culturing the PBMCs in a hematopoietic stem cell (HSC) expansion medium for a period of time to expand blood progenitor cells; and transfecting the cultured PBMCs with a combination of transcription factors such that the cells are induced to overexpress the transcription factors, wherein the transfected cultured PBMCs are reprogrammed into iPSCs, the transfecting step and the transfecting step are repeated at least twice. the step of transferring the transfected cells one day later into a container containing a plurality of feeder cells; transferring the cells to a second container only one day after the transferring step; performing at least one of adding medium to the cells, exchanging medium in the cells, and adding one or more cell growth factors to the cells a selected number of days after the transferring to the second container step; passaging the cells by placing each colony in a third container coated with an extracellular matrix until first cell colonies appear; and expanding the first cell colonies for use in one or more of in vitro testing, in vivo testing, or generation of a genomic library.

[0008] In some aspects, in methods related to the technology described herein, the expanding step includes washing the iPSCs with phosphate buffered saline (PBS) and incubating the iPSCs with a cell dissociation solution.

[0009] In some aspects, in methods related to the technology described herein, incubation occurs at about 37° C. for a period of time.

[0010] In some aspects, in methods related to the technology described herein, the expanding step comprises aspirating and dissociating the iPSCs into a single cell suspension.

[0011] In some aspects, methods related to the technology described herein further comprise differentiating the iPSCs into primary lineages, the primary lineages including at least one of ectoderm, mesoderm, and endoderm.

[0012] In some aspects, methods to which the technology described herein pertains further comprise differentiating primary lineages into end-point tissues.

[0013] In some aspects, methods related to the technology described herein further include generating an individualized genome assembly for the at least one individual.

[0014] In some aspects, methods to which the technology described herein pertains further comprise extracting large amounts of high molecular weight (HMW) DNA from the obtained PBMCs by a process that purifies archival quality DNA.

[0015] In some aspects, methods related to the technology described herein further include preparing a genomic library from the extracted HMW DNA, sequencing long read fragments of the HMW DNA, and assembling and annotating the long read fragments to reconstruct the genome of at least one individual from whom the PBMCs were obtained.

[0016] In some aspects, in methods related to the technology described herein, the transcription factor is selected from the group consisting of c-myc, Klf4, Sox2, Oct3 / 4, Klf2, Nanog, Tfcp2L1, and Stat3.

[0017] In some aspects, in methods related to the technology described herein, the plurality of feeder cells are mouse embryonic fibroblast (MEF) or SNL feeder cells.

[0018] In some aspects, in methods related to the technology described herein, the step of transferring the transfected cells to a vessel containing a plurality of feeder cells further comprises adding a tankyrase 1 / 2 inhibitor in the range of about 1 μM to about 3 μM.

[0019] In some aspects, in methods involving the technology described herein, the ratio of transfected cells to the plurality of feeder cells is about 1:3 to about 1:7.

[0020] In some aspects, in methods related to the technology described herein, the transfecting step uses a Sendai virus vector.

[0021] In some aspects, in methods related to the technology described herein, the one or more cell growth factors comprises basic fibroblast growth factor.

[0022] In some aspects, in methods related to the technology described herein, passaging the cells by placing each colony in a third container coated with extracellular matrix includes adding a serine / threonine kinase inhibitor to the medium in the third container.

[0023] In some aspects, the technology described herein relates to a method of preclinical drug evaluation, the method comprising an individualized genome assembly of a subject and induced pluripotent stem cells (iPSCs) generated from the subject.

[0024] In some aspects, methods related to the technology described herein further include transplanting cells or tissue generated from iPSCs from the subject into the subject and assessing functionality of the transplanted cells or tissue in the subject.

[0025] In some aspects, methods related to the technology described herein further include generating one or more panels and predicting whether a particular population of animals is suitable for drug testing based on the one or more panels.

[0026] In some aspects, methods related to the technology described herein further include generating a guide RNA or a gene editing tool using iPSCs generated from the subject, and performing in vivo testing of the guide RNA or gene editing tool in the subject.

[0027] In some aspects, the technology described herein is a method for preclinical testing of a human therapeutic, the method including obtaining tissue or blood from at least one individual of a population comprising a single species of non-human primate, isolating PBMCs from the at least one individual, generating iPSCs from the PBMCs from the at least one individual, extracting DNA from the PBMCs from the at least one individual, preparing a DNA library using, at least in part, the extracted DNA, preparing a whole genome assembly of the at least one individual, and performing a computational analysis of the whole genome assembly.

[0028] In some aspects, in methods related to the technology described herein, the computer analysis includes identifying one or both of major histocompatibility complex associated genes or minor histocompatibility complex associated genes.

[0029] In some aspects, in methods related to the technology described herein, the computer analysis includes identifying immune system related genes.

[0030] In some aspects, in methods related to the technology described herein, the computer analysis includes identifying genes associated with an anti-drug antibody (ADA) response.

[0031] In some aspects, in methods related to the technology described herein, the computational analysis comprises performing preclinical genotyping.

[0032] In some aspects, in methods related to the technology described herein, the computer analysis includes identifying liver toxicity-associated genes.

[0033] In some aspects, in methods involving the technology described herein, the DNA is high molecular weight DNA.

[0034] In some aspects, in methods involving the technology described herein, the DNA is archival quality DNA.

[0035] In some aspects, in methods related to the technology described herein, preparing a DNA library includes performing long-read sequencing on the extracted DNA.

[0036] In some aspects, in methods related to the technology described herein, preparing a whole genome assembly comprises recreating the genome of the at least one individual based on the prepared DNA library.

[0037] In some aspects, the technology described herein relates to a method for preclinical testing of a human therapeutic comprising obtaining tissue or blood from at least one individual of a population comprising a single species of non-human primate, isolating PBMCs from the at least one individual, generating iPSCs from the PBMCs from the at least one individual, performing in vitro testing on the iPSCs generated from the at least one individual, deriving in vitro test results from the in vitro testing, and correlating the in vitro test results with the genotype of the at least one individual.

[0038] In some aspects, in methods related to the technology described herein, the step of conducting an in vitro test comprises designing a guide RNA and transfecting the guide RNA into iPSCs in vitro.

[0039] In some aspects, in methods related to the technology described herein, deriving in vitro test results comprises running a panel to detect gene editing efficiency of guide RNAs in vitro in iPSCs.

[0040] In some aspects, the technology described herein relates to a method for preclinical testing of human therapeutics, the method comprising obtaining tissue or blood from at least one individual of a population comprising a single species of non-human primate, isolating PBMCs from the at least one individual, generating iPSCs from the PBMCs from the at least one individual, performing an in vivo test in the at least one individual, deriving an in vivo test result from the in vivo test, and correlating the in vivo test result of the in vivo test with the genotype of the at least one individual.

[0041] In some aspects, in methods related to the technology described herein, generating iPSCs includes culturing PBMCs in hematopoietic stem cell (HSC) expansion medium for a period of time to expand blood progenitor cells; transfecting the cultured PBMCs with a combination of transcription factors such that the cells are induced to overexpress the transcription factors, whereby the transfected cultured cells are reprogrammed into iPSCs; and transferring the transfected cells to a vessel containing a plurality of feeder cells no more than one day after the transfecting step. transferring the cells to a second container no more than one day after the transferring step; at least one of adding medium to the cells, exchanging medium in the cells, and adding one or more cell growth factors to the cells a selected number of days after the transferring to the second container step; passaging the cells by placing each colony in a third container coated with extracellular matrix until first cell colonies appear; and expanding the first cell colonies for use in one or more of in vitro testing, in vivo testing, or generation of a genomic library.

[0042] In some aspects, in methods related to the technology described herein, performing the in vivo testing comprises autologously transplanting or injecting a subset of iPSCs into the at least one individual.

[0043] In some aspects, methods to which the technology described herein pertains further comprise labeling the iPSCs prior to autotransplantation or autoinjection.

[0044] In some aspects, in methods related to the technology described herein, labeling comprises fluorescent labeling.

[0045] In some aspects, in methods related to the technology described herein, deriving in vivo test results comprises running a panel to detect anti-drug antibodies.

[0046] In some aspects, in methods related to the technology described herein, deriving in vivo test results includes running a panel to detect liver toxicity.

[0047] In some aspects, the technology described herein relates to a method of preclinical testing of a human therapeutic comprising obtaining tissue or blood from at least one individual of a population comprising a single species of non-human primate, isolating PBMCs from the at least one individual, generating iPSCs from the PBMCs from the at least one individual, performing in vitro testing on a subset of iPSCs generated from the at least one individual, deriving in vitro test results from the in vitro testing, auto-injecting or auto-transplanting a second subset of iPSCs generated from the at least one individual into the at least one individual, performing in vivo testing in the at least one individual, deriving in vivo test results from the in vivo testing, and correlating the in vivo and in vitro test results.

[0048] In some aspects, in methods related to the technology described herein, generating iPSCs includes culturing PBMCs in hematopoietic stem cell (HSC) expansion medium for a period of time to expand blood progenitor cells; transfecting the cultured PBMCs with a combination of transcription factors such that the cells are induced to overexpress the transcription factors, whereby the transfected cultured cells are reprogrammed into iPSCs; and transferring the transfected cells to a vessel containing a plurality of feeder cells no more than one day after the transfecting step. transferring the cells to a second container no more than one day after the transferring step; at least one of adding medium to the cells, exchanging medium in the cells, and adding one or more cell growth factors to the cells a selected number of days after the transferring to the second container step; passaging the cells by placing each colony in a third container coated with extracellular matrix until first cell colonies appear; and expanding the first cell colonies for use in one or more of in vitro testing, in vivo testing, or generation of a genomic library.

[0049] In some aspects, in methods related to the technology described herein, performing the in vitro testing comprises designing a guide RNA and transfecting the guide RNA into a subset of iPSCs in vitro.

[0050] In some aspects, in methods related to the technology described herein, deriving in vitro test results comprises running a panel to detect in vitro gene editing efficiency of guide RNAs in a subset of iPSCs.

