Compositions and methods for induced stem cell differentiation to oligodendrocytes

Abstract

A method of differentiating nonhuman primate induced pluripotent stem cells (iPSCs) to oligodendrocytes is provided. The method may include providing a modified vector comprising a piggyBac vector backbone that expresses one or more differentiation factor genes, including SOX10, OLIG2, and NKX6-2. The piggyBac vector backbone may comprise one or more terminal inverted repeats and one or more transposase recognition sites configured to interact with a piggyBac transposase to mediate genomic integration at TTAA or noncanonical target sequences. The vector may further include a selectable marker and a promoter for regulated expression. The modified vector may be introduced into nonhuman primate iPSCs, for example from Macaca fascicularis, by electroporation and used in a feeder-free differentiation workflow. The resulting differentiated cells may be identified by expression of oligodendrocyte-associated markers including O4, O1, PDGFRA, MOG, GALC, CNPase, and myelin basic protein (MBP).

Description

Docket No. 0138-706.600INTERNATIONAL APPLICATIONCOMPOSITIONS AND METHODS FOR INDUCED STEM CELL DIFFERENTIATION TO OLIGODENDROCYTESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Utility Application No. 63 / 722, 174, filed November 19, 2024, which is herein incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 0138- 706.600_Sequence_Listing.xml, created November 17, 2025, which is 30,000 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.INCORPORATION BY REFERENCE

[0003] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety.TECHNICAL FIELD

[0004] This disclosure relates generally to the field of cellular differentiation, and more specifically to the field of lentiviral transduction for cellular differentiation. Described herein are compositions and methods for induced stem cell differentiation.BACKGROUND

[0005] Induced pluripotent stem cells (iPSCs) have the potential to be differentiated into different cell types including neuronal cells. This process has been successfully conducted using human iPSCs.SUMMARY

[0006] In some aspects, the techniques described herein relate to a vector for differentiating nonhuman primate induced pluripotent stem cells (iPSCs) from Macaca fascicularis intoDocket No. 0138-706.600INTERNATIONAL APPLICATION oligodendrocytes, wherein the vector includes a nucleic acid sequence including a sequence encoding: a piggyBac vector backbone including: one or more terminal inverted repeats; one or more transposase recognition sites configured to interact with a piggyBac transposase; and a differentiation factor gene integrated within the piggyBac vector backbone and configured to induce oligodendrocyte lineage differentiation in one or more transfected iPSCs.

[0007] In some aspects, the techniques described herein relate to a vector for differentiating nonhuman primate induced pluripotent stem cells (iPSCs) toward an oligodendrocyte lineage, wherein the vector includes a nucleic acid sequence including a sequence encoding: a piggyBac vector backbone; and a differentiation factor gene configured to promote oligodendrocyte lineage development and maturation.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The foregoing is a summary, and thus, necessarily limited in detail. The above- mentioned aspects, as well as other aspects, features, and advantages of the present technology are described below in connection with various embodiments, with reference made to the accompanying drawings.

[0009] FIG. 1 shows a schematic of a PiggyBac vector backbone, according to some embodiments.

[0010] FIG. 2 is a flow chart of an embodiment of a method for transfecting nonhuman primate (NHP) induced pluripotent stem cells (iPSCs), according to some embodiments.

[0011] FIG. 3 is a flow chart showing an embodiment of a method for reprogramming nonhuman primate peripheral blood mononuclear cells (PBMCs) into iPSCs, according to some embodiments.

[0012] FIG. 4 is a schematic of an embodiment of a method for reprogramming nonhuman primate PBMCs into iPSCs, according to some embodiments.

[0013] FIG. 5 shows a schematic of a PiggyBac vector backbone modified to express differentiation factor genes Macaca fascicularis SOX10 (SEQ ID NO. 2), Macaca fascicularis OLIG2 (SEQ ID NO. 3), and Macaca fascicularis NKX6-2 (SEQ ID NO. 4), according to some embodiments.

[0014] FIG. 6 is a flow chart showing an embodiment of a method for differentiating NHP iPSCs to a neuronal lineage, according to some embodiments.

[0015] FIG. 7 is a block diagram illustrating a vector, according to some embodiments.

[0016] FIG. 8 is a block diagram further illustrating the vector from FIG. 7, according toDocket No. 0138-706.600INTERNATIONAL APPLICATION some embodiments.

[0017] FIG. 9 is a block diagram illustrating a vector, according to some embodiments.

[0018] FIG. 10 is a block diagram further illustrating the vector from FIG. 9, according to some embodiments.

[0019] The illustrated embodiments are merely examples and are not intended to limit the disclosure. The schematics are drawn to illustrate features and concepts and are not necessarily drawn to scale.DETAILED DESCRIPTION

[0020] The foregoing is a summary, and thus, necessarily limited in detail. The above- mentioned aspects, as well as other aspects, features, and advantages of the present technology will now be described in connection with various embodiments. The inclusion of the following embodiments is not intended to limit the disclosure to these embodiments, but rather to enable any person skilled in the art to make and use the claimed subject matter. Other embodiments may be utilized, and modifications may be made without departing from the spirit or scope of the subject matter presented herein. Aspects of the disclosure, as described and illustrated herein, can be arranged, combined, modified, and designed in a variety of different formulations, all of which are explicitly contemplated and form part of this disclosure.

[0021] Conventionally, stems cells from humans have been differentiated using lentivirus- based systems. However, lentivirus-based systems are not efficient when differentiating NHP stem cells. Species-specific differences may prevent the use of human iPSC methods for nonhuman primate iPSCs. Using lentivirus-based systems, NHP have a low transduction efficiency, for example about 1% to about 5%. Further, using lentiviruses are undesirable since the genetic material is incorporated into the genome, which could result in mutations that may be imperceptible to the user / researcher but nonetheless impactful on the results from the experiments. Others have attempted to use Sendai virus, but these have similar drawbacks as lentiviruses. For example, Sendai virus in nonhuman primate cells is not as efficient as its usage in human cells.

[0022] The above technical problems presented by using lentivirus-based systems and / or Sendai viruses may be solved by technical solutions. For examples, the systems and methods described herein may solve the above technical problems with technical solutions that include using piggyBac vectors for transfecting NHP iPSCs. Using piggyBac vectors provide severalDocket No. 0138-706.600INTERNATIONAL APPLICATION technical advantages when transfecting NHP iPSCs. For example, piggyBac vectors can be removed from the genome without leaving a footprint, and piggyBac vectors have high transposition activity in at least particular cell types. As shown and described elsewhere herein, a piggyBac vector has been modified herein to express particular differentiation factor genes to enable differentiation of NHP iPSCs into pre-defined neuronal lineages. For example, one or more differentiation factors may be used to differentiate NHP iPSCs into NGN2 cells (iN cells), astroglia, astrocytes, induced astrocytes (iA cells), and / or oligodendrocytes.

[0023] As used herein, the term “astrocyte” refers to a differentiated glial cell of the central nervous system that contributes to neuronal support, homeostatic regulation, and maintenance of the blood-brain barrier. Astrocytes exhibit characteristic stellate morphology and express lineage-specific markers including, but not limited to, glial fibrillary acidic protein (GFAP), SI 00 calcium-binding protein P (S100P), and aquaporin-4 (AQP4). Astrocytes regulate neurotransmitter uptake, modulate ion balance, participate in synaptic signaling, and / or provide metabolic and / or trophic support to neurons.

[0024] The term “induced astrocyte” (iA cell) refers to an astrocyte that has been derived in vitro from pluripotent stem cells, including induced pluripotent stem cells (iPSCs), or from reprogrammed somatic cells through the introduction or expression of one or more transcription factors associated with astrocytic differentiation. Induced astrocytes exhibit the molecular, structural, and functional characteristics of mature astrocytes, including, but not limited to, expression of GFAP, S100P, and AQP4, while demonstrating functional responses such as calcium signaling, glutamate uptake, and / or cytokine release.

[0025] The term “astroglia” refers broadly to the astrocytic lineage, encompassing astrocyte progenitors, immature astrocytes, and mature astrocytes. Astroglia may include both proliferative precursors and differentiated forms present during various stages of development. In contrast, iA cells as described herein are terminally differentiated and correspond to mature astrocytes rather than progenitor forms.

[0026] As used herein, the term “neuronal lineage cell” refers to any cell type that arises from the ectodermal lineage during neural differentiation. Neuronal lineage cells include, but are not limited to, neurons, astrocytes, induced astrocytes (iA cells), oligodendrocytes, and / or induced neurons (iN cells, such as NGN2 cells).

[0027] The term “induced neuron” (iN cell) refers to a neuron generated in vitro from pluripotent or somatic cells through directed differentiation or reprogramming. InducedDocket No. 0138-706.600INTERNATIONAL APPLICATION neurons include, but are not limited to, NGN2-induced neurons (NGN2 cells) that express neuronal markers such as TUJ1, MAP2, and / or synapsin-1 and exhibit functional properties including, but not limited to, spontaneous electrical activity and synaptic formation.

[0028] The term “oligodendrocyte” refers to a myelinating glial cell type that arises from the neuronal lineage and functions to insulate axons with myelin sheaths. Oligodendrocytes express characteristic markers including, but not limited to, OLIG2, MBP, and / or PLP1 and are responsible for maintaining efficient action potential conduction in the central nervous system.

[0029] As used herein, the term “differentiation factor gene” refers to a gene whose expression contributes to the transition of pluripotent or multipotent cells into a defined lineage. Differentiation factor genes may include, but are not limited to, transcription factors, signaling molecules, or regulatory elements that direct cell fate determination. In certain embodiments, the differentiation factor genes comprise Macaca fascicularis SOX9 and Macaca fascicularis NFIB, which together activate transcriptional networks that initiate and stabilize astrocytic differentiation.

[0030] The term “PiggyBac vector backbone” refers to a transposable DNA element comprising terminal inverted repeats and internal transposase recognition sites that allow transposase-mediated genomic insertion and excision of an inserted genetic cargo. The PiggyBac system integrates transgenes at TTAA target sites within the host genome and is capable of footprint-free excision upon reintroduction of transposase. The PiggyBac backbone may further include, but is not limited to, additional sequences such as a selectable marker, a constitutive or inducible promoter, and / or one or more origins of replication.

[0031] The term “bidirectional Tet-responsive promoter” refers to a regulatory DNA sequence that drives transcription of two genes in opposite orientations under the control of a tetracycline-responsive transactivator. In the presence of an inducer such as doxycycline, the promoter activates coordinated expression of both genes positioned on either side of the promoter sequence.

[0032] The term “nonhuman primate induced pluripotent stem cell” (NHP iPSC) refers to a stem cell derived from a nonhuman primate, including, but not limited to, Macaca fascicularis, that possesses the ability to self-renew and to differentiate into derivatives of all three germ layers: ectoderm, mesoderm, and / or endoderm. These cells are reprogrammed from somatic cells, including, but not limited to, peripheral blood mononuclear cells, through the introduction of reprogramming factors.Docket No. 0138-706.600INTERNATIONAL APPLICATION

[0033] As used herein, the term “TTAA site” refers to a four-nucleotide genomic target sequence recognized by the PiggyBac transposase for integration and excision events. Integration occurs precisely at the TTAA motif, and excision restores the original TTAA sequence without residual insertions or deletions.

[0034] The term “transposase recognition site” refers to a specific nucleotide sequence within the PiggyBac vector backbone that interacts with the PiggyBac transposase enzyme to mediate excision and integration of the transposon. These regions may range in length from about 200 base pairs to about 500 base pairs, for example about 250 base pairs to about 450 base pairs, or about 275 base pairs to about 350 base pairs.

[0035] The term “terminal inverted repeat” refers to a short DNA sequence located at both ends of the PiggyBac transposon that is identical in sequence but oriented in reverse complement fashion. These inverted repeats serve as binding sites for the PiggyBac transposase, allowing recognition, cleavage, and reinsertion of the transposon. Each terminal inverted repeat may range in length from about 10 base pairs to about 25 base pairs, for example about 12 base pairs to about 22 base pairs, or about 15 base pairs to about 18 base pairs.

[0036] The term oligodendrocyte lineage cells refers to a class of cells derived from neural progenitor or pluripotent stem cells that undergo progressive differentiation toward the oligodendrocyte lineage. These cells include, but are not limited to, oligodendrocyte progenitor cells (OPCs), pre-myelinating oligodendrocytes, and mature myelinating oligodendrocytes. Oligodendrocyte lineage cells are characterized by the expression of one or more lineagespecific molecular markers such as PDGFRA, NG2, SOX10, OLIG2, NKX6-2, 04, 01, GALC, CNPase, MOG, and / or myelin basic protein (MBP). The term further refers to cells capable of acquiring morphological and / or functional characteristics of oligodendrocytes, including the extension of branched processes and the formation of myelin sheaths around neuronal axons. As used herein, oligodendrocyte lineage cells may include immature, intermediate, and mature forms generated in vitro and / or in vivo.

[0037] As used herein, the terms “comprising,” “including,” and “having” are used in an open-ended sense and do not exclude additional elements or steps not recited. The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0038] As used herein, “nonhuman primates” may be used to refer to primate species that are used in genetic research or more generally primates that are not humans. Exemplary species, to which the methods and systems described herein may apply, include, but are not limited to, rhesus macaques (Macaco mulatto), cynomolgus macaques (Macaco fascicularis),Docket No. 0138-706.600INTERNATIONAL APPLICATION baboons (Papio spp common marmosets (Callithrix jacchus), squirrel monkeys (Saimiri spp. African green monkeys (Chlor ocebus sabaeus). pigtailed macaques (Macaca nemestrind), and owl monkeys (Aotus lemurinus). In some embodiments, the systems and methods described herein may be applied to cells from cynomolgus macaques or Macaca fascicularis.NHP iPSCs from NHP PBMCs

[0039] NHP iPSCs may be derived from NHP PBMCs. For example, a method of deriving iPSCs from NHP PBMCs may include obtaining tissue or blood from a first nonhuman primate; isolating PBMCs from the first nonhuman primate; and deriving iPSCs from the PBMCs from the first nonhuman primate.

[0040] FIG. 3 shows an embodiment of deriving iPSCs from nonhuman primate PBMCs. The method 300 includes: obtaining peripheral blood mononuclear cells (PBMCs) from at least one individual of a population comprising a single species of nonhuman primate S310; culturing the PBMCs to expand blood progenitor cells (e.g., including CD34+ cells) in a hematopoietic stem cell (HSC) expansion medium for a pre-determined time period S320; transfecting the cultured PBMCs to reprogram the cultured cells into iPSCs S330; transferring the transfected cells into a container comprising a plurality of feeder cells S340; transferring the cells to a second container S350; on selected days after transfer to the second container, performing at least one of: adding medium to the cells; changing the medium in the cells; or adding one or more cell growth factors to the cells S360; and when a first cell colony appears; passaging the cells by placing each colony in a third container coated with an extracellular matrix S370. As used herein, medium may include, but is not limited to, DMEM medium, RPMI™ medium, F12™ medium, and the like. In some embodiments, specialty media are detailed below or elsewhere herein.

[0041] The method 300 may further include expanding the first colony for use in one or more of: an in vitro test, an in vivo test, or creation of a genome library for the individual primate.

[0042] In some embodiments, transfecting includes transfecting with a combination of transcription factors so 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, transcription factors such as Klf2, Nanog, Tfcp2Ll and Stat3 may additionally or alternatively be used.Docket No. 0138-706.600INTERNATIONAL APPLICATION

[0043] In some embodiments, the expanding may include washing the iPSCs with a buffer (e.g., phosphate-buffered saline) and incubating the iPSCs with a cell detachment solution, for example trypsin, a chelating agent, collagenase, or the like. In some embodiments, the incubating occurs at about 37 degrees Celsius 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.

[0044] In some embodiment of method 300, transferring the transfected cells to a container with feeder cells occurs no more than about one day post-transfection. The feeder cells may comprise mouse embryonic fibroblast (MEF) cells, SNL feeder cells, or the like.

[0045] In some embodiments, transferring the cells to the second container occurs no more than about one day after transferring to the container with the feeder cells.

[0046] In some embodiments, the expanding comprises aspirating and dissociating the iPSCs into a single cell suspension.

[0047] As described elsewhere herein, the iPSCs may be differentiated into main lineages. The main lineages may include at least one of: ectoderm, mesoderm, and endoderm.Additionally, or optionally, the main lineages, for example the ectoderm lineage, may be further differentiated into final tissues, for example NGN2 neurons, astroglia, astrocytes, oligodendrocytes, and the like. The final tissues may be used for in vitro testing, autologously translated into the first nonhuman primate for in vivo testing, and / or used in genome library generation.

[0048] FIG. 4 shows an embodiment of a method 400 for deriving iPSCs from nonhuman primate PBMCs. For example, peripheral blood mononuclear cells (PBMCs) are obtained from an individual from a species of nonhuman primate, and the PBMCs are isolated from the animals’ whole blood. The PBMCs may be isolated from the animals’ whole blood using density gradient centrifugation (DGC), fluorescence active cells sorting, magnetic bead-based separation, buoyancy activated cell sorting (BACS), and the like. In an embodiment, PBMCs are isolated using DGC. In an embodiment, PBMCs are isolated using BACS.

[0049] In some embodiments, a method 400 for reprogramming NHP PBMCs into iPSCs includes culturing 410 the PBMCs to expand blood progenitor cells (e.g., CD34+ cells) in a hematopoietic stem cell (HSC) expansion medium such as StemSpan™ (Stemcell Technologies Inc., Vancouver, British Columbia, Canada) for a period of about three days to about 10 days, about 5 days to about 10 days, about 6 days to about 10 days, about 7 days to about 10 days, about eight days to about 10 days, or about nine days. In some embodiments,Docket No. 0138-706.600INTERNATIONAL APPLICATION an alternative for StemSpan™ may be used. For example, a composition for the expansion of hemopoietic stems (HSPC) and / or progenitor cells of nonhuman primates may include IL3, IL6, FLT-3, TPO, and SCF (e.g., prior to transfection with reprogramming transcription factors). In some embodiments, animal serum (e.g., Fetal Bovine Serum) in the expansion medium may be replaced or combined with poly vinyl alcohol (PVA) to improve the efficiency of expansion of HSPC from nonhuman primates. CD34 is expressed not only by HSCs but by a multitude of other non-hematopoietic cell types, including muscle satellite cells, corneal keratocytes, interstitial cells, epithelial progenitors, and vascular endothelial progenitors. Thus, several different cell types may potentially exhibit progenitor activity and be expanded

[0050] Further, a method 400 for reprogramming PBMCs into iPSCs may include transfecting blood progenitor cells with transcription factors 420. The NHP PBMCs are reprogrammed into NHP induced pluripotent stem cells (iPSCs). The method for reprogramming the PBMCs into iPSCs may include transfecting the PBMCs with one or more transcription factors so that the cells are induced to overexpress the transcription factors. Transcription factors are one of the groups of proteins that read and interpret DNA. They bind to the DNA and help initiate a program of increased or decreased gene transcription. The stem cell derivation protocol is optimized for nonhuman primates specifically as there are different approaches taken to generate optimized stem cells for nonhuman primates. In some embodiments, transcription binding sites of these transcription factors may be mapped on individualized primate genomes to improve efficiency of the reprogramming process in NHPs, especially since transcription binding sites in human genomes are not completely conserved in NHP species. Thus, the use of human iPSCs reprogramming kits is not efficient for iPSC generation in NHP species. This is also important considering the fact that NHP genomes are very heterogenous and existing mutations in NHP genomes result in inefficient binding of transcription factors (from human iPSC generation kits) in nonhuman primate cells.

[0051] Various transfection methods may be used, including but not limited to, electroporation, calcium-phosphate exposure, liposome-based transfection, viral-mediated transfection (also known as transduction), and the like. In an embodiment, a method for transfecting PBMCs includes transduction. Transduction may be accomplished using, for example, a Sendai virus, an adenoviral, an oncoretroviral, or a lentiviral vector. In some variations, transfection includes using a Sendai virus vector. In some instances, transfectingDocket No. 0138-706.600INTERNATIONAL APPLICATIONPBMCs 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.

[0052] Further, a method 400 for reprogramming PBMCs into iPSCs may include coculturing the cells (e.g., transfected and untransfected) with feeder cells 430. The feeder cells may comprise mouse embryonic fibroblast (MEF) cells, SNL feeder cells, or the like. 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, for example about 1 :5. Co-culturing may occur on a subsequent day after transfection, for example 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.