[0051] In some aspects, methods related to the technology described herein further comprise transfecting iPSCs with guide RNA for autologous transplantation or autologous injection.

[0052] In some aspects, in methods related to the technology described herein, deriving in vivo test results includes running a panel to detect liver toxicity.

[0053] In some aspects, in methods related to the technology described herein, deriving in vivo test results comprises running a panel to detect in vivo gene editing efficiency of guide RNAs in a second subset of iPSCs.

[0054] In some aspects, in methods related to the technology described herein, the correlating step includes comparing in vitro gene editing efficiency with in vivo gene editing efficiency.

[0055] In some aspects, in methods related to the technology described herein, performing the in vitro testing comprises treating the iPSCs in vitro with a therapy.

[0056] In some aspects, in methods related to the technology described herein, deriving in vitro test results comprises running a panel to detect in vitro toxicity of the therapy.

[0057] In some aspects, in methods related to the technology described herein, conducting an in vivo test result comprises treating the at least one individual with the therapy.

[0058] In some aspects, in methods related to the technology described herein, deriving in vivo test results includes running a panel to detect in vivo toxicity of the therapy.

[0059] In some aspects, in methods related to the technology described herein, the correlating step includes comparing the in vitro toxicity with the in vivo toxicity of the therapy.

[0060]

[0023] As this is a summary, details are by definition limited. The above aspects, as well as other aspects, features, and advantages of the present technology, will now be described with reference to various embodiments and with reference to the accompanying drawings.

[0061] The illustrated embodiments are examples only and are not intended to limit the disclosure.The drawings are intended to illustrate features and concepts and are not necessarily drawn to scale. [Brief description of the drawings]

[0062] [Figure 1] FIG. 1 is a schematic diagram illustrating one embodiment of a method for preclinical testing of human therapeutics using induced pluripotent stem cells (iPSCs) from non-human primates, genome assembly, and associated genomic computing. [Diagram 2] FIG. 1 is a schematic showing the differentiation of primate iPSCs into various tissues and cells for the purpose of evaluating the efficacy, functionality, and / or toxicity of candidate therapies in vitro and subsequently in the same non-human primate from which the iPSCs were generated. [Figure 3A] 1 is a flow chart illustrating one embodiment of a method for reprogramming non-human primate peripheral blood mononuclear cells (PBMCs) into iPSCs. [Figure 3B] FIG. 1 is a schematic diagram of one embodiment of a method for reprogramming non-human primate peripheral blood mononuclear cells (PBMCs) into iPSCs. [Figure 4] FIG. 1 is a schematic diagram showing one embodiment of a method for in vitro testing of drugs on tissues and / or cells generated from non-human primate iPSCs prior to evaluating the efficacy, functionality, or safety of a therapeutic in vivo in the same non-human primate from which the iPSC cells and / or tissues were generated. [Diagram 5]FIG. 1 is a schematic diagram of one embodiment of a method for in vitro testing of gene therapy products on iPSCs or cells generated from iPSCs to assess efficacy, functionality, and / or toxicity prior to in vivo testing in the same non-human primate from which the iPSC cells and / or tissues were generated. [Figure 6A] FIG. 1 is a schematic diagram showing one embodiment of a method for autologous transplantation of iPSCs or cells and / or tissues generated from iPSCs into the same non-human primate from which the iPSCs were generated, as a model for preclinical testing of cell and / or gene therapy methods in humans. [Figure 6B] 1 shows labeling of iPSCs for further studies. [Figure 6C] 1 shows gene editing in iPSC cells or tissues. [Figure 7A] 4x magnification of macaque iPSCs generated using conventional compositions and methods developed for human iPSCs. [Figure 7B] 10x magnification of macaque iPSCs generated using conventional compositions and methods developed for human iPSCs. [Figure 7C] 4X magnification of macaque iPSCs generated using the compositions and methods for non-human primate iPSCs described herein. [Figure 7D] 10x magnification of macaque iPSCs generated using the compositions and methods for non-human primate iPSCs described herein. [Figure 7E] 1 is a graph showing a comparison of the efficiency of iPSC generation using conventional compositions and methods developed for human iPSCs versus the compositions and methods described herein for non-human primate iPSCs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] The above is a summary, and therefore, by definition, limited in detail. The above aspects, as well as other aspects, features, and advantages of the technology, will now be described with reference to various embodiments. The inclusion of the following embodiments in this specification is not intended to limit the disclosure to those embodiments, but rather to enable one of ordinary skill in the art to make and use the contemplated embodiments. Other embodiments may be utilized, and modifications may be made without departing from the spirit or scope of the subject matter. The aspects of the disclosure described and illustrated herein may be arranged, combined, modified, and designed in various ways, all of which are expressly contemplated and part of the disclosure.

[0064] Traditionally, in vitro testing is performed on human cell lines, followed by basic in vivo testing in rodents such as mice and rats. Correlation of mouse results with human results is difficult and unpredictable. This is because mice and humans share at least about 70% of the same protein gene sequences, and these genes make up about 1.5% of their respective genomes. After rodent testing, which may or may not represent how the human body will react, testing moves to non-human primates. However, because of potential discrepancies between human cell line testing and rodent in vivo testing, a large number of non-human primates are typically used. According to FDA regulations, it is often not possible to jump from rodent in vivo testing to human in vivo testing in a single step.

[0065] Furthermore, according to a report from the United States Department of Agriculture, 820,812 animals were used in research in the United States in 2016, with over 71,000 of these animals being non-human primates. Furthermore, the use of non-human primates for vaccine development has increased significantly during the 2020 COVID-19 pandemic, resulting in a shortage of animals worldwide. The main cause of this shortage is the fact that China, the main supplier of non-human primates to the pharmaceutical industry in the United States and other countries, stopped exporting NHPs to the United States in 2020. Furthermore, there are growing concerns about the use of NHPs in biomedical research.

[0066] Thus, new methods are needed to improve the correlation between in vitro and in vivo results, to increase the likelihood of correlation between preclinical trials and human clinical trials, and to reduce the use of non-human primates.

[0067] Several groups have attempted to use human in vitro iPSC models to generate and combine in vitro and in silico data for drug discovery. However, methods based on human iPSC in vitro systems lack in vivo data from humans to verify the accuracy of in vitro test results. Using personalized iPSCs from NHPs that match each animal allows for a personalized in vitro-in vivo combined approach that is not currently possible using human iPSCs. Although non-human primates share about 95% genomic similarity with humans, they are not a perfect model for humans. However, this combined approach is useful for drug discovery because it is possible to combine both in vitro data from NHP iPSCs and tissues and in vivo data from NHPs (which, unlike humans, can be used for in vivo experiments).

[0068] In addition, several research groups are studying the process of reprogramming and iPSC generation to understand the process of rejuvenation and aging using human cells and mice. The process of iPSC generation in NHPs is intriguing and not as simple as that in humans and mice. In addition, different species of non-human primates have different life spans. For example, chimpanzees live up to about 55 years, and macaques live up to about 30 years. Conducting comparative studies of iPSCs among chimpanzees, humans, macaques, and other NHPs will help us understand the process of aging and rejuvenation.

[0069] In general, the methods described herein improve upon conventional methods for preclinical evaluation of drugs and therapeutics in non-human primate models prior to FDA approval. For example, the described methods include using non-human primate induced pluripotent stem cells (iPSCs), using personalized genome assemblies from the same animals from which the iPSCs were generated, and performing in vivo testing for preclinical evaluation on the same animals from which the iPSCs and personalized genome assemblies were generated, thereby increasing the correlation of results between in vitro and in vivo testing, improving genetic mapping of safety and therapeutic response, which leads to more selective (higher correlation focused) drug research and a gradual reduction in the use of non-human primates compared to conventional methods for preclinical evaluation of drugs and therapeutics in non-human primate models.

[0070] In some embodiments, the methods described herein may provide the technical effect and / or advantage of avoiding the problem of immune rejection because experimental designs may be made to utilize iPSCs, individualized genomes, and autologous transplantation of tissues and cells in specific and individualized non-human primates (NHPs). For example, cells and tissues are derived from a specific animal's own cells at various stages of testing, so that valid conclusions can be drawn and / or valid comparisons can be made that may better inform human testing and anticipated human responses.

[0071] As used herein, the terms drug, therapeutic agent, pharmaceutical, gene therapy, genome editing tool, treatment, and the like may be used interchangeably in that the methods described herein may be used for the preclinical evaluation of any of these and others.

[0072] Practical Applications Practical applications of the methods described herein include improved methods for preclinical evaluation of Good Laboratory Practice (GLP), efficacy, functionality, and drug safety, anti-drug antibody (ADA) responses and associated genetics, genome editing methods including efficacy and functionality, toxicity testing of gene therapy using iPSCs and / or cells or tissues generated from iPSCs, immunotherapy and cell therapy including chimeric antigen receptor (CAR) T cells for cancer treatment, monoclonal antibodies for cancer and other non-infectious or infectious diseases (which provides a rapid and non-terminal method of interrogating the status of genetically validated targets), and iPSC labeling for imaging. Various practical applications of the compositions and methods may be further described elsewhere herein.

[0073] The methods described herein are believed to be particularly applicable to the preclinical evaluation of human drugs, therapies, pharmaceuticals, and the like, because human DNA is, on average, 96% identical to the DNA of our most distant primate relatives, and approximately 99% identical to our closest relatives, chimpanzees and bonobos.

[0074] During drug development, from preclinical to post-marketing stages, evaluations of a drug's level of safety are made. Measures of safety usually refer to the type and amount of adverse events associated with the use of the product compared to expected rates. Good Laboratory Practices (GLP) are designed to facilitate the development of quality test data. GLP provides tools and methodologies to ensure the right approach to controlling laboratory testing during the preclinical stages of drug development.