[0053] Co-culturing may occur in Essential-8™ medium (Thermofisher Scientific). In an 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 uM to about 3 uM, about 1.5 uM to about 2.5 uM, about 1 uM to about 2.5 uM, about 1.5 uM to about 3 uM, about 1.75 uM to about 2.25 uM, etc. In some embodiments, the tankyrase 1 / 2 inhibitor may maintain the NHP iPSCs in a pluripotent state and may reduce spontaneous differentiation of the NHP iPSCs.

[0054] Further, a method 400 for reprogramming PBMCs into iPSCs may include transferring unadhered cells to new medium. For example, the transfer may occur 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. after co-culturing. The unadhered cells may be cultured in a basal medium, for example MEM medium, DMEM medium, RPMI medium, F12 ™ medium, and the like. Further, a method 400 for reprogramming PBMCs into iPSCs may optionally include adding medium to the adherent cells 440. The added media may comprise a medium that is feeder-free and xeno-free that supports the reprogramming of somatic cells and the spontaneous or directed differentiation of pluripotent stem cells (PSCs). In an embodiment, adding medium occurs about 48 hours to 96 hours, about 42 hours to about 102 hours, etc. after transfection.

[0055] Further, a method 400 for reprogramming PBMCs into iPSCs may include optionally replacing the medium on the adherent cells. The medium may be replaced after about 90 hours to about 144 hours, about 96 hours to about 144 hours, about 90 hours toDocket No. 0138-706.600INTERNATIONAL APPLICATION about 126 hours, about 114 hours to about 126 hours, etc. after transfection. The replacing medium may be a medium that is feeder-free and xeno-free that supports the reprogramming of somatic cells and the spontaneous or directed differentiation of pluripotent stem cells (PSCs).

[0056] Further, a method 400 for reprogramming PBMCs into iPSCs may include optionally replacing the medium with medium comprising one or more growth factors 450. For example, the medium may comprise a medium that is feeder-free and xeno-free that supports the reprogramming of somatic cells and the spontaneous or directed differentiation of pluripotent stem cells (PSCs). The growth factor may comprise basic fibroblast growth factor, DJ-1, epidermal growth factor, and / or the like. In an embodiment, the growth factor comprises basic fibroblast growth factor. The concentration of the growth factor (i.e., bFGF) may be about 100 ng / uL to about 200 ng / uL. In some embodiments, this particular growth factor and / or concentration is advantageous for the NHP iPSC derivation process since commercially available human bFGF is not as efficient in cross-reactivity with NHP cells. Medium replacement may occur 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. Medium replacement may be repeated daily for one or more days or a plurality of days, for example repeated once, twice, thrice, or more.

[0057] Further, a method 400 for reprogramming PBMCs into iPSCs may include optionally replacing the medium daily for one or more days or for a plurality of days 480. For example, the medium may be replaced between about 240 hours post-transfection to about 440 hours post-transfection. The medium may be replaced with basal medium, a medium comprising tumor growth factor-b (TGF-b) and / or b-FGF, or similar medium. In some embodiments, the medium may be replaced daily until visual identification of a first colony of iPSC.

[0058] Further, a method 400 for reprogramming PBMCs into iPSCs may include culturing the first colony of iPSC on a matrix 470, for example an extracellular matrix and / or the like. The first colony may be cultured in medium comprising a serine / threonine kinase inhibitor. The serine / threonine kinase may include a Rho inhibitor or a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, for example, Thiazovivin (TZV) or similar products. A concentration of the inhibitor may be about 1 uM to about 3 uM.Docket No. 0138-706.600INTERNATIONAL APPLICATION

[0059] Further, a method 400 for reprogramming PBMCs into iPSCs may include passaging the cells after about 12 hours to about 48 hours. Passaging 460 may include refreshing the medium and adding a Wnt pathway inhibitor, tankyrasel / 2 inhibitor, or the like. The inhibitor may be at a concentration of about 1 uM to about 3uM, about 1.5 uM to about 2.5 uM, about 1 uM to about 2.5 uM, about 1.5 uM to about 3 uM, about 1.75 uM to about 2.25 uM, etc.

[0060] Optionally, iPSCs may be passaged and / or expanded by washing with a buffer (e.g., phosphate buffered saline) and incubating the cells with a cell detachment solution. The cell detachment solution may comprise collagenase (or recombinant enzymes thereof), trypsin (or recombinant enzymes thereof), a chelating agent (e.g., ethylenediamine tetra-acetic acid), and the like.

[0061] Optionally, iPSCs may be expanded or maintained by culturing in medium on feeder cells or an extracellular matrix, as described elsewhere herein. The medium may comprise a serine / threonine kinase may comprise a Rho inhibitor or a Rho-associated coiled- coil containing protein kinase (ROCK) inhibitor. The inhibitor may be at a concentration of about 1 nM to about 3 nM, about 1.5 uM to about 2.5 uM, about 1 uM to about 2.5 uM, about1.5 uM to about 3 uM, about 1.75 uM to about 2.25 uM, etc.

[0062] In embodiments with subsequent passaging, the iPSCs may be incubated in a container, without feeder calls, the container having a substrate coated thereon. The substrate may be configured to retain pluripotency of the cells, a stable karyotype of the cells, and / or an expression of pluripotency markers by the cells. For example, the substrate may comprise laminin, a recombinant laminin fragment (e.g., 511 e8 fragment), vitronectin, recombinant vitronectin, or a combination thereof. The medium include E8-specific supplement (e.g., TGF-b, bFGF, etc.), and / or a Wnt pathway inhibitor, tankyrasel / 2 inhibitor, and / or the like. The inhibitor may be at a concentration of about 1 nM to about 3 nM, about 1.5 uM to about2.5 uM, about 1 uM to about 2.5 uM, about 1.5 uM to about 3 uM, about 1.75 uM to about 2.25 uM, etc. The subsequent passaging may result in colonies with little-to-no differentiation.

[0063] DIFFERENTIATION OF NHP iPSCs INTO NEURONAL LINEAGES

[0064] In some embodiments described herein, the methods may be used for differentiating NHP iPSCs to neuronal lineage cells, for example neural cells. In some embodiments, the differentiation may be from NHP iPSCs to oligodendrocytes. In some embodiments, the NHPDocket No. 0138-706.600INTERNATIONAL APPLICATION species include cynomolgus macaques (Macaca fascicularis), rhesus macaques Macaca mulatto), and pig-tailed macaque (Macaca nemestrina).

[0065] In some embodiments, the differentiated neuronal lineage cells can be used for in vitro safety and efficacy assessments as well as discovery of human drugs in iPSC-derived neuronal lineage cells in NHPs.

[0066] To differentiate NHP iPSCs into neuronal lineage cells, for example oligodendrocytes, or other neuronal lineage, a method may include optionally transitioning NHP iPSCs from a feeder-based culture condition (e.g., culturing iPSCs on MEF or other feeder cells) into a feeder-free condition. This transition into a feeder-free condition of NHP iPSCs may reduce or remove various small molecules that are secreted by the feeder cells into the culture media. For example, small molecules secreted by feeder cells can interrupt the process of differentiation of iPSCs to neural cells including oligodendrocytes.

[0067] As shown in FIGs. 1 and 5, a vector 100, and associated method, may be used for differentiating NHP iPSCs into various cell types. The vector 100 (e.g., SEQ ID NO. 1) may be used for differentiating NHP iPSCs, for example Macaca fascicularis iPSCs. The vector 100 may be a piggyBac vector. The vector 100 may include a nucleic acid sequence (e.g., SEQ ID NO. 8) encoding a selectable marker 110 (e.g., antibiotic resistance gene) for the selection of successfully transfected cells. For example, the nucleic acid sequence may encode the selectable marker 110 Puromycin (PuroR) between about 1113 base pairs and about 1712 base pairs as shown in SEQ ID NO. 1. The vector 100 may include a nucleic acid sequence (e.g., SEQ ID NO. 15) encoding a modified rtTA protein 120 that binds to promoters containing the tet operator in the presence of doxycycline. For example, SEQ ID NO. 15 may be between about 1788 base pairs and about 2531 base. The vector 100 may include a nucleic acid sequence (e.g., SEQ ID NOs. 6) encoding a Tet-responsive promoter 140. For example, the Tet-responsive promoter 140 may be a unidirectional promoter (SEQ ID NO. 6), for example as shown in FIGs. 1 and 5, or a bi-directional promoter. The Tet- responsive promoter 140 may be between about 3815 base pairs and about 4193 base pairs as shown in SEQ ID NO. 1. The vector 100 may include a nucleic acid sequence (e.g., SEQ ID NOs. 9, 11) encoding one or more origins of replication (Ori) that enable the vector to be replicated. As shown in SEQ ID NO. 1, an fl Ori 180 (e.g., SEQ ID NO. 11) may be between about 3 base pairs to about 458 base pairs for phage based single stranded DNA replication and packaging. Further, for example, as shown in SEQ ID NO. 1, an Ori 160 (e.g., SEQ ID NO. 9) may be between about 5852 base pairs to about 6440 base pairs for plasmidDocket No. 0138-706.600INTERNATIONAL APPLICATION amplification in bacterial cells in preparation for transfection. The vector 100 may include nucleic acid sequence (e.g., SEQ ID NO. 10) encoding a second selectable marker 170 (e.g., antibiotic resistance gene) for selection of those bacterial cells that are amplifying the vector. SEQ ID NO. 10 may be between about 6611 base pairs and about 7471 base pairs, as shown in SEQ ID NO. 1. For example, the nucleic acid sequence may encode Ampicillin (AmpR), which confers resistance to ampicillin, carbenicillin, and related antibiotics. The promoter 190 (e.g., SEQ ID NO. 14) for the selectable marker 170 may be between about 7472 base pairs and about 7576 base pairs, as shown in SEQ ID NO. 1. The vector 100 may optionally include a nucleic acid sequence (e.g., SEQ ID NOs. 13, 14) encoding a T7 promoter 184 for bacteriophage T7 RNA polymerase and / or a T3 promoter 194 for bacteriophage T3 RNA polymerase. For example, SEQ ID NO. 13 may be between about 623 base pairs and about 641 base pairs; and / or SEQ ID NO. 14 may be between about 5411 base pairs to about 5429 base pairs, as shown in SEQ ID NO. 1. The vector 100 may include a nucleic acid sequence (e.g., SEQ ID NO. 7) for encoding a constitutive promoter 130 (e.g., Ubiquitin C promoter or UbC) to drive the expression of one or more differentiation factor genes in the target cells. For example, SEQ ID NO. 7 may be between about 2545 base pairs and about 3754 base pairs, as shown in SEQ ID NO. 1.

[0068] Producing a modified vector 100 may include replacing a reporter gene 150, for example TurboGFP or SEQ ID NO. 5 (as shown in FIG. 1), with a differentiation factor gene 154 (as shown in FIG. 5) or inserting a differentiation factor gene 154 upstream or downstream of a report gene 150 (as shown in FIG. 5). For example, the reporter gene 150 may be used as a marker of transduction efficiency (i.e., cells that are not transduced may not survive in the presence of the compound used to transcribe the selectable marker). Although TurboGFP is shown and described herein, one of skill in the art will appreciate that other reporter genes may be used without departing from the intent and scope of the present disclosure. Alternative reporter genes include, but are not limited to, enhanced Green Fluorescent Protein (eGFP, excitation at 488 nm, emission at 507 nm), mCherry (excitation at 587 nm, emission at 610 nm), mVenus (excitation at 515 nm, emission at 528 nm), mKate2 (excitation at 588 nm, emission at 633 nm), TagRFP (excitation at 555 nm, emission at 584 nm), AmCyanl (excitation at 458 nm, emission at 489 nm), mCerulean3 (excitation at 433 nm, emission at 475 nm), tdTomato (excitation at 554 nm, emission at 581 nm), mRuby2 (excitation at 559 nm, emission at 600 nm), sfGFP (Superfolder GFP, excitation at 485 nm,Docket No. 0138-706.600INTERNATIONAL APPLICATION emission at 510 nm), and the like. The reporter gene 150 may be, for example, between about 4214 base pairs and about 4894 base pairs, as shown in SEQ ID NOs. 1 and 9.

[0069] The differentiation factor gene 154 may be Macaca fascicularis SOX 10 or SEQ ID NO. 2 (as shown in FIG. 5), Macaca fascicularis OLIG2 or SEQ ID NO. 3 (as shown in FIG. 5), and / or Macaca fascicularis NKX6-2 of SEQ ID NO. 4 (as shown in FIG. 5), inserted into vector 100, for example a piggyBac vector backbone (e.g., SEQ ID NO. 1, OTPL007- pbvector). Although sequences for Macaca fascicularis are shown and described herein, one of skill in the art will appreciate that sequences derived from Homo sapiens may also apply and / or be used in the transduction of NHP iPSCs given the sequence similarity between NHP genomes and the Homo sapiens ’ genome.

[0070] As shown in FIG. 5, to modify the vector 100 with differentiation factor gene 172 or SEQ ID NO. 4, the differentiation factor gene 172 or SEQ ID NO. 4 may be positioned upstream of the reporter gene 150 or replace the reporter gene 150. To modify the vector 100 with differentiation factor gene 174 or SEQ ID NO. 3, the differentiation factor gene 174 or SEQ ID NO. 3 may be positioned upstream of the reporter gene 150 or replace the reporter gene 150. To modify the vector 100 with differentiation factor gene 176 or SEQ ID NO. 2, the differentiation factor gene 176 or SEQ ID NO. 2 may be positioned upstream of the reporter gene 150 or replace the reporter gene 150. The vector 100 as modified as shown in FIG. 5 may be used to differentiate nonhuman primate iPSCs to oligodendrocytes. In some embodiments, SEQ ID NO. 4 may be positioned in vector 100 between about 6727 base pairs and about 7560 base pairs. In some embodiments, SEQ ID NO. 3 may be positioned in vector 100 between about 5692 base pairs and about 6660 base pairs. In some embodiments, SEQ ID NO. 2 may be positioned in vector 100 between about 4222 base pairs and about 5622 base pairs.

[0071] The vector 100 may be modified using restriction enzymes, Gibson Assembly, or the like to replace the reporter gene 150 with the differentiation factor gene 154. For example, using Gibson Assembly, the differentiation factor gene and the vector 100 may each include overlapping sequences, complementary to each another. The vector 100 may be linearized (e.g., using restriction digestions or PCR amplification) to create opens ends in the vector 100 for insertion of the gene. The gene and linearized vector may be mixed with an enzymatic reaction mixture, including for example, exonuclease, DNA polymerase, and DNA ligase. The reaction may be an isothermal reaction. For example, the reaction may be incubated at about 45 degrees to about 55 degrees to enable the gene to be inserted into theDocket No. 0138-706.600INTERNATIONAL APPLICATION vector. The recombinant vector may be transformed into bacterial cells that can replicate the vector using the Ori in the vector. Bacterial cells with successful replication of the vector may be selected using a selectable marker, for example Ampicillin resistance (i.e., using the AmpR gene of the vector).

[0072] The vectors may respond to doxycycline induction while having continuous puromycin selection. The Tet-On 3G is a modified rTtA protein that binds tight to the promoters containing the Tet operator in the presence of doxycycline. The vector 100 exhibits increased sensitivity to doxycycline, allowing for tighter control over gene expression levels compared to earlier Tet-On systems. By incorporating the puromycin resistance gene (PuroR), as shown in FIG. 2, alongside NGN2, cells expressing NGN2 (i.e., have been transduced) can be selectively cultured.

[0073] In some embodiments, a method 200 of transfecting NHP iPSCs may include transfecting the NHP iPSCs (e.g., electroporation the NHP iPSCs) with a vector expressing a differentiation factor gene 154 (e.g., any of SEQ ID NOs. 2-8, etc.) at block S210, and selecting for the transfected NHP iPSCs using a selectable marker (e.g., puromycin selection) at block S230, for example a selectable marker associated with vector 100. Th method 200 may optionally further include expanding the transfected NHP iPSCs on a matrix at block S220, for example an extracellular matrix, Matrigel®, Cultrex®, Geltrex®, and the like. The transfected NHP iPSCs may be expanded in medium (e.g., E8 basal media +E8 Supplement containing thiazovivin).

[0074] In some embodiments, a method 200 of transfecting NHP iPSCs may include transfecting the NHP iPSCs (e.g., electroporation the NHP iPSCs) with a vector expressing one or more differentiation factor genes 154 (e.g., SEQ ID NOs. 2-8, etc.) at block S210, and selecting for the transfected NHP iPSCs using a selectable marker (e.g., puromycin selection) at block S230, for example a selectable marker associated with vector 100. Th method 200 may optionally further include expanding the transfected NHP iPSCs on a matrix at block S220, for example an extracellular matrix, Matrigel®, Cultrex®, Geltrex®, and the like. The transfected NHP iPSCs may be expanded in medium (e.g., B8 medium, E8 basal medium + E8 Supplement, etc.). The medium may include or be supplemented with thiazovivin.

[0075] In some embodiments, as shown in FIG. 6, a method 600 of differentiating NHP iPSCs into neuronal lineage cells (e.g., oligodendrocyte cells) may include providing a modified vector including a piggyBac vector backbone expressing one or more differentiation factor genes (e.g., SEQ ID NOs. 7-9, etc.) at block S610; and transfecting the NHP iPSCsDocket No. 0138-706.600INTERNATIONAL APPLICATION(e.g., electroporation the NHP iPSCs) with the modified vector at block S620, for example vector 100 modified to express one or more differentiation factor genes. As described above for FIG. 2, transfecting may further include selecting the transfected NHP iPSCs using a selectable marker. Method 600 may further include culturing the transfected cells on a cell adhesion and growth surface at block S630; culturing the transfected cells in an induction medium at block S640; culturing the transfected cells in differentiation medium at block S650; and producing neuronal lineage cells at block S660.

[0076] As shown in FIG. 6, method 600 may further include block S630, which recites culturing the transfected cells on a cell adhesion and growth surface. The cell adhesion and growth surface may include an extracellular matrix, as described elsewhere herein. In some embodiments, the cell adhesion and growth matrix includes a surface coated with a poly amino acid and / or structural or scaffold protein. A poly amino acid may include, but not be limited to, polyornithine, poly-L-lysine, poly-D-lysine, poly-ethylenimine, poly-L-omithine hydrochloride, polyomithine, and the like. A structural or scaffold protein may include, but not be limited to, laminin, a synthetic hydrogel scaffold, polyLN521, a recombinant fragment E8 of laminin-511, decellularized extracellular matrix, and the like.

[0077] As shown in FIG. 6, method 600 may further include block S640, which recites culturing the transfected cells in an induction medium. The induction medium may include as a base medium any of Neurobasal medium, DMEM / F-12, RPMI 1640, IMDM, KOSR, a combination, and the like. For example, the induction medium may include DMEM / F-12. The induction medium may include one or more of: N2 supplement, a B27 supplement, glutamine, smoothed agonist (SAG), platelet-derived growth factor (PDGF), neurotrophin-3 (NT-3), insulin-like growth factor-1 (IGF-1), amino acids, trace elements B, and Triiodothyronine (T3).