[0075] To address issues regarding GLP safety measures, non-human primate genomes may be used to establish possible correlations between genotypes and safety of drugs during testing in the same non-human primates. The assembled genomic information may determine whether a particular genotype of a non-human primate is associated with the development of drug resistance, drug toxicity, and anti-drug antibodies (ADA) in the same NHP, as well as potential issues with drugs and / or therapies in the same NHP. The assembled genomic information may enable the creation of gene panels (e.g., genetic assays for detecting one or more gene mutations or genetic markers), which may be used to predict whether a new colony of animals is suitable for testing a particular drug and / or therapy. As an example, there is the issue of animals developing ADA during preclinical testing of drugs. Even if animals are screened for existing antibodies against the drug of interest, a certain population of animals still develop ADA during preclinical testing. Genetic panels based on the whole genome of animals for immunoglobulin receptor regions, major histocompatibility complex (MHC), minor histocompatibility complex (MiHC), killer cell immunoglobulin-like receptors (KIR), and other immune genes would be useful to exclude animals with ADA during preclinical testing. Furthermore, iPSC lines personalized for each NHP in the colony are suitable tools to evaluate the safety of drugs and / or therapies in vitro pre-GLP and / or before using the drugs and / or therapies in vivo, thereby reducing the number of NHPs used.

[0076] As shown in FIG. 2, iPSCs 200 from NHPs 210 can be differentiated by the methods described herein into the major lineages, i.e., ectoderm 240, mesoderm 230, and endoderm 220, and then into end-state tissues, i.e., liver 250, pancreas 260, neurons 270, cardiomyocytes 280, hematopoietic system, immune cells, etc. Thus, drug safety may be first evaluated through in vitro assays on tissues generated from individualized animal iPSCs and then evaluated in vivo on the same animal. The methods described herein may be used to perform pre-analysis steps (e.g., testing) on ​​iPSC-derived cells and tissues that are highly homologous to humans and directly related to the individual animal under safety evaluation. That is, performing testing and / or pre-analysis of such extracted aspects of individual animals and then performing experiments using the same animals is advantageous over conventional systems of performing in vitro testing of a first species to evaluate safety, etc., performing in vivo testing of a second species to evaluate safety, etc., and then performing clinical studies of a target species (e.g., human) different from the first and second species. One example of an advantage of the method described herein is that data is generated that provides a direct correlation between the iPSCs being pre-analyzed, the animal being experimented on, and the genetic characteristics of the same animal. This is because the exact same individual animals and their respective genomes are used for both steps. In other words, using the exact same individual animals for in vitro and in vivo evaluations and genetic relationships provides a direct correlation between pre-analysis data and experimental outcome data, which can be directly related to humans in the case of high homology between humans and NHPs. Examples of extracted aspects include efficacy, functionality, and safety measures associated with iPSC-derived cells and tissues generated from personalized iPSCs. In particular, such aspects are predictive of efficacy, functionality, and safety measures established in the same animal prior to in vivo testing in that same animal.

[0077] As shown in FIG. 6B, cells and tissues generated from the iPSC line 650 (e.g., cells or tissues) of each individual animal may be labeled with fluorescent proteins or other cell labeling methods to track within the animal during autologous transplantation. Cells and tissues generated from iPSCs may be labeled as shown in FIG. 2 and autologous transplantation or injection into the same animal is a practical tool to evaluate the functionality of the cells as shown in FIG. 6A. In some embodiments, a preclinical test method using the methods and compositions described herein includes autologous transplantation of labeled cells and tissues generated from iPSCs from a first NHP into the first NHP. The preclinical test method may further include imaging the cells. Imaging may be performed to identify trafficking patterns, localization, efficacy, cell or tissue viability, and other parameters. Labeling and imaging of cells and tissues derived from iPSCs transplanted into the same animal may be performed to analyze the effect of treatments being tested on the NHP. For example, labeling and imaging allows transplanted cells to be isolated after a period of time, and allows cells to be tracked in vivo, for example using fluorescence activated cell sorting, to assess cell viability, among other assessments. By using the same non-human primate during various steps of preclinical treatment testing, it is possible to ensure a 1:1 evaluation and / or correlation regarding safety, efficacy, etc. Furthermore, by using the same non-human primate during various steps of preclinical treatment testing, it is possible to result in fewer NHPs being used.

[0078] Methods and Compositions overview Generally, methods are described herein for generating induced pluripotent stem cells (iPSCs) from non-human primates, including, for example, cynomolgus monkeys, pig-tailed macaques (e.g., Macaca nemestrina, Macaca leonine, and Macaca sylvanusi), baboons, African green monkeys, rhesus monkeys, chimpanzees, and the like.

[0079] Generally, the present specification describes methods for sequencing the genomic DNA of an individualized non-human primate, as well as de novo assembly of the sequenced genomic fragments of the non-human primate to obtain an individualized reference genome for the individual non-human primate. For example, assembly of the genomic fragments can include using long-read technologies, but can also include short-read technologies, as described elsewhere herein.

[0080] As used herein, media may include, but are not limited to, DMEM media, RPMI™ media, F12™ media, etc. In some embodiments, specialty media are detailed below or elsewhere herein.

[0081] One embodiment of the method of preclinical evaluation includes generating induced pluripotent stem cells (iPSCs) from at least one individual of a population that includes a single species of non-human primate, and generating an individual reference genome for the at least one individual. In such an embodiment, the same at least one individual (i.e., non-human primate) is used in a subsequent preclinical study, and the in vivo results from the same non-human primate are compared with the in vitro results of iPSCs from the same non-human primate. The answer obtained by this method is that the use of iPSCs and a reference genome from the same non-human primate provides a solid foundation for preclinical studies that reduces the risk level of in vivo testing.

[0082] 1 is a schematic diagram illustrating one embodiment of a method for preclinical testing of human therapeutics using non-human primate (NHP) iPSCs, genome assembly, and associated genomic analysis and / or genomic computation. For example, method 100 may include obtaining tissue or blood from a first non-human primate (110), isolating PBMCs from the first non-human primate (120), generating iPSCs from the PBMCs from the first non-human primate (130), extracting DNA from the PBMCs from the first non-human primate (140), preparing a DNA library 150 using at least some DNA sequencing method (160), preparing a whole genome assembly of the first non-human primate (170), and optionally (shown in dashed lines) performing computational analysis (180). One example of genomic analysis is in the field of gene therapy preclinical trials. In the process of gene therapy, an individualized genome is desirable for designing guide RNA (gRNA) to prevent off-target effects and for precise insertion of target genes into target loci using AAV vectors by precise design of homology arms in the AAV vector. A personal genome of NHPs matched to iPSCs from the same animal would be an ideal tool for preclinical testing of gene therapy and other cell therapies (e.g., CAR-T cells, which require personalized genomic information such as T cell receptor information). The computer analysis may include identification of major histocompatibility complex genes and / or minor histocompatibility complex genes, and / or immune system related genes. The computer analysis may also include anti-drug antibody (ADA) response genotyping. The analysis may also include performing preclinical genotyping. In an exemplary embodiment, the method may further include performing in vitro testing on the iPSCs generated from the first non-human primate and correlating the results of the in vitro testing with the genotype of the first non-human primate. One example is liver toxicity in animals and humans due to AAV vectors or other cellular gene and therapeutic methods.An initial test to predict the level of immunogenicity of the AAV vector to hepatocytes or immune cells (e.g., PBMCs) generated from iPSCs in vitro may be used as a predictor of liver toxicity in NHPs in vivo. In vivo liver toxicity may be predicted based on correlating the in vitro results with the genotype of the same NHP. In one embodiment, the method may include performing an in vivo test in a first non-human primate and correlating the results of the in vivo test with the results of the in vitro test and / or the genotype of the first non-human primate.

[0083] Generation of non-human primate iPSCs 3A illustrates one embodiment of generating iPSCs from PBMCs of a non-human primate. Method 700 includes obtaining peripheral blood mononuclear cells (PBMCs) from at least one individual of a population that includes a single species of non-human primate (S710), culturing the PBMCs in hematopoietic stem cell (HSC) expansion medium for a predetermined period of time to expand blood progenitor cells (e.g., including CD34+ cells) (S720), transfecting the cultured PBMCs to reprogram the cultured cells into iPSCs (S730), and transferring the transfected cells to a container that includes a plurality of feeder cells. The method includes the steps of: (S740), transferring the cells to a second container (S750), and after a selected number of days from transfer to the second container, at least one of adding medium to the cells, replacing medium in the cells, or adding one or more cell growth factors to the cells (S760), and upon appearance of first cell colonies, passaging the cells by placing each colony in a third container coated with extracellular matrix (S770).

[0084] Method 700 may further include expanding the first colony for use in one or more of in vitro testing, in vivo testing, or creating a genomic library for the individual primate.

[0085] In some embodiments, the transfecting step includes transfecting a combination of transcription factors such that the cells are induced to overexpress the transcription factors. For example, the transcription factors may include c-myc, KLf4, Sox2, or Oct3 / 4. Additional or alternative transcription factors may be used and are within the scope of the present disclosure. For example, additional or alternative transcription factors such as Klf2, Nanog, Tfcp2L1, and Stat3 may be used.

[0086] In some embodiments, the expanding step may include washing the iPSCs with a buffer (e.g., phosphate buffered saline) and incubating the iPSCs with a cell dissociation solution (e.g., trypsin, a chelating agent, collagenase, etc.). In some embodiments, the incubation is at about 37° C. for a predetermined time period. The predetermined time period may be about 30 seconds to about 10 minutes, about 1 minute to about 60 minutes, about 1 minute to about 5 minutes, etc.

[0087] In some embodiments of method 700, the step of transferring the transfected cells to a container with feeder cells occurs no more than about one day after transfection. The feeder cells may include mouse embryonic fibroblasts (MEFs), SNL feeder cells, and the like.

[0088] In some embodiments, the step of transferring the cells to the second container occurs no more than about one day after transfer to the container with the feeder cells.

[0089] In some embodiments, the expanding step comprises aspirating and dissociating the iPSCs into a single cell suspension.