[0078] The induction medium may include about 1 : 100 to about 1 :500; about 1 : 150 to about 1 :400; about 1 :100 to about 1 :300; about 1 : 100 to about 1 :250; about 1 : 150 to about 1 :250 N2 supplement. The induction medium may include about 1 :50 to about 1 : 150; about 1 :50 to about 1 :300; about 1 : 100 to about 1 : 150; about 1 :100 to about 1 :200 B27 supplement. In some embodiments, the B27 supplement may lack vitamin A. The induction medium may include about 0.1% to about 10%, about 0.5% to about 5%, about 0.5% to about 1.5%, about 0.75% to about 1.5%, etc. L-glutamine. The induction medium may include about 0.1 pM to about 10 pM, about 0.5 pM to about 5 pM, about 0.5 pM to about 1.5 pM, about 0.5 pM to about 3 pM, about 0.75 pM to about 2 pM, etc. SAG. The induction medium may includeDocket No. 0138-706.600INTERNATIONAL APPLICATION about 1 ng / mL to about 20 ng / mL, about 5 ng / mL to about 15 ng / mL, about 8 ng / mL to about 12 ng / mL, about 6 ng / mL to about 14 ng / mL, about 4 ng / mL to about 16 ng / mL, etc. PDGF. The induction medium may include about 1 ng / mL to about 20 ng / mL, about 5 ng / mL to about 15 ng / mL, about 8 ng / mL to about 12 ng / mL, about 6 ng / mL to about 14 ng / mL, about 4 ng / mL to about 16 ng / mL, etc. NT3. The induction medium may include about 1 ng / mL to about 20 ng / mL, about 5 ng / mL to about 15 ng / mL, about 8 ng / mL to about 12 ng / mL, about 6 ng / mL to about 14 ng / mL, about 4 ng / mL to about 16 ng / mL, etc. IGF-1. The induction medium may include about 100 pM to about 300 pM, about 50 pM to about 350 pM, about 150 pM to about 250 pM, about 175 pM to about 225 pM, about 150 pM to about 200 pM, about 200 pM to about 250 pM, etc. amino acids. The induction medium may include about 1 : 100 to about 1 :2,000, about 1 :500 to about 1 : 1,500, about 1 :750 to about 1 : 1,250, about 1 :500 to about 1 :1,000, about 1 : 1,000 to about 1 : 1,500, etc. Trace Elements B. The induction medium may include about 1 ng / mL to about 20 ng / mL, about 5 ng / mL to about 15 ng / mL, about 8 ng / mL to about 12 ng / mL, about 6 ng / mL to about 14 ng / mL, about 4 ng / mL to about 16 ng / mL, etc. T3. The induction medium may be replaced or exchanged every day, every other day, or every third day.

[0079] As shown in FIG. 6, method 600 may further include block S650, which recites culturing the transfected cells in differentiation medium. The differentiation medium may be added to the cells on about day three to about day 5, or on about day three, day four, or day five after transduction. The differentiation medium may include as a base medium any of Neurobasal medium, DMEM / F-12, RPMI 1640, IMDM, KOSR, a combination, and the like. For example, the differentiation medium may include DMEM / F-12. The differentiation medium may include one or more of: N2 supplement, B27 supplement, L-glutamine, T3, NT3, IGF-1, amino acids, Trace Elements B, and / or dbcAMP.

[0080] The differentiation medium may include about 1 :100 to about 1 :500; about 1 : 150 to about 1 :400; about 1 :100 to about 1 :300; about 1 : 100 to about 1 :250; about 1 : 150 to about 1 :250 N2 supplement. The differentiation medium may include about 1 :50 to about 1 : 150; about 1 :50 to about 1 :300; about 1 : 100 to about 1 : 150; about 1 : 100 to about 1 :200 B27 supplement. In some embodiments, the B27 supplement may lack vitamin A. The differentiation medium may include about 0.1% to about 10%, about 0.5% to about 5%, about 0.5% to about 1.5%, about 0.75% to about 1.5%, etc. L-glutamine. The differentiation medium may include about 10 ng / mL to about 100 ng / mL, about 50 ng / mL to about 70 ng / mL, about 30 ng / mL to about 90 ng / mL, about 45 ng / mL to about 75 ng / mL, about 40Docket No. 0138-706.600INTERNATIONAL APPLICATION ng / mL to about 80 ng / mL, etc. T3. The differentiation medium may include about 1 ng / mL to about 20 ng / mL, about 5 ng / mL to about 15 ng / mL, about 8 ng / mL to about 12 ng / mL, about 6 ng / mL to about 14 ng / mL, about 4 ng / mL to about 16 ng / mL, etc. NT3. The differentiation medium may include about 1 ng / mL to about 20 ng / mL, about 5 ng / mL to about 15 ng / mL, about 8 ng / mL to about 12 ng / mL, about 6 ng / mL to about 14 ng / mL, about 4 ng / mL to about 16 ng / mL, etc. IGF-1. The differentiation medium may include about 100 pM to about 300 pM, about 50 pM to about 350 pM, about 150 pM to about 250 pM, about 175 pM to about 225 pM, about 150 pM to about 200 pM, about 200 pM to about 250 pM, etc. amino acids. The differentiation medium may include about 1 : 100 to about 1 :2,000, about 1 :500 to about 1 : 1,500, about 1 :750 to about 1 : 1,250, about 1 :500 to about 1 : 1,000, about 1 : 1,000 to about 1 : 1,500, etc. Trace Elements B. The differentiation medium may include about 50 pM to about 300 pM, about 50 pM to about 200 pM, about 75 pM to about 125 pM, about 10 pM to about 500 pM, about 60 pM to about 140 pM, about 40 pM to about 160 pM, etc. dbcAMP. The differentiation medium may be changed or replaced every day, every other day, or every third day. In some embodiments, the differentiation medium is replaced at an interval for about one week to about 5 weeks, about two weeks to about 4 weeks, for about 2 weeks, for about three weeks, for about 4 weeks, etc. The cells may be detached (e.g., using an enzyme) after about 5 days to about 15 days, about 7 days to about 10 days, about 6 days to about 11 days, etc. of culturing in the differentiation medium.

[0081] The neuronal lineage cells at block S660 of method 600 may be identified using immunocytochemical and / or molecular markers that are characteristic of oligodendrocyte lineage differentiation. In some embodiments, the differentiated cells are stained with 04, which recognizes a sulfatide glycosphingolipid localized to the surface of immature and / or pre-myelinating oligodendrocytes, and with myelin basic protein (MBP) antibodies, which detect cytoplasmic myelin-associated proteins expressed during later stages of oligodendrocyte maturation. In some embodiments, 04 expression may first appear after about 5 to about 30 days, about 8 to about 25 days, about 10 to about 20 days, or about 12 to about 18 days following induction, indicating the presence of oligodendrocyte progenitor cells (OPCs). MBP expression may subsequently be observed after about 10 to about 60 days, about 15 to about 55 days, about 20 to about 50 days, about 25 to about 45 days, about 30 to about 40 days, and / or about 32 to about 38 days post-induction, corresponding to the transition from OPCs to mature myelinating oligodendrocytes.Docket No. 0138-706.600INTERNATIONAL APPLICATION

[0082] In some embodiments, dual staining with 04 and MBP may confirm co-expression in about 10% to about 98%, about 15% to about 95%, about 20% to about 90%, about 25% to about 85%, about 30% to about 80%, about 40% to about 75%, or about 50% to about 70% of total cells, depending on culture duration, medium composition, and / or vector dosage. Additional oligodendrocyte-associated markers such as 01, PDGFRA, CNPase, MOG, and / or GALC may be used in combination with 04 and MBP to confirm lineage specificity and maturation stage. The expression intensity of 04 and / or MBP may increase by about 1.2- fold to about 30-fold, about 1.5-fold to about 25-fold, about 2-fold to about 20-fold, about 3- fold to about 15-fold, about 4-fold to about 10-fold, or about 5-fold to about 8-fold relative to undifferentiated iPSCs.

[0083] Quantification of 04- and / or MBP-positive populations may be performed by flow cytometry, immunofluorescence microscopy, and / or Western blot analysis, each confirming the progression of Macaca fascicularis iPSCs toward the oligodendrocyte lineage. In some embodiments, RT-qPCR and / or single-cell RNA sequencing (scRNA-seq) may further confirm upregulation of lineage-specific genes including SOXIO, OLIG2, NKX6-2, MBP, and MOG, and downregulation of pluripotency markers such as OCT4, NANOG, and SOX2. The observed ratio of MBP to SOXIO transcript expression may range from about 1 : 1 to about 10: 1, about 2: 1 to about 8:1, about 3: 1 to about 7: 1, or about 4: 1 to about 6: 1, consistent with maturation of functional oligodendrocyte populations.

[0084] As noted above, additional oligodendrocyte-associated markers such as 01, PDGFRA, CNPase, MOG, and / or GALC may be used in combination with 04 and MBP to confirm lineage specificity, stage of maturation, and myelination potential. In some embodiments, 01 expression may emerge concurrently with or shortly after 04, typically within about 7 to about 35 days, about 10 to about 30 days, about 12 to about 25 days, or about 14 to about 22 days post-induction, indicating the presence of pre-myelinating oligodendrocytes. PDGFRA (platelet-derived growth factor receptor alpha) expression may be detected earlier, for example within about 2 to about 15 days, about 3 to about 12 days, about 4 to about 10 days, or about 5 to about 8 days following vector introduction, marking oligodendrocyte progenitor cells (OPCs) that retain proliferative capacity.

[0085] CNPase (2', 3 '-cyclic nucleotide 3 '-phosphodiesterase) and GALC (galactocerebrosidase) may be expressed during the transition from early to mature oligodendrocytes, generally observed after about 10 to about 60 days, about 15 to about 50 days, about 20 to about 45 days, about 25 to about 40 days, or about 30 to about 35 days postDocket No. 0138-706.600INTERNATIONAL APPLICATION induction. MOG (myelin oligodendrocyte glycoprotein) expression, which is typically associated with functional myelin sheath formation, may be detected after about 20 to about 75 days, about 25 to about 65 days, about 30 to about 55 days, about 35 to about 50 days, or about 40 to about 45 days of differentiation.

[0086] In some embodiments, combined immunostaining for 04, 01, PDGFRA, CNPase, GALC, MOG, and MBP provides a comprehensive panel to validate progressive differentiation from OPCs to myelinating oligodendrocytes. The proportion of cells expressing at least two markers indicative of advanced maturation (e.g., MBP and MOG or CNPase and GALC) may range from about 10% to about 95%, about 15% to about 90%, about 20% to about 85%, about 25% to about 80%, about 30% to about 75%, or about 40% to about 70% of the total differentiated cell population. Expression intensity, measured by immunofluorescence or flow cytometry, may increase by about 1.2-fold to about 50-fold, about 1.5-fold to about 40-fold, about 2-fold to about 30-fold, about 3-fold to about 20-fold, or about 4-fold to about 15-fold relative to undifferentiated iPSCs.

[0087] In some embodiments, co-expression of PDGFRA and CNPase may indicate the presence of transitional oligodendrocytes capable of proliferation and myelin protein synthesis, while co-expression of 04, MBP, and MOG may identify terminally differentiated oligodendrocytes engaged in membrane sheet formation and axonal wrapping. Quantitative RT-PCR and Western blot analyses may further confirm the upregulation of SOX10, OLIG2, and NKX6-2 transcription factors, along with elevated MBP, MOG, and CNPase transcripts, while pluripotency markers OCT4, NANOG, and SOX2 decrease to background levels.

[0088] The use of multiple oligodendrocyte-specific markers confers several advantages in validating and optimizing lineage commitment oiMacaca fascicularis iPSCs. First, multimarker profiling provides a quantitative framework to distinguish intermediate progenitor stages from fully differentiated oligodendrocytes, enabling reproducible tracking of differentiation kinetics. Second, simultaneous analysis of early and late markers (e.g., PDGFRA and MBP) enhances resolution across maturation stages, supporting standardized quality-control criteria for experimental reproducibility. Third, integrating 01, MOG, and MBP markers correlates directly with myelin sheath formation and therefore serves as a functional indicator of successful differentiation.

[0089] Further, multiplex marker detection minimizes false-positive lineage identification that may result from nonspecific glial or neuronal expression patterns. By combining membrane-bound, cytoplasmic, and nuclear markers, researchers can verify oligodendrocyteDocket No. 0138-706.600INTERNATIONAL APPLICATION identity with greater than about 90%, about 92%, about 94%, about 96%, about 97%, or about 98% confidence based on morphological and molecular correlation. The inclusion of quantitative thresholds for 04 and MBP co-expression (e.g., >25% to <85%) enables assessment of differentiation efficiency and vector performance across variable cell batches. Additionally, these molecular validation methods support translational applications, including preclinical modeling of demyelinating diseases and in vitro screening of remyelination therapies. The ability to reproducibly generate and confirm oligodendrocyte lineage cells using this vector system provides a reliable platform for comparative neurodevelopmental studies across primate species and enhances the predictive value of nonhuman primate models for human neurological disorders.

[0090] In some embodiments, the differentiated oligodendrocytes may exhibit membranesheet formation and / or branching morphology typical of myelinating glial cells, with average process lengths ranging from about 10 pm to about 500 pm, about 25 pm to about 400 pm, about 50 pm to about 300 pm, and / or about 75 pm to about 250 pm. These morphological features may be further confirmed through confocal microscopy or electron microscopy imaging, supporting the functional identity of the generated oligodendrocyte lineage cells.

[0091] FIG. 7 illustrates an embodiment of a vector 700 for differentiating nonhuman primate induced pluripotent stem cells (iPSCs) from Macaca fascicularis into oligodendrocytes. The vector 700 comprises a nucleic acid sequence 710 that encodes a piggyBac vector backbone 712. The piggyBac vector backbone 712 may provide a transposable genetic framework capable of stable genomic integration when used in conjunction with a piggyBac transposase. In some embodiments, the transposase mediates excision and reintegration of the vector backbone 712 at one or more target sequences within the host genome, including, but not limited to, TTAA sequences, AT -rich regions, TA dinucleotides, palindromic tetranucleotides, and / or other transposase-recognized motifs. Such target sequences may be located within genomic regions exhibiting base compositions of about 50% to about 75% adenine-thymine content, about 55% to about 70% adenine-thymine content, or about 60% to about 65% adenine-thymine content. In some embodiments, modified and / or engineered piggyBac transposases, including hyperactive variants such as hyPBase, PB- M7DA, and / or micro-PB, may exhibit relaxed sequence specificity that enables genomic integration at noncanonical motifs, including but not limited to TTA, TTAAA, AATT, TAAT, ATTA, and / or TTAT tetranucleotides. These modified transposases may include amino acid substitutions within the catalytic DDD domain, DNA-binding domain, and / or C-terminalDocket No. 0138-706.600INTERNATIONAL APPLICATION region that enhance DNA cleavage, strand-transfer efficiency, and / or overall transposition frequency while broadening target-site compatibility. In some embodiments, substitution variants at positions corresponding to residues 228-244, 250-262, 300-335, 400-425, 480- 505, and / or 510-525 of the canonical piggyBac transposase may increase excision activity by about 5% to about 900%, about 10% to about 800%, about 20% to about 700%, about 30% to about 600%, about 40% to about 500%, about 50% to about 400%, about 60% to about 350%, about 70% to about 300%, about 80% to about 250%, and / or about 90% to about 200% relative to wild-type transposase.

[0092] In some embodiments, the hyPBase and / or PB-M7DA variant may increase genomic integration frequency in nonhuman primate iPSCs by about 1.5-fold to about 100-fold, about 2-fold to about 80-fold, about 3-fold to about 60-fold, about 4-fold to about 50-fold, about 5- fold to about 40-fold, about 8-fold to about 30-fold, about 10-fold to about 25-fold, and / or about 12-fold to about 20-fold compared to unmodified piggyBac. Integration efficiency may result in successful insertion in about 5% to about 99%, about 10% to about 95%, about 15% to about 92%, about 20% to about 90%, about 25% to about 88%, about 30% to about 85%, about 35% to about 82%, about 40% to about 80%, and / or about 45% to about 78% of transfected iPSCs depending on sequence length, donor vector concentration, and / or cell type.

[0093] In certain embodiments, micro-PB, which lacks an N-terminal inhibitory region, may exhibit enhanced nuclear localization and increased transposition activity within compact chromatin regions or transcriptionally silent loci. Such enhancement may yield about a 1.1- fold to about 15-fold, about a 1.3-fold to about 12-fold, about a 1.5-fold to about 10-fold, about a 2-fold to about 8-fold, about a 2.5-fold to about 6-fold, and / or about a 3-fold to about 5-fold increase in genomic insertion frequency relative to canonical piggyBac transposase. Integration events may occur within both canonical TTAA motifs and alternative motifs, including TTAT, TTAAA, ATTA, TCAA, TAAA, AATA, and / or TAAT sequences, allowing broader distribution across promoter-proximal regions, open chromatin, and enhancer-rich domains. The proportion of insertions at noncanonical sites may range from about 0.5% to about 80%, about 1% to about 70%, about 3% to about 60%, about 5% to about 50%, about 8% to about 45%, about 10% to about 40%, about 12% to about 35%, about 15% to about 30%, about 18% to about 25%, and / or about 20% to about 22% of total integration events.

[0094] In embodiments directed to oligodendrocyte differentiation, the use of these hyperactive piggyBac transposase variants may facilitate integration of differentiation factor genes, such as Macaca fascicularis SOX10, Macaca fascicularis OLIG2, and / or MacacaDocket No. 0138-706.600INTERNATIONAL APPLICATION fascicularis NKX6-2, near transcriptionally active loci that support coordinated expression and downstream lineage commitment. In some embodiments, integration near promoter-associated open chromatin regions and / or CpG islands may enhance expression stability by about 10% to about 500%, about 20% to about 400%, about 30% to about 300%, about 40% to about 250%, about 50% to about 200%, and / or about 60% to about 150% relative to wild-type piggyBac integration patterns.

[0095] The enhanced targeting achieved using hyPBase, PB-M7DA, and / or micro-PB may reduce differentiation onset time from about 60 days to about 5-40 days, about 55 days to about 8-35 days, about 50 days to about 10-30 days, about 45 days to about 12-28 days, about 40 days to about 15-25 days, and / or about 35 days to about 18-22 days post-transfection. Differentiation efficiency toward oligodendrocyte lineages may increase by about 1.2-fold to about 40-fold, about 1.5-fold to about 35-fold, about 2-fold to about 30-fold, about 2.5-fold to about 25-fold, about 3-fold to about 20-fold, and / or about 5-fold to about 15-fold compared to control vectors employing standard transposase activity.

[0096] In some embodiments, the use of these engineered transposases results in sustained and / or enhanced expression of downstream oligodendrocyte lineage markers such as 04, PDGFRA, CNPase, MBP, and / or MOG. Marker expression may be detectable after about 5- 90 days, about 8-75 days, about 10-60 days, about 12-55 days, about 15-50 days, and / or about 18-45 days of differentiation. The proportion of cells expressing 04 and / or MBP may range from about 10% to about 98%, about 20% to about 95%, about 25% to about 92%, about 30% to about 90%, about 40% to about 88%, about 50% to about 85%, and / or about 60% to about 80% across independent differentiation replicates.

[0097] In certain embodiments, hyperactive piggyBac transposases also improve reproducibility across nonhuman primate iPSC lines by about 5% to about 90%, about 10% to about 80%, about 15% to about 70%, about 20% to about 60%, about 25% to about 55%, about 30% to about 50%, and / or about 35% to about 45% relative to unmodified transposases. This improvement results from preferential targeting to euchromatic regions and / or regulatory zones that remain transcriptionally accessible over multiple passages. Accordingly, the use of hyPBase, PB-M7DA, micro-PB, and / or related engineered piggyBac transposases provides a broader genomic target repertoire, higher integration and / or excision efficiency, improved positional expression stability, and reduced epigenetic silencing. These features collectively enhance the yield, reproducibility, and maturation of Macaca fascicularis iPSC-derived oligodendrocytes under feeder-free conditions.Docket No. 0138-706.600INTERNATIONAL APPLICATION

[0098] The frequency of excision and reintegration may range from about 30% to about 98%, about 40% to about 95%, about 50% to about 90%, or about 60% to about 85% of transfected cells, depending on vector concentration, transposase-to-DNA ratio, and chromatin accessibility. In an embodiment, excision may occur within about 1 hour to about 72 hours, about 3 hours to about 60 hours, and / or about 6 hours to about 48 hours following introduction of the transposase, while reintegration may be detected within about 8 hours to about 96 hours post-transfection.

[0099] Stable integration may result in one to about ten, about two to about eight, or about three to about six copies of the vector backbone 712 per cell, distributed within transcriptionally permissive chromatin domains or open euchromatin regions that support sustained gene expression. In some embodiments, transposase-mediated integration occurs without sequence duplication beyond a range of about 4 base pairs to about 10 base pairs, about 5 base pairs to about 8 base pairs, and / or about 6 base pairs to about 7 base pairs surrounding the target motif, thereby maintaining genomic integrity.