[0090] In one application of method 700, as shown in Figure 2, the iPSCs are capable of differentiating into primary lineages. The primary lineages may include at least one of ectoderm 240, mesoderm 230, and endoderm 220. Additionally or optionally, the primary lineages are capable of further differentiating into terminal tissues. The terminal tissues may be used for in vitro testing, may be autografted into a first non-human primate for in vivo testing, and / or may be used for genomic library generation.

[0091] Method 700 may further include generating an individualized genome assembly of at least one individual that is the same individual from whom the PBMCs were harvested or obtained. Generating the genome assembly may include extracting a large amount of high molecular weight (HMW) DNA from the obtained PBMCs by a process that purifies archival quality DNA, as described elsewhere herein, sequencing long read fragments of the HMW DNA, and assembling and annotating the long read fragments to recreate the genome of the at least one individual from whom the PBMCs were obtained.

[0092] In one embodiment, a method of preclinical drug evaluation may include an individualized genome assembly of a subject and generating induced pluripotent stem cells (iPSCs) from the subject. Cells and / or tissues generated from the iPSCs from the subject may be transplanted into the subject to assess the functionality of the cells in the subject.

[0093] In one embodiment, one or more panels may be generated to predict whether a particular animal or a particular population of animals is suitable for a particular drug experiment based on one or more panels, for example, these panels include small molecules or gene therapy AAV vectors tested on tissues (e.g., hepatocytes) and / or primary cells (e.g., PBMCs) generated from iPSCs.

[0094] In one embodiment, cells and / or tissues generated from iPSCs from a subject may be used to generate guide RNAs or gene editing tools. Guide RNAs or gene editing tools may be used to perform in vivo testing of the guide RNAs or gene editing tools in the subject.

[0095] 3B illustrates one embodiment of a method 300 for generating iPSCs from PBMCs of a non-human primate. For example, peripheral blood mononuclear cells (PBMCs) are obtained from an individual of a non-human primate of some kind, and the PBMCs are separated from the whole blood of the animal. Separating the PBMCs from the whole blood of the animal may be performed using density gradient centrifugation (DGC), fluorescence activated cell sorting, magnetic bead-based separation, buoyancy activated cell sorting (BACS), and the like. In one embodiment, the PBMCs are separated by DGC. In one embodiment, the PBMCs are separated by BACS.

[0096] In some embodiments, a method 300 of reprogramming PBMCs of an NHP into iPSCs includes culturing PBMCs to expand blood progenitor cells (e.g., CD34+ cells) in hematopoietic stem cell (HSC) expansion medium, such as StemSpan™ (Stemcell Technologies Inc., Vancouver, BC, Canada), for a period of about 3 to about 10 days, about 5 to about 10 days, about 6 to about 10 days, about 7 to about 10 days, about 8 to about 10 days, or about 9 days (310). In some embodiments, alternatives to StemSpan™ may be used. For example, compositions for expansion of non-human primate hematopoietic stem and / or progenitor cells (HSPCs) (e.g., prior to transfection of reprogramming transcription factors) include IL3, IL6, FLT-3, TPO, and SCF. In some embodiments, animal serum (e.g., fetal bovine serum) in the expansion medium may be replaced with or combined with polyvinyl alcohol (PVA) to increase the efficiency of expansion of HSPCs from non-human primates.CD34 is not only expressed by HSCs, but also by many other non-hematopoietic cell types, such as muscle satellite cells, keratocytes, stromal cells, epithelial progenitor cells, and endothelial progenitor cells.Thus, multiple different cell types can potentially express progenitor cell activity and can be expanded.

[0097] Further, the method 300 of reprogramming PBMCs to iPSCs may include a step of transfecting blood progenitor cells with transcription factors (320). PBMCs of an NHP are reprogrammed into NHP induced pluripotent stem cells (iPSCs). The method of reprogramming PBMCs to iPSCs may include a step of transfecting PBMCs with one or more transcription factors such that the cells are induced to overexpress the transcription factors. Transcription factors are a group of proteins that read and interpret DNA. They bind to DNA and help initiate a program of increased or decreased gene transcription. Stem cell induction protocols are specifically optimized for non-human primates, as various approaches are taken to generate stem cells optimized for non-human primates. In some embodiments, the transcription binding sites of these transcription factors may be mapped on the individualized primate genome to increase the efficiency of the reprogramming process in NHPs, especially since the transcription binding sites in the human genome are not entirely conserved in NHP species. Thus, the use of human iPSC reprogramming kits is not efficient for iPSC generation in NHP species, given the fact that the NHP genome is highly diverse and pre-existing mutations within the NHP genome cause inefficient binding of transcription factors (from human iPSC generation kits) in non-human primate cells.

[0098] A variety of transfection methods may be used, including, but not limited to, electroporation, calcium phosphate exposure, liposome-based transfection, viral-mediated transfection (also referred to as transduction), and the like. In one embodiment, the method of transfecting PBMCs includes transduction. Transduction can be accomplished, for example, using a Sendai virus vector, an adenovirus vector, an oncoretrovirus vector, or a lentivirus vector. In some variations, transfection includes the use of a Sendai virus vector. In some cases, transfecting PBMCs includes liposome-mediated transfection. For example, the transcription factors may include c-myc, KLf4, Sox2, or Oct3 / 4. Additional or alternative transcription factors may be used and are within the scope of the present disclosure.

[0099] Further, the method of reprogramming PBMCs to iPSCs may include a step of co-culturing the cells (e.g., transfected and untransfected cells) with feeder cells (330). The feeder cells may include mouse embryonic fibroblasts (MEFs), SNL feeder cells, etc. The ratio of transfected cells to feeder cells may be about 1:2 to about 1:8, about 1:3 to about 1:7, about 1:4 to about 1:6, e.g., about 1:5. The co-culturing may be performed at a later date after transfection, e.g., about 20 hours to about 36 hours, about 18 hours to about 30 hours, about 18 hours to about 36 hours, about 22 hours to about 26 hours, etc. after transfection.

[0100] Co-culture may be performed in Essential-8™ medium (Thermofisher Scientific). In one embodiment, the Essential-8™ medium may include a tankyrase 1 / 2 inhibitor. The tankyrase 1 / 2 inhibitor may be present in the medium at a concentration of about 1 μM to about 3 μM, about 1.5 μM to about 2.5 μM, about 1 μM to about 2.5 μM, about 1.5 μM to about 3 μM, about 1.75 μM to about 2.25 μM, etc. In some embodiments, the tankyrase 1 / 2 inhibitor is capable of maintaining NHP iPSCs in a pluripotent state and significantly reducing spontaneous differentiation of NHP iPSCs.

[0101] Further, the method 300 of reprogramming PBMCs to iPSCs may include a step of transferring the non-adherent cells to new medium. For example, the transferring step may be performed after about 24 hours to about 72 hours, about 18 hours to about 78 hours, about 18 hours to about 30 hours, about 30 hours to about 42 hours, about 42 hours to about 52 hours, about 52 hours to about 66 hours, about 66 hours to about 78 hours, etc. of co-culture. The non-adherent cells may be cultured in a basal medium, for example, MEM medium, DMEM medium, RPMI medium, F12™ medium, etc. Further, the method 300 of reprogramming PBMCs to iPSCs may optionally include a step (340) of adding medium to the adherent cells. The added medium may include a feeder-free and xeno-free medium that supports the reprogramming of somatic cells as well as the spontaneous or induced differentiation of pluripotent stem cells (PSCs). For example, the medium to be added may be Essential-6® medium (Thermo Fisher Scientific). In one embodiment, the medium may be added about 48 hours to about 96 hours, about 42 hours to about 102 hours, etc. after transfection.

[0102] Further, the method 300 of reprogramming PBMCs to iPSCs may optionally include a step of replacing the medium on the adherent cells. The medium replacement may be performed about 90 hours to about 144 hours, about 96 hours to about 144 hours, about 90 hours to about 126 hours, about 114 hours to about 126 hours, etc. after transfection. The medium to be replaced may be a feeder-free and xeno-free medium that supports the reprogramming of somatic cells as well as spontaneous or induced differentiation of pluripotent stem cells (PSCs). For example, the medium to be added may be Essential-6® medium (Thermo Fisher Scientific).

[0103] Further, the method 300 of reprogramming PBMCs to iPSCs may optionally include a step (350) of replacing the medium with a medium comprising one or more growth factors. For example, the medium may comprise a feeder-free and xeno-free medium that supports the reprogramming of somatic cells as well as spontaneous or induced differentiation of pluripotent stem cells (PSCs). For example, the supplemented medium may be Essential-6® medium (Thermo Fisher Scientific). The growth factors may be basic fibroblast growth factor, DJ-1, epidermal growth factor, and the like. In one embodiment, the growth factor comprises basic fibroblast growth factor. The concentration of the growth factor (i.e., bFGF) may be about 100 ng / μL to about 200 ng / μL. In some embodiments, this particular growth factor and / or concentration is advantageous for NHP iPSC generation process since commercially available human bFGF is not as efficient in cross-reactivity with NHP cells. The medium change may be performed about 144 hours to about 192 hours, about 160 hours to about 250 hours, about 162 hours to about 174 hours, about 188 hours to about 198 hours, about 210 hours to about 222 hours, about 232 hours to about 246 hours, etc. after transfection. The medium change may be repeated daily for one or more days or for multiple days, for example, once, twice, three times, or more.

[0104] Additionally, the method 300 of reprogramming PBMCs to iPSCs may optionally include a step (380) of performing a medium change daily for one or more or multiple days. For example, the medium change may occur between about 240 hours post-transfection and about 440 hours post-transfection. The medium may be changed to basal medium, Essential-6®, Essential-8®, medium containing transforming growth factor-b (TGF-b) and / or b-FGF, or a similar medium. In some embodiments, the medium may be changed daily until first colonies of iPSCs are visually identified.