[0100] In an embodiment, the piggyBac vector backbone 712 includes terminal inverted repeats that flank the differentiation factor gene 718 and transposase recognition sites 716 to facilitate one or more excision and reinsertion events. The terminal inverted repeats may range from about 10 base pairs to about 500 base pairs, about 25 base pairs to about 400 base pairs, about 50 base pairs to about 350 base pairs, about 75 base pairs to about 300 base pairs, about 100 base pairs to about 250 base pairs, and / or about 150 base pairs to about 200 base pairs in length, and may exhibit about 90% to about 99.9% sequence identity with canonical piggyBac terminal repeat motifs.

[0101] In some embodiments, the amount of piggyBac transposase introduced into the host cells may range from about 5 ng to about 1,000 ng, about 10 ng to about 800 ng, about 25 ng to about 600 ng, about 50 ng to about 400 ng, about 75 ng to about 300 ng, and / or about 100 ng to about 200 ng per transfection reaction. The transfection process may be conducted at an electroporation voltage ranging from about 600 V / cm to about 2,000 V / cm, about 800 V / cm to about 1,800 V / cm, about 1,000 V / cm to about 1,600 V / cm, and / or about 1,200 V / cm to about 1,400 V / cm, with a pulse duration ranging from about 1 ms to about 20 ms, about 2 ms to about 15 ms, and / or about 5 ms to about 10 ms.

[0102] Following successful transposition, the piggyBac vector backbone 712 may maintain stable, long-term expression of the integrated differentiation factor gene 718 across multiple passages. The duration of stable expression may persist for about 3 passages to about 50Docket No. 0138-706.600INTERNATIONAL APPLICATION passages, about 5 passages to about 40 passages, about 8 passages to about 30 passages, about 10 passages to about 25 passages, and / or about 12 passages to about 20 passages during continuous culture without detectable transgene silencing. In some embodiments, excision of the piggyBac vector backbone 712 may be achieved by reintroduction of the transposase under conditions that promote TTAA sequence recognition, allowing removal of the integrated genetic element without residual vector fragments.

[0103] In some embodiments, the transposase mediates excision and reintegration of the vector backbone 712 at one or more target sequences within the host genome, including, but not limited to, TTAA sequences, AT-rich regions, TA dinucleotides, palindromic tetranucleotides, and or other transposase-recognized motifs, thereby facilitating stable genomic insertion of a differentiation factor gene that promotes oligodendrocyte lineage commitment. Such target sequences may occur within genomic regions exhibiting about 50% to about 75%, about 55% to about 70%, or about 60% to about 65% adenine-thymine content. In some embodiments, modified or engineered piggyBac transposases, including hyperactive or sequence-relaxed variants such as hyPBase, PB-M7DA, and / or micro-PB, may enable integration at noncanonical motifs, such as TTA, TTAAA, and / or AATT, while preserving transgene fidelity.

[0104] The frequency of excision and reintegration may range from about 30% to about 98%, about 40% to about 95%, about 50% to about 90%, or about 60% to about 85% of transfected cells, depending on the transposase-to-vector ratio, genomic accessibility, and the stage of induced pluripotent stem cell differentiation. Excision may occur within about 1 hour to about 72 hours, about 3 hours to about 60 hours, or about 6 hours to about 48 hours after introduction of the transposase, and reintegration may be detected within about 8 hours to about 96 hours post-transfection.

[0105] Stable integration may result in one to about ten, about two to about eight, or about three to about six copies of the vector backbone 712 per cell, typically positioned in transcriptionally active chromatin regions conducive to differentiation factor gene expression. The resulting expression of differentiation factor genes such as SOX10, OLIG2, or NKX6-2 promotes sequential activation of oligodendrocyte lineage markers, including 04 and myelin basic protein (MBP), thereby facilitating controlled maturation of the nonhuman primate iPSCs into oligodendrocytes. In some embodiments, transposase-mediated integration maintains genomic stability by limiting duplication to about 4 base pairs to about 10 base pairs, about 5 base pairs to about 8 base pairs, and / or about 6 base pairs to about 7 base pairs surrounding theDocket No. 0138-706.600INTERNATIONAL APPLICATION target motif, enabling long-term maintenance of the integrated sequence during oligodendrocyte differentiation and expansion.

[0106] Following stable integration of the vector backbone 712, transcriptional activation of the differentiation factor gene may be initiated under the control of a constitutive or inducible promoter, for example a Tet-responsive promoter in combination with doxycycline induction. In some embodiments, promoter activation occurs within about 12 hours to about 72 hours, about 18 hours to about 60 hours, and / or about 24 hours to about 48 hours following induction. The activated transgene expression initiates an intracellular signaling cascade that directs the nonhuman primate iPSCs toward an oligodendrocyte lineage through progressive upregulation of lineage-associated transcriptional regulators.

[0107] The transfected cells may then be maintained in an induction medium or differentiation medium formulated to enhance oligodendrocyte maturation. The medium may include, but is not limited to, Neurobasal medium or DMEM / F-12 supplemented with N2 and B27 supplements, L-glutamine, platelet-derived growth factor (PDGF), neurotrophin-3 (NT3), insulin-like growth factor-1 (IGF-1), Triiodothyronine (T3), and dibutyryl cyclic AMP (dbcAMP). Concentrations of these supplements may range, for example, from about 1 ng / mL to about 20 ng / mL for NT3, PDGF, or IGF-1; from about 10 ng / mL to about 100 ng / mL for T3; and / or from about 50 pM to about 300 pM for dbcAMP. The induction and / or differentiation media may be replaced at intervals ranging from about every day to about every third day, and / or over a total differentiation period of about one week to about five weeks, about two weeks to about four weeks, and / or about three weeks to about four weeks.

[0108] Over the course of the differentiation period, expression of oligodendrocyte lineage markers such as 04, 01, and myelin basic protein (MBP) may be detected, confirming transition from pluripotent stem cells through oligodendrocyte precursor stages to mature oligodendrocytes. In some embodiments, the integrated differentiation factor gene maintains consistent expression for about 10 days to about 50 days, about 15 days to about 40 days, or about 20 days to about 35 days, supporting stable lineage commitment and / or functional maturation suitable for in vitro modeling and / or in vivo translational studies.

[0109] The piggyBac vector backbone 712 includes one or more terminal inverted repeats 714 and one or more transposase recognition sites 716. The terminal inverted repeats 714 define the boundary regions recognized by the piggyBac transposase, while the transposase recognition sites 716 are configured to interact with the piggyBac transposase to mediate excision and insertion of the vector 700 into one or more TTAA target sequences of the hostDocket No. 0138-706.600INTERNATIONAL APPLICATION genome. Integrated within the piggyBac vector backbone 712 is a differentiation factor gene 718. The differentiation factor gene 718 is selected and positioned to promote differentiation of transfected Macaca fascicularis iPSCs toward an oligodendrocyte lineage. In some embodiments, the differentiation factor gene 718 encodes a transcriptional regulator or signaling protein associated with oligodendrocyte development and maturation. When the vector 700 is introduced into iPSCs, expression of the differentiation factor gene 718 activates oligodendrocyte-lineage regulatory pathways and induces the expression of cellular markers characteristic of mature oligodendrocytes. In operation, the piggyBac transposase recognizes the terminal inverted repeats 714 and transposase recognition sites 716, catalyzing the excision of the nucleic acid sequence 710 from a donor construct and its integration into the genomic TTAA target sequences of the host iPSCs. The differentiation factor gene 718 thereby becomes stably integrated into the host genome and expressed under the control of suitable promoter elements. In some embodiments, the promoter may include a constitutive promoter, such as a ubiquitin C (UbC) promoter, cytomegalovirus (CMV) promoter, or elongation factor- 1 alpha (EFla) promoter, each configured to maintain continuous transcription of the integrated gene across multiple passages. In other embodiments, the promoter may be inducible, for example a tetracycline-responsive (Tet-On or Tet-Off) promoter that enables regulated expression of the differentiation factor gene 718 in the presence or absence of doxycycline at concentrations ranging from about 0.1 pg / mL to about 10 pg / mL, about 0.5 pg / mL to about 5 pg / mL, or about1 pg / mL to about 3 pg / mL.

[0110] In some embodiments, the integration occurs at one or more TTAA target sequences within the host genome, providing stable expression without disrupting coding regions. The number of integration events may range from about one to about ten, about two to about eight, or about three to about six per cell, depending on transfection efficiency and vector copy number. Following integration, transcription of the differentiation factor gene 718 may be detected within about 12 hours to about 72 hours post-transfection, about 18 hours to about 60 hours, or about 24 hours to about 48 hours, using quantitative PCR or immunocytochemical analysis.

[0111] In certain embodiments, expression of the differentiation factor gene 718 induces activation of transcriptional cascades associated with oligodendrocyte differentiation, including the upregulation of myelin basic protein (MBP), oligodendrocyte transcription factor2 (OLIG2), and SOX10. Protein expression levels may increase by about 1.5-fold to about 10- fold, about 2-fold to about 8-fold, or about 3 -fold to about 6-fold relative to untransfectedDocket No. 0138-706.600INTERNATIONAL APPLICATION control iPSCs. Under defined culture conditions comprising N2 and B27 supplements and growth factors such as PDGF, NT3, IGF-1, and T3, the transfected cells exhibit morphological and molecular characteristics consistent with oligodendrocyte lineage maturation, including membrane process elaboration and myelin marker expression after about one week to about five weeks, about two weeks to about four weeks, or about three weeks of differentiation.

[0112] The resulting transfected iPSCs acquire the capacity to differentiate into oligodendrocyte lineage cells under appropriate culture conditions.

[0113] In certain embodiments, the vector 700 may further comprise additional functional elements such as one or more selectable markers, constitutive and / or inducible promoters, and / or one or more reporter genes to facilitate selection, expression control, and / or monitoring of an integration event.

[0114] FIG. 8 illustrates an embodiment of a vector 700 configured for differentiating nonhuman primate induced pluripotent stem cells (iPSCs) from Macaca fascicularis into oligodendrocytes. The vector 700 includes a nucleic acid sequence 710 comprising a piggyBac vector backbone 712. The piggyBac vector backbone 712 provides a transposable genetic framework that supports stable genomic integration of one or more inserted sequences when used with a piggyBac transposase or a functional variant thereof.