[0105] Further, the method 300 of reprogramming PBMCs to iPSCs may include culturing (370) a first colony of iPSCs on a matrix (e.g., extracellular matrix, Matrigel®, Cultrex®, Geltrex®, etc.). The first colony may be cultured in a medium containing a serine / threonine kinase inhibitor. For example, the medium may contain Essential-8®. The serine / threonine kinase may include a Rho inhibitor or a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., thiazovivin (TZV) or similar product). The concentration of the inhibitor may be from about 1 μM to about 3 μM.

[0106] Additionally, the method 300 of reprogramming PBMCs to iPSCs may include passaging the cells after about 12 hours to about 48 hours. The passaging step (360) may include refreshing the medium and adding a Wnt pathway inhibitor, tankyrase 1 / 2 inhibitor, etc. The inhibitor may have a concentration of about 1 μM to about 3 μM, about 1.5 μM to about 2.5 μM, about 1 μM to about 2.5 μM, about 1.5 μM to about 3 μM, about 1.75 μM to about 2.25 μM, etc.

[0107] Optionally, passaging and / or expansion of iPSCs may be performed by washing with a buffer (e.g., phosphate buffered saline) and incubating the cells with a cell dissociation solution, which may include collagenase (or a recombinant enzyme thereof), trypsin (or a recombinant enzyme thereof), a chelating agent (e.g., ethylenediaminetetraacetic acid), and the like.

[0108] Optionally, the iPSCs may be expanded or maintained by culturing in medium on feeder cells or on an extracellular matrix as described elsewhere herein. The medium may include a serine / threonine kinase, which may include a Rho inhibitor or a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor. The inhibitor may have a concentration of about 1 nM to about 3 nM, about 1.5 μM to about 2.5 μM, about 1 μM to about 2.5 μM, about 1.5 μM to about 3 μM, about 1.75 μM to about 2.25 μM, etc.

[0109] In embodiments where subsequent passaging is performed, the iPSCs may be incubated in a substrate-coated vessel without feeder cells. The substrate may be configured to preserve the pluripotency of the cells, a stable karyotype of the cells, and / or expression of pluripotency markers by the cells. For example, the substrate may include laminin, recombinant laminin fragments (e.g., 511 e8 fragment), vitronectin, recombinant vitronectin, or combinations thereof. The medium may include Essential-8®, E8-specific supplements (e.g., TGF-b, bFGF, etc.), as well as Wnt pathway inhibitors, tankyrase 1 / 2 inhibitors, etc. The inhibitors may have concentrations of about 1 nM to about 3 nM, about 1.5 μM to about 2.5 μM, about 1 μM to about 2.5 μM, about 1.5 μM to about 3 μM, about 1.75 μM to about 2.25 μM, etc. Subsequent passaging results in colonies with little or no differentiation.

[0110] Genome assembly using primate high molecular weight (HMW) DNA and long-read sequencing Returning to FIG. 1, the method includes steps of extracting DNA from PBMCs from a first non-human primate (140), preparing a DNA library 150 using at least some DNA sequencing method (160), and preparing a whole genome assembly of the first non-human primate (170). Although PBMCs are shown and described in FIG. 1, other cells or tissues may be used, such as neuronal progenitor cells, keratinocytes, hepatocytes, B cells, or fibroblasts. Genomic DNA with a large number of base pairs is referred to as high molecular weight DNA. This is because the DNA fragments resulting from the large number of base pairs in long reads have a high molecular weight, whereas short reads, in contrast, have much fewer base pairs and therefore a low molecular weight. Typically, long reads may contain at least about 10,000 base pairs, but may also contain about 5,000 to about 50,000 base pairs. Although long reads are mentioned herein, short reads are also contemplated.

[0111] To perform personalized genome assembly on non-human primate animal colonies that are matched with individualized iPSC lines (described above), DNA is extracted using a process that purifies archival quality DNA, as shown in block 140. DNA extraction may be performed by enzyme-based extraction or chloroform-based extraction to avoid mechanical damage of cells and / or DNA. In one embodiment, the protocol implemented in the Gentra® Puregene® Cell Kit (QIAGEN, INC., Germantown, MD, USA) may be used to extract DNA, e.g., high molecular weight DNA. As will be appreciated by those skilled in the art, other protocols for extracting DNA may also be applicable, e.g., the protocol implemented in the QuiAmp® Genomic DNA Kit (QIAGEN, INC.).

[0112] As shown in Figure 1, method 100 includes preparing a DNA library (160) using at least some DNA sequencing methodology. DNA sequencing may include using long-read sequencing technology, such as technology that directly sequences single molecules of DNA in real time, with or without amplification (e.g., technology available from Illumina®, PacBio®, Nanopore®), etc. Other long-read sequencing methods may also be used, as genome assembly utilizes long-read methods.

[0113] In some embodiments, library preparation and sequencing can resolve more variable regions of the genome, such as within the region of the major histocompatibility complex (MHC) locus. Additionally, in one embodiment, genomic DNA above a predetermined base pair threshold may be purified according to size, which may be done, for example, using beads, electrophoretic purification, etc. The purified DNA may be used in a genome assembly pipeline, at block 170 of FIG. 1, using a software-based assembler for single molecule sequencing tools. For example, any of a variety of assembly tools (e.g., Flye, Redbean, Canu, etc.) may be used to create a draft genome assembly from the purified DNA sequences.

[0114] In some embodiments, the assembled genome can be used in combination with the corresponding iPSC line (from the corresponding animal) to design guide RNAs (matched to the individual animal based on its corresponding single nucleotide polymorphisms (SNPs). Guide RNAs may be used in combination with gene editing methods such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) or Adeno-Associated Virus (AAV) based gene editing methods.

[0115] Predicting the formation (or magnitude) of ADA (anti-drug antibody) responses using whole-genome long-read sequencing In some embodiments, a genome-wide based survey may be conducted to identify NHPs capable of anti-drug antibodies (ADA). Some animals develop anti-drug antibodies during the process of evaluating the safety of a drug. ADA binds to the drug and masks its potential effect. Detection of antibodies against the drug using ELISA or other similar antibody detection methods may be performed in the non-human primates injected with the drug. Some animals are genetically more prone to develop ADA. Identification of such genetic regions may be performed by performing whole genome sequencing of ADA-capable animals compared to ADA-noncapable animals. Given the genetic variability of non-human primates, such identified genetic regions may contribute to the ADA response of NHPs and may be correlated with similar genetic regions in humans.

[0116] Whole genome sequencing assemblies of individual animals may be used to predict possible anti-drug antibody (ADA) responses of animals. In one embodiment, phenotypic data collected from ADA-capable animals can be correlated with the corresponding whole genome data of each animal to screen for candidate loci that are correlated with ADA. Specifically, reconstruction of variable immune regions, including but not limited to B cell receptor (BCR) genomic loci and possible genomic loci for immunoglobulin coding regions, major histocompatibility (MHC) regions, and T cell receptor regions, can identify the basic genetic elements of ADA in some animals that can be resolved by whole genome information data. One limitation of such approaches has been the use of short-read sequencing, which makes it difficult to reconstruct or assemble these long variable regions in the genome of NHPs. Using a long-read sequencing approach, it is possible to assemble these variable regions of the innate and adaptive immune systems (including BCR, MHC, KIR) to find correlations between ADA and the genotype of the animal. These individual genomes can then be used to profile the B cell receptor repertoire and T cell receptor repertoire of each animal using single cell RNA sequencing, and these regions can be aligned to the individual genome to aid in the prediction of ADA for each animal.

[0117] In some embodiments, one or more genetic assays may be developed based on genetic variation in the B cell receptor (BCR) genomic region and other candidate regions (locuses) using genome-wide association studies (GWAS) focused on target loci of interest.

[0118] If retrospective data on ADA responses of colony animals is available, such information can be used to correlate with genotypes identified by the colony's whole genome assembly.

[0119] Alternatively, in the absence of a record of the ADA response of an animal colony, the genomic information can be used to track animals over time for future studies to determine whether they develop ADA. In such embodiments, there can be a correlation between the phenotypic information and the assembled genotype of each animal.

[0120] Whole genome assembly and annotation of individual animals within a colony for subsequent application of personalized therapeutics and drug safety testing in non-human primates In some embodiments, the PBMCs isolated in block 130 of FIG. 1 may be used to assemble an individual genome for each NHP in the colony in block 170, which may be compatible with the corresponding NHP from which the iPSCs were generated. A technical effect and / or advantage of having a personalized genome for the NHP is that autologous transplantation of tissues and cells can be performed in the animal without immune rejection. Because those cells and tissues are generated from that particular animal's own cells, the major histocompatibility complex (MHC) and any other immune components involved in transplant immune rejection will match the cells being injected. Furthermore, the personalized genome from each animal may allow for designing individual guide RNAs for future gene therapy or gene editing or cell labeling that minimize off-target effects. Because iPSCs have self-renewal over multiple passages, they can be used for multiple cell culture passages to optimize guide RNAs and evaluate the efficacy and functionality of gene therapy vectors (e.g., AAV vectors) to take advantage of the personalized genome.

[0121] Evaluating Drug Safety in Complex Non-Human Primates in In Vitro Models When evaluating drug and / or treatment safety measures with respect to GLP, the genome of the animal is useful for correlating genotype with drug and / or treatment safety. The genomic information generated can solve such problems and can create panels that allow predicting whether a new colony of animals is suitable for testing a particular drug and / or treatment.

[0122] Furthermore, as shown in Figures 4-5, animal-personalized iPSC lines are suitable tools to assess the safety of drugs in vitro pre-GLP and / or before their in vivo use.

[0123] As shown in Figure 2, because iPSCs can differentiate into multiple lineages (ectoderm 240, mesoderm 230, and endoderm 220) and end tissues (liver 250, neural cells 270, hematopoietic and immune cells, pancreatic cells 260, etc.), safety assessments of drugs and / or therapies can be performed on such personalized animal iPSC-derived tissues by in vitro assays on iPSC-derived tissues prior to use in animals in vivo. Safety measures for personalized iPSC-derived tissues are predictive of safety in animals prior to in vivo experiments, as shown in Figures 4-5.

[0124] For example, Figure 4 illustrates a method 400 for evaluating drug safety, which evaluates efficacy, functionality, safety, and / or toxicity of a therapeutic in vitro using individual iPSCs combined with an individual reference genome prior to use of the therapeutic in a live animal. Method 400 includes performing an in vitro toxicity assay on iPSCs generated from a first NHP (410), analyzing results of the in vitro toxicity assay (420), and if the results indicate toxicity (e.g., the results are above a predetermined threshold), stopping or altering one or more parameters of the in vitro toxicity assay (430), and if the results indicate limited toxicity (e.g., the results are below a predetermined threshold), performing an in vivo toxicity assay on the first NHP from which the iPSCs were generated (440). In vitro toxicity assays may include, but are not limited to, cytotoxicity assays (e.g., ATP-based), cell viability assays (e.g., using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (or MTT), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (or MTS), neutral red, etc.), inflammatory response assays (e.g., using enzyme-linked immunosorbent assays (ELISA)), cell interaction assays (e.g., using multicompartment perfusion systems, etc.). In vivo toxicity testing may include, but are not limited to, evaluation of drug lethality (e.g., acute lethality, subacute lethality, subchronic lethality, chronic lethality, etc.), performing proteomic, metabolomic, and / or toxicogenomic studies and assays, etc.

[0125] Further, for example, as shown in FIG. 5, a method 500 for evaluating efficacy of a gene editing tool includes performing an in vitro efficacy assay on iPSCs generated from a first NHP (510), analyzing results of the in vitro efficacy assay (520), and if the results indicate efficacy below a predetermined threshold, aborting or altering one or more parameters of the in vitro efficacy assay (530), and if the results indicate efficacy above a predetermined threshold, performing an in vivo efficacy assay on the first NHP from which the iPSCs were generated (540).

[0126] In another embodiment, a method 500 of assessing toxicity of a gene editing tool includes performing an in vitro toxicity assay on iPSCs generated from a first NHP (510), analyzing results of the in vitro toxicity assay (520), and if the results are indicative of toxicity (e.g., if toxicity is above a predetermined threshold), stopping or altering one or more parameters of the in vitro toxicity assay (530), and if the results are indicative of toxicity (e.g., if toxicity is below a predetermined threshold), performing an in vivo toxicity assay on the first NHP from which the iPSCs were generated (540).

[0127] In another embodiment, the gene editing tool comprises an adeno-associated vector (AAV).In another embodiment, the gene editing tool comprises a CRISPR-based tool.

[0128] The personalized genome of the animal colony provides accurate genomic information for CRISPR-based gene editing, including but not limited to guide RNA and / or AAV design. In contrast to the human genome, the published reference genomes of most NHP species (e.g., cynomolgus monkeys) are poorly annotated. Furthermore, due to high intra-species genetic variation in most NHPs (e.g., cynomolgus monkeys), a single reference genome does not provide the most accurate information for designing guide RNA for genome editing. Diverse individuals within one NHP species (e.g., cynomolgus monkeys) may cause off-target effects of genome editing. The personalized genome of the animal colony and iPSCs may provide complementary feasible tools for accurate design of guide RNA and avoidance of off-target effects. Guide RNA design may be performed in vitro (using the personalized genome of the animal) on either iPSCs, cells generated from iPSCs, or primary cells (e.g., PBMCs). This approach will result in precise / personalized design of genome editing tools (e.g., guide RNAs and AAV homology arms that facilitate GLP and improved AAV efficacy and functionality in in vivo studies) while simultaneously lowering the cost of testing, minimizing the use of live animals in testing, and / or saving animal lives, saving costs in therapeutic development.

[0129] In one embodiment, a method for evaluating the safety and / or toxicity of a drug or gene therapy tool includes measuring the level of an immune response (e.g., an immune response to an AAV vector used for gene therapy) in peripheral blood mononuclear cells (PBMCs) in vitro. Measuring the immune response generated in PBMCs (e.g., in response to the AAV vector) may be performed by quantitative polymerase chain reaction (qPCR), a bead-based assay (e.g., Luminex®), and / or ELISA. The method may further include measuring the level of an immune response in PBMCs in vitro. Expression of some innate immune genes (e.g., Toll-like receptors (TLRs) and interleukins) may be predictive of the level of toxicity of an AAV vector in a live animal.

[0130] Preclinical testing of drugs or therapies (autologous therapies) in the same animals using iPSCs and personalized genomes In one embodiment, cells and tissues generated from the iPSC line in each individual animal may be tracked by in vivo imaging methods. For example, as shown in FIG. 6B, cells and tissues 650 generated from iPSCs may be labeled (e.g., with fluorescent proteins or other markers). As shown in FIG. 6A, cells or tissues derived from labeled iPSCs may be autografted or injected into the same animal to assess cell functionality.

[0131] Individualized iPSCs for each animal in the colony provide a tool to label / edit iPSCs to indicate levels of safety or efficacy of therapy or to optimize gene editing for any purpose. Data from in vitro assays may be used as a preliminary assay prior to in vivo testing of animals. Such assays include evaluating the immune response of PBMCs or other cells / tissues to stimuli (e.g., AAV) as described elsewhere herein. Successfully gene-edited iPSC lines (with toxicity assessment results that are safe for the target cells / tissues) may be used for subsequent in vivo testing and / or for later autologous transplantation of cells into animals for imaging and functional assays, as shown in Figures 4-6C.

[0132] For example, a method 600 of autologous transplantation of cells or tissue generated from iPSCs in an NHP includes obtaining tissue or blood from at least one individual of a population comprising a single species of non-human primate (610), generating iPSCs from the tissue or blood (620), differentiating the iPSCs into a tissue or cell type of interest (630), and autologously transplanting the tissue or cell type of interest into the at least one individual (640).

[0133] In some embodiments, method 600 further includes performing in vitro testing on a subset of iPSCs generated from the at least one individual. In vitro testing may include assessing cell viability, evaluating efficacy of treatment (e.g., inhibition or activation of a target protein, inhibition or activation of a target pathway, inhibition or activation of the cell cycle, etc.), evaluating cell toxicity, etc.

[0134] In some embodiments, autoinjecting or autotransplanting includes autoinjecting or autotransplanting a second subset of iPSCs generated from the at least one individual into the at least one individual, which includes performing an in vivo test in the at least one individual, deriving an in vivo test result from the in vivo test, and correlating the in vivo test result with the in vitro test result. An example is liver toxicity in animals and humans from AAV vectors or other cell and gene therapy methods. An initial test to predict the level of immunogenicity of AAV vectors to hepatocytes or immune cells (e.g., PBMCs) generated from iPSCs in vitro can be used as a predictor of liver toxicity in NHPs in vivo.

[0135] The first subset of iPSCs generated can be during a first period of in vitro expansion of the iPSCs, and the second subset of iPSCs generated can be during a second period of in vitro expansion of the iPSCs.

[0136] In one embodiment, method 600 may include performing gene editing on the generated iPSCs, or a differentiated tissue or cell type of interest, as shown in FIG. 6C and described elsewhere herein.

[0137] Individualized iPSCs offer a rapid and non-terminal method to query genetically validated targets in NHPs. iPSCs in NHPs have two important features: self-renewal and pluripotency.

[0138] With regard to self-renewal, iPSCs can be passaged and expanded repeatedly in vitro.

[0139] With regard to pluripotency, iPSCs are capable of differentiating into various tissues and cells. For example, iPSCs may be used to design guide RNAs (gRNAs) and perform gene editing studies using various tools including AAVs, as shown in Figure 5, which may be particularly relevant for NHP species with highly diverse intra-species populations (e.g., cynomolgus and rhesus macaques). Individualized iPSC lines coupled with individualized assembled genomes of animals provide a viable tool for a fast and non-terminal method of querying genetically validated targets in NHPs. Such assays are useful prior to performing in vivo experiments.

[0140] The self-renewal capacity (immortality for multiple in vitro passages) and pluripotent features of iPSCs make them a viable tool for designing guide RNAs and performing gene editing studies using various tools including AAV (adeno-associated virus). This may be especially true for NHP species with highly diverse populations, e.g., cynomolgus and rhesus macaques.

[0141] Finally, robust iPSC and genomic tools, including a) individualized iPSC lines and b) individualized genome assemblies, may provide an in vitro system for measuring drug safety levels, for designing gene editing tools that can prevent off-target effects, and for safe delivery of autologous cells and tissues for a wide variety of studies and imaging. This individualized iPSC genomic platform provides a strong foundation for evaluating drug safety in vitro, reducing the risks of in vivo evaluation, which will reduce costs in the long run and save animal lives.

[0142] Experimental Results 7A-7B are brightfield microscopy images (4x and 10x, respectively) of macaque iPSCs generated using conventional primate iPSC culture conditions according to the protocol in Takahashi et al., "Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors," Cell 131, 861-782, November 30, 2007, which is incorporated herein by reference in its entirety.

[0143] Briefly, in this conventional method and composition, iPSCs were generated and maintained in Primate ES medium (ReproCELL, Japan) supplemented with 4 ng / ml recombinant human basic fibroblast growth factor (bFGF, WAKO, Japan). For passaging, human iPSCs were washed once with PBS and then incubated at 37°C in DMEM / F12 containing 1 mg / ml collagenase IV (Invitrogen). When colonies at the edge of the dish started to separate from the bottom, the DMEF / F12 / collagenase was removed and washed with Primate ES cell culture. Cells were scraped off, an appropriate amount of medium was added, and the contents were transferred onto SNL feeder cells in a new dish. The split ratio was 1:3 as specified. The cells were transfected with pMXs vector and Fugene 6 transfecting reagent (Roche). 24 hours after transfection, the medium was collected as the first virus-containing supernatant and replaced with fresh medium. This new medium was collected 24 hours later as the second virus-containing supernatant. Human fibroblasts expressing the mouse Slc7a1 gene were seeded the day before transduction. The virus-containing supernatant was filtered and 4mg / ml polybrene was added. Equal volumes of the supernatants containing each of the four retroviruses were mixed, transferred to a fibroblast dish, and incubated overnight. 24 hours after transduction, the virus-containing medium was replaced with the second supernatant. Six days after transduction, fibroblasts were harvested by trypsinization and replated on SNL feeder layers. The next day, the medium was replaced with Primate ES cell medium supplemented with 4ng / ml bFGF. The medium was replaced every other day. 30 days after transduction, colonies were picked and transferred to 0.2ml Primate ES cell medium. Colonies were mechanically separated into small clumps by aspirating and dispensing with a pipette. The cell suspension was transferred onto SNL feeders in 24-well plates. As shown in Figures 7A-7B, under conventional culture conditions, the majority of macaque iPSCs differentiated spontaneously when observed under microscopic images.

[0144] Meanwhile, Figures 7C-7D show bright field microscopy images (4x and 10x, respectively) of macaque iPSCs generated using the compositions and methods described in the Methods and Compositions section and shown in Figures 3A-3B. As shown in Figures 7C-7D, iPSC cultures using the improved compositions and methods described herein exhibit iPSCs with rounded edges and retain their pluripotent function in the improved culture conditions.

[0145] Working Example Example 1. A method of generating induced pluripotent stem cells (iPSCs) from individuals in a population of non-human primates (NHPs), comprising obtaining peripheral blood mononuclear cells (PBMCs) from at least one individual of the population comprising a single species of non-human primate; culturing the PBMCs in hematopoietic stem cell (HSC) expansion medium for a period of time to expand blood progenitor cells; transfecting the cultured PBMCs with a combination of transcription factors such that the cells are induced to overexpress the transcription factors, wherein the transfected cultured PBMCs are reprogrammed into iPSCs; and, no more than one day after the transfecting step, selecting the transfected cells from the HSC expansion medium to expand the blood progenitor cells. transferring the cells to a container containing a plurality of feeder cells; transferring the cells to a second container no more than one day after the transferring step; and performing at least one of adding medium to the cells, exchanging medium in the cells, and adding one or more cell growth factors to the cells a selected number of days after the transferring to the second container; passaging the cells by placing each colony in a third container coated with extracellular matrix until first cell colonies appear; and expanding the first cell colonies for use in one or more of in vitro testing, in vivo testing, or creation of a genomic library.

[0146] Example 2. The method of any one of the preceding examples, wherein the expanding step comprises washing the iPSCs with phosphate buffered saline (PBS) and incubating the iPSCs with a cell dissociation solution.

[0147] Example 3. The method of any one of the preceding examples, wherein the incubating step occurs at about 37° C. for a period of time.

[0148] Example 4. The method of any one of the preceding examples, wherein the expanding step comprises aspirating and dissociating the iPSCs into a single cell suspension.

[0149] Example 5. The method of any one of the preceding examples, further comprising differentiating the iPSCs into primary lineages, the primary lineages including at least one of ectoderm, mesoderm, and endoderm.

[0150] Example 6. The method of any one of the preceding examples, further comprising differentiating primary lineages into end-point tissues.

[0151] Example 7. The method of any one of the preceding examples, further comprising generating an individualized genome assembly of the at least one individual.

[0152] Example 8. The method of any one of the preceding examples, further comprising extracting a large amount of high molecular weight (HMW) DNA from the obtained PBMCs by a process for purifying archival quality DNA.

[0153] Example 9. The method of any one of the preceding examples, further comprising preparing a genomic library from the extracted HMW DNA, sequencing long read fragments of the HMW DNA, and assembling and annotating the long read fragments to recreate the genome of at least one individual from whom the PBMCs were obtained.

[0154] Example 10. The method of any one of the preceding examples, wherein the transcription factor is selected from the group consisting of c-myc, Klf4, Sox2, Oct3 / 4, Klf2, Nanog, Tfcp2L1, and Stat3.

[0155] Example 11. The method of any one of the preceding examples, wherein the plurality of feeder cells are mouse embryonic fibroblasts (MEFs) or SNL feeder cells.

[0156] Example 12. The method of any one of the preceding examples, wherein the step of transferring the transfected cells to a vessel containing a plurality of feeder cells further comprises adding a tankyrase 1 / 2 inhibitor in the range of about 1 μM to about 3 μM.

[0157] Example 13. The method of any one of the preceding examples, wherein the ratio of the transfected cells to the plurality of feeder cells is about 1:3 to about 1:7.

[0158] Example 14. The method of any one of the preceding examples, wherein the transfecting step uses a Sendai virus vector.

[0159] Example 15. The method of any one of the preceding examples, wherein the one or more cell growth factors comprises basic fibroblast growth factor.

[0160] Example 16. The method of any one of the preceding examples, wherein passaging the cells by placing each colony in a third container coated with an extracellular matrix comprises adding a serine / threonine kinase inhibitor to the medium in the third container.

[0161] Example 17. A method of preclinical drug evaluation comprising a personalized genome assembly of a subject and induced pluripotent stem cells (iPSCs) generated from the subject.

[0162] Example 18. The method of Example 17, further comprising transplanting cells and tissue generated from iPSCs from the subject into the subject and assessing functionality of the transplanted cells and tissue in the subject.

[0163] Example 19. The method of any one of Examples 17-18, further comprising generating one or more panels and predicting whether a particular population of animals is suitable for drug testing based on the one or more panels.

[0164] Example 20. The method of any one of Examples 17-19, further comprising generating a guide RNA or a gene editing tool using iPSCs generated from the subject, and performing in vivo testing of the guide RNA or the gene editing tool in the subject.

[0165] Example 21. A method for preclinical testing of a human therapeutic comprising obtaining tissue or blood from at least one individual of a population comprising a single species of non-human primate, isolating PBMCs from the at least one individual, generating iPSCs from the PBMCs from the at least one individual, extracting DNA from the PBMCs from the at least one individual, preparing a DNA library using at least some of the extracted DNA, preparing a whole genome assembly of the at least one individual, and performing a computational analysis of the whole genome assembly.

[0166] Example 22. The method of example 21, wherein the computer analysis includes identifying one or both of major histocompatibility complex associated genes or minor histocompatibility complex associated genes.

[0167] Example 23. The method of any one of Examples 21-22, wherein the computer analysis includes identifying immune system related genes.

[0168] Example 24. The method of any one of Examples 21-23, wherein the computer analysis comprises identifying anti-drug antibody (ADA) response-associated genes.

[0169] Example 25. The method of any one of Examples 21-24, wherein the computational analysis comprises performing preclinical genotyping.

[0170] Example 26. The method of any one of Examples 21-25, wherein the computer analysis comprises identifying liver toxicity-associated genes.

[0171] Example 27. The method of any one of Examples 21 to 26, wherein the DNA is high molecular weight DNA.

[0172] Example 28. The method of any one of Examples 21 to 27, wherein the DNA is archival quality DNA.

[0173] Example 29. The method of any one of Examples 21 to 28, wherein preparing a DNA library comprises performing long-read sequencing on the extracted DNA.

[0174] Example 30. The method of any one of Examples 21 to 29, wherein preparing a whole genome assembly comprises recreating the genome of the at least one individual based on the prepared DNA library.

[0175] Example 31. A method of preclinical testing of a human therapeutic comprising obtaining tissue or blood from at least one individual of a population comprising a single species of non-human primate, isolating PBMCs from the at least one individual, generating iPSCs from the PBMCs from the at least one individual, performing in vitro testing on the iPSCs generated from the at least one individual, deriving in vitro test results from the in vitro testing, and correlating the in vitro test results with the genotype of the at least one individual.

[0176] Example 32. The method of Example 31, wherein the step of performing the in vitro test comprises designing a guide RNA and transfecting the guide RNA into iPSCs in vitro.

[0177] Example 33. The method of any one of Examples 31-32, wherein the step of deriving an in vitro test result comprises running a panel to detect gene editing efficiency of guide RNAs in vitro in iPSCs.

[0178] Example 34. A method of preclinical testing of a human therapeutic comprising obtaining tissue or blood from at least one individual of a population comprising a single species of non-human primate, isolating PBMCs from the at least one individual, generating iPSCs from the PBMCs from the at least one individual, performing in vivo testing in the at least one individual, deriving in vivo test results from the in vivo testing, and correlating the in vivo test results of the in vivo testing with the genotype of the at least one individual.

[0179] Example 35. The steps of generating iPSCs include culturing PBMCs in hematopoietic stem cell (HSC) expansion medium for a period of time to expand blood progenitor cells; transfecting the cultured PBMCs with a combination of transcription factors such that the cells are induced to overexpress the transcription factors, whereby the transfected cultured cells are reprogrammed into iPSCs; and transferring the transfected cells to a vessel containing a plurality of feeder cells no more than one day after the transfecting step; and transferring the cells to a vessel containing a plurality of feeder cells no more than one day after the transferring step. 35. The method of claim 34, comprising: transferring the cells to a second container; performing at least one of adding medium to the cells, exchanging medium in the cells, and adding one or more cell growth factors to the cells a selected number of days after the transferring to the second container; passaging the cells by placing each colony in a third container coated with extracellular matrix until first cell colonies appear; and expanding the first cell colonies for use in one or more of in vitro testing, in vivo testing, or generation of a genomic library.

[0180] Example 36. The method of any one of Examples 34-35, wherein performing the in vivo testing comprises autologously transplanting or injecting a subset of iPSCs into the at least one individual.

[0181] Example 37. The method of any one of Examples 34-36, further comprising labeling the iPSCs prior to autologous transplantation or injection.

[0182] Example 38. The method of any one of Examples 34 to 37, wherein the labeling comprises fluorescent labeling.

[0183] Example 39. The method of any one of Examples 34-38, wherein deriving an in vivo test result comprises running a panel to detect anti-drug antibodies.

[0184] Example 40. The method of any one of Examples 34-39, wherein deriving an in vivo test result comprises running a panel to detect liver toxicity.

[0185] Example 41. A method of preclinical testing of a human therapeutic comprising obtaining tissue or blood from at least one individual of a population comprising a single species of non-human primate, isolating PBMCs from the at least one individual, generating iPSCs from the PBMCs from the at least one individual, performing in vitro testing on a subset of iPSCs generated from the at least one individual, deriving in vitro test results from the in vitro testing, auto-injecting or auto-transplanting a second subset of iPSCs generated from the at least one individual into the at least one individual, performing in vivo testing in the at least one individual, deriving in vivo test results from the in vivo testing, and correlating the in vivo and in vitro test results.

[0186] Example 42. The steps of generating iPSCs include culturing PBMCs in hematopoietic stem cell (HSC) expansion medium for a period of time to expand blood progenitor cells; transfecting the cultured PBMCs with a combination of transcription factors such that the cells are induced to overexpress the transcription factors, whereby the transfected cultured cells are reprogrammed into iPSCs; and transferring the transfected cells to a vessel containing a plurality of feeder cells no more than one day after the transfecting step; and transferring the cells to a vessel containing a plurality of feeder cells no more than one day after the transferring step. 42. The method of Example 41, comprising: transferring the cells to a second container; performing at least one of adding medium to the cells, exchanging medium in the cells, and adding one or more cell growth factors to the cells a selected number of days after the transferring to the second container; passaging the cells by placing each colony in a third container coated with extracellular matrix until first cell colonies appear; and expanding the first cell colonies for use in one or more of in vitro testing, in vivo testing, or generation of a genomic library.

[0187] Example 43. The method of any one of Examples 41-42, wherein the step of performing an in vitro test comprises designing a guide RNA and transfecting the guide RNA into a subset of iPSCs in vitro.

[0188] Example 44. The method of any one of Examples 41-43, wherein the step of deriving an in vitro test result comprises running a panel to detect in vitro gene editing efficiency of guide RNAs in a subset of iPSCs.

[0189] Example 45. The method of any one of Examples 41-44, further comprising transfecting iPSCs with guide RNA for autologous transplantation or autologous injection.

[0190] Example 46. The method of any one of Examples 41-45, wherein deriving an in vivo test result comprises running a panel to detect liver toxicity.

[0191] Example 47. The method of any one of Examples 41-46, wherein the step of deriving an in vivo test result comprises running a panel to detect in vivo gene editing efficiency of guide RNAs in a second subset of iPSCs.

[0192] Example 48. The method of any one of Examples 41-47, wherein the correlating step comprises comparing in vitro gene editing efficiency with in vivo gene editing efficiency.

[0193] Example 49. The method of any one of Examples 41-48, wherein performing the in vitro testing comprises treating the iPSCs in vitro with a therapy.

[0194] Example 50. The method of any one of Examples 41-49, wherein the step of deriving in vitro test results comprises running a panel that detects in vitro toxicity of the therapy.

[0195] Example 51. The method of any one of Examples 41-50, wherein the step of conducting an in vivo test result comprises treating the at least one individual with the therapy.

[0196] Example 52. The method of any one of Examples 41-51, wherein the step of deriving in vivo test results comprises running a panel to detect in vivo toxicity of the therapy.

[0197] Example 53. The method of any one of Examples 41-52, wherein the correlating step comprises comparing the in vitro toxicity with the in vivo toxicity of the therapy.

[0198] The description of the embodiments written thus far is intended to enable one of ordinary skill in the art to utilize the methods described, but one of ordinary skill in the art will understand and appreciate that there are variations, combinations, and equivalents of the specific embodiments, methods, and examples described herein. Thus, the present specification should not be limited by the embodiments, methods, and examples described above, but rather by all embodiments and methods that fall within the scope and spirit of the appended claims.

[0199] As used herein and in the claims, "a day" or "one day" may be used. "A day" or "one day" may include, for example, about 24 hours plus or minus about 6 hours.

[0200] In the specification and claims, the singular forms "a," "an," and "the" include both singular and plural references unless the context clearly contradicts. For example, the term "nonhuman primate" may include, and is intended to include, a plurality of nonhuman primates. At times, the claims and disclosure may include the phrases "a plurality," "one or more," or "at least one," but the absence of such words does not mean that a plurality is not envisioned and should not be interpreted to mean so.

[0201] The words "about" or "approximately," when used before a numerical designation or range (e.g., defining a length or pressure), indicate approximations that may vary by ±5%, ±1%, or ±0.1%. All numerical ranges given herein are inclusive of the beginning and ending values ​​stated. The word "substantially" refers to almost all (i.e., greater than 50%) or substantially all of a device, material, or composition.

[0202] As used herein, the words "comprising" or "comprises" shall mean that the devices, systems, and methods include the recited elements and may further include any other elements. "Consisting essentially of" shall mean that the devices, systems, and methods include the recited elements and exclude other elements that have an essential significance to the combination for the purposes of the description. Thus, a system or method consisting essentially of the elements defined herein will not exclude other materials, features, or steps that do not materially affect the basic and novel characteristics of the claimed disclosure. "Consisting of" shall mean that the devices, systems, and methods include the recited elements and exclude all or more insignificant or insignificant elements or steps. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0203] The examples and specific examples contained herein are illustrative, not limiting, of specific embodiments in which the subject matter may be practiced. Other embodiments may be utilized or derived therefrom, such that structural or logical substitutions or changes may be made without departing from the scope of the present disclosure. Such embodiments of the subject matter may be referred to individually herein or collectively under the term "the present invention," which is merely for convenience and is not intended to spontaneously limit the scope of the present application to any one invention or inventive concept, even if more than one is actually disclosed. Thus, although specific embodiments have been shown and described herein, the specific embodiments shown may be substituted with any configuration designed to achieve the same purpose. The present disclosure encompasses any adaptations or variations of the various embodiments. Combinations of the above-described embodiments, as well as other embodiments not specifically described herein, will become apparent to those skilled in the art upon review of the above description.

Claims

1. 1. A method for generating induced pluripotent stem cells (iPSCs) from individuals within a population of non-human primates (NHPs), comprising: obtaining peripheral blood mononuclear cells (PBMCs) from at least one individual of a population comprising a single species of non-human primate; Culturing the PBMCs in hematopoietic stem cell (HSC) expansion medium for a predetermined period of time to expand blood progenitor cells; transfecting the cultured PBMCs with a combination of transcription factors such that the cells are induced to overexpress the transcription factors, wherein the transfected cultured PBMCs are reprogrammed into iPSCs; transferring the transfected cells to a container containing a plurality of feeder cells within one day after the transfecting step; transferring the cells to a second container within one day after the transferring step; a selected number of days after the step of transferring to the second container; adding medium to the cells; replacing the medium within the cells; and adding one or more growth factors to said cells; and performing at least one of: until the first cell colonies appear. passaging the cells by placing each colony in a third container coated with extracellular matrix; and expanding the first cell colony for use in one or more of in vitro testing, in vivo testing, or generating a genomic library; A method comprising:

2. 10. The method of claim 1, wherein the expanding step comprises washing the iPSCs with phosphate buffered saline (PBS) and incubating the iPSCs in a cell dissociation solution.

3. 3. The method of claim 2, wherein the incubating step is performed at about 37°C for a predetermined period of time.

4. 3. The method of claim 2, wherein the expanding step comprises aspirating and dissociating the iPSCs into a single cell suspension.

5. 10. The method of claim 1, further comprising differentiating the iPSCs into primary lineages, wherein the primary lineages include at least one of ectoderm, mesoderm, and endoderm.

6. The method of claim 5 , further comprising differentiating the primary lineages into end-point tissues.

7. 10. The method of claim 1, further comprising generating an individualized genome assembly for the at least one individual.

8. 8. The method of claim 7, further comprising extracting a large amount of high molecular weight (HMW) DNA from the obtained PBMCs by an archival quality DNA purification process.

9. 9. The method of claim 8, further comprising preparing a genomic library from the extracted HMW DNA, sequencing long read fragments of the HMW DNA, and assembling and annotating the long read fragments to recreate the genome of the at least one individual from whom the PBMCs were obtained.

10. 2. The method of claim 1, wherein the transcription factor is selected from the group consisting of c-myc, Klf4, Sox2, Oct3 / 4, Klf2, Nanog, Tfcp2L1, and Stat3.

11. 2. The method of claim 1, wherein the plurality of feeder cells are mouse embryonic fibroblasts (MEFs) or SNL feeder cells.

12. 10. The method of claim 1, wherein said step of transferring said transfected cells to a vessel containing said plurality of feeder cells further comprises adding a tankyrase 1 / 2 inhibitor in the range of about 1 μM to about 3 μM.

13. 2. The method of claim 1, wherein the ratio of transfected cells to the plurality of feeder cells is about 1:3 to about 1:

7.

14. 2. The method of claim 1, wherein the transfecting step uses a Sendai virus vector.

15. The method of claim 1 , wherein the one or more cell growth factors comprises basic fibroblast growth factor.

16. 2. The method of claim 1, wherein the step of passaging the cells by placing each colony in the third container coated with the extracellular matrix comprises the step of adding a serine / threonine kinase inhibitor to the medium in the third container.

17. The method of claim 1, further comprising a step of recovering PBMCs from the whole blood of at least one individual of a population including cynomolgus monkeys.

18. The method of claim 1, further comprising culturing PBMCs in a hematopoietic stem cell (HSPC) growth medium containing one or more of interleukin-3 (IL-3), interleukin-6 (IL-6), FMS-like tyrosine kinase 3 ligand (FLT-3), thrombopoietin (TPO), and stem cell factor (SCF) to expand hematopoietic stem cells.

19. The method described in claim 1, wherein the predetermined period is before transfection.

20. The method of claim 1, further comprising the step of transfecting a first subset of the cultured PBMCs and a second subset of the PBMCs with a combination of transcription factors such that the cells are induced to overexpress the transcription factors, wherein the transfected cultured PBMCs of each subset are reprogrammed into iPSCs and the results are compared with iPSCs obtained from the second transfected subset.

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