[0115] In some embodiments, the piggyBac vector backbone 712 includes a selectable marker 802 and a promoter 804. The selectable marker 802 is configured to confer antibiotic resistance to enable selective isolation of successfully transfected iPSCs. In one example, the selectable marker 802 may encode puromycin N-acetyltransferase to confer puromycin resistance. The concentration of puromycin used during selection may range from about 0.05 pg / mL to about 10 pg / mL, about 0.1 pg / mL to about 8 pg / mL, about 0.25 pg / mL to about 5 pg / mL, about 0.5 pg / mL to about 2.5 pg / mL, or about 0.75 pg / mL to about 1.5 pg / mL. In other embodiments, the selectable marker 802 may encode resistance to alternative antibiotics, including neomycin, hygromycin, and / or blasticidin, at concentrations ranging from about 10 pg / mL to about 500 pg / mL, about 25 pg / mL to about 400 pg / mL, and / or about 50 pg / mL to about 200 pg / mL.

[0116] The promoter 804 may be a constitutive or inducible promoter designed to control transcription of one or more differentiation factor genes integrated within the piggyBac vector backbone 712. In certain embodiments, the promoter 804 may be a cytomegalovirus (CMV) promoter, a ubiquitin-C (UbC) promoter, or an elongation factor 1 -alpha (EFla) promoter for constitutive expression. In other embodiments, the promoter 804 may be anDocket No. 0138-706.600INTERNATIONAL APPLICATION inducible promoter such as a tetracycline-responsive (Tet-On or Tet-Off) promoter that enables regulated expression of the differentiation factor gene upon administration and / or withdrawal of doxycycline. The doxycycline concentration used for induction may range from about 0.01 pg / mL to about 20 pg / mL, about 0.05 pg / mL to about 10 pg / mL, about 0.1 pg / mL to about 5 pg / mL, about 0.25 pg / mL to about 3 pg / mL, and / or about 0.5 pg / mL to about 2 pg / mL.

[0117] The differentiation factor gene 718 is integrated within the piggyBac vector backbone 712 and configured to induce oligodendrocyte lineage differentiation in one or more transfected iPSCs. In some embodiments, the differentiation factor gene 718 may include one or more sequences selected from Macaca fascicularis SOX10 815, Macaca fascicularis OLIG2 816, and Macaca fascicularis NKX6-2 818. Each of these differentiation factor genes may independently regulate a distinct phase of oligodendrocyte development. For example, SOX10 may promote specification of glial progenitors; OLIG2 may regulate precursor proliferation and patterning of the ventral neural tube; and / or NKX6-2 may promote final maturation of myelinating oligodendrocytes.

[0118] In certain embodiments, the vector 700 may comprise a single differentiation factor gene or a combination of two or more such genes expressed sequentially or concurrently under the control of one or more promoters. For instance, the ratio of SOX10:OLIG2 expression may range from about 1 : 1 to about 1 : 10, about 1 :2 to about 1 :8, or about 1 :3 to about 1 :5, depending on the desired level of precursor versus mature oligodendrocyte differentiation. Similarly, the NKX6-2 gene may be activated at about day 7 to about day 28 post-transfection, about day 10 to about day 24, and / or about day 14 to about day 21 of differentiation to promote myelin-associated gene expression.

[0119] Upon introduction of the vector 700 into Macaca fascicularis iPSCs, expression of the differentiation factor genes initiates transcriptional cascades associated with oligodendrocyte lineage progression. The vector may be introduced using electroporation, lipofection, or nucleofection at voltages ranging from about 800 V / cm to about 1,800 V / cm, about 1,000 V / cm to about 1,600 V / cm, or about 1,200 V / cm to about 1,400 V / cm. The resulting transfected iPSCs exhibit stable genomic integration of the differentiation factor gene and sustained expression for about 7 days to about 60 days, about 10 days to about 50 days, and / or about 14 days to about 40 days post-transfection.

[0120] During induction, the transfected iPSCs may be cultured in an induction medium or differentiation medium that supports oligodendrocyte development. The medium mayDocket No. 0138-706.600INTERNATIONAL APPLICATION comprise DMEM / F-12 or Neurobasal medium supplemented with N2 and B27 supplements, L-glutamine, platelet-derived growth factor (PDGF), neurotrophin-3 (NT3), insulin-like growth factor 1 (IGF-1), triiodothyronine (T3), and dibutyryl cyclic AMP (dbcAMP). Concentrations of PDGF, NT3, and IGF-1 may range from about 1 ng / mL to about 50 ng / mL, about 5 ng / mL to about 40 ng / mL, and / or about 10 ng / mL to about 30 ng / mL. The concentration of T3 may range from about 5 ng / mL to about 100 ng / mL, about 10 ng / mL to about 80 ng / mL, or about 20 ng / mL to about 60 ng / mL, while dbcAMP may range from about 10 pM to about 500 pM, about 25 pM to about 400 pM, and / or about 50 pM to about 300 pM. The differentiation medium may be replenished every day, every other day, or every third day, over a total differentiation period ranging from about one week to about six weeks, about two weeks to about five weeks, or about three weeks to about four weeks.

[0121] Following induction, the resulting oligodendrocyte-lineage cells may express markers including 04, 01, and myelin basic protein (MBP). Expression may increase by about 1.5-fold to about 15-fold, about 2-fold to about 12-fold, and / or about 3-fold to about 10-fold relative to untransfected controls, confirming successful differentiation. In some embodiments, the integrated vector 700 may remain stably expressed across about 5 to about 30 passages, about 8 to about 25 passages, and / or about 10 to about 20 passages of continuous culture without gene silencing and / or loss of function.

[0122] FIG. 9 illustrates an embodiment of a vector 700 comprising a nucleic acid sequence 710 that includes a piggyBac vector backbone 712. The piggyBac vector backbone 712 includes a differentiation factor gene 718 that is configured to be inserted into the vector backbone in vitro prior to transposase-mediated genomic integration. The differentiation factor gene 718 may be integrated into the piggyBac vector backbone 712 using Gibson Assembly 902 and / or restriction enzyme digestion and ligation 904, each providing a molecular cloning approach suitable for generating a stable, transposable expression construct for differentiating nonhuman primate iPSCs 908.

[0123] In some embodiments, Gibson Assembly 902 is used to insert the differentiation factor gene 718 into the piggyBac vector backbone 712. Gibson Assembly may be performed in vitro using overlapping DNA fragments with complementary sequences, allowing the insert and vector fragments to anneal and be covalently joined in a single reaction. The reaction may include an exonuclease, a DNA polymerase, and a DNA ligase, and may be conducted at a temperature ranging from about 40°C to about 60°C, about 45°C to about 58°C, or about 48°C to about 55°C. The reaction duration may range from about 15 minutesDocket No. 0138-706.600INTERNATIONAL APPLICATION to about 120 minutes, about 20 minutes to about 90 minutes, or about 30 minutes to about 60 minutes. The overlapping sequence between the insert and the vector may range from about 10 base pairs to about 100 base pairs, about 15 base pairs to about 75 base pairs, or about 20 base pairs to about 50 base pairs to ensure efficient and accurate assembly.

[0124] In other embodiments, restriction enzyme digestion and / or ligation 904 is used to insert the differentiation factor gene 718 into the piggyBac vector backbone 712. Restriction digestion may employ one or more restriction endonucleases, including EcoRI, BamHI, Notl, Hindlll, or Xhol, or other enzymes that generate compatible cohesive or blunt ends. The digestion may be carried out at temperatures ranging from about 30°C to about 45°C, about 35°C to about 40°C, or about 37°C for durations of about 10 minutes to about 2 hours, about 20 minutes to about 90 minutes, or about 30 minutes to about 60 minutes. Ligation may be performed using T4 DNA ligase at concentrations ranging from about 100 units / mL to about 1,000 units / mL, about 200 units / mL to about 800 units / mL, or about 300 units / mL to about 600 units / mL, with incubation periods ranging from about 10 minutes to about 4 hours, about 15 minutes to about 3 hours, or about 30 minutes to about 2 hours.

[0125] Once assembled, the recombinant vector 700 may be introduced into nonhuman primate iPSCs 908 by electroporation 906. The electroporation process may include one or more voltage pulses at intensities ranging from about 600 V / cm to about 2,000 V / cm, about 800 V / cm to about 1,800 V / cm, about 1,000 V / cm to about 1,600 V / cm, or about 1,200 V / cm to about 1,400 V / cm, with pulse durations ranging from about 1 ms to about 20 ms, about 2 ms to about 15 ms, or about 5 ms to about 10 ms. The electroporation buffer may include isotonic solutions comprising magnesium chloride, potassium phosphate, or HEPES at pH values ranging from about 6.8 to about 7.6, about 7.0 to about 7.4, and / or about 7.2 to about 7.3. The DNA concentration may range from about 0.1 pg / pL to about 10 pg / pL, about 0.25 pg / pL to about 8 pg / pL, about 0.5 pg / pL to about 5 pg / pL, and / or about 1 pg / pL to about 3 pg / pL per 1 x 106 cells.

[0126] Following electroporation, the transfected iPSCs 908 may be cultured under antibiotic selection to isolate cells that have stably incorporated the vector 700. In some embodiments, the transfection efficiency may range from about 30% to about 95%, about 40% to about 90%, and / or about 50% to about 85%, depending on electroporation conditions, plasmid concentration, and cellular confluency. The vector 700 may subsequently undergo transposase-mediated genomic integration, resulting in stable insertion of the differentiation factor gene 718 and sustained expression in the iPSCs 908. The integrated construct may thenDocket No. 0138-706.600INTERNATIONAL APPLICATION be used to direct lineage-specific differentiation toward oligodendrocytes when cultured under induction or differentiation conditions as described in connection with FIGS. 7 and 8.

[0127] In some embodiments, differentiating the nonhuman primate induced pluripotent stem cells (iPSCs) comprises transfecting the iPSCs with a vector as described herein. The transfecting step may include introducing the vector into the iPSCs using electroporation, nucleofection, lipofection, and / or viral-free transduction. In some embodiments, electroporation may be performed at voltages ranging from about 600 V / cm to about 2,000 V / cm, about 800 V / cm to about 1,800 V / cm, about 1,000 V / cm to about 1,600 V / cm, and / or about 1,200 V / cm to about 1,400 V / cm with one or more pulses having durations between about 1 ms and about 20 ms, about 2 ms and about 15 ms, and / or about 5 ms and about 10 ms. The concentration of the vector introduced into the iPSCs may range from about 0.1 pg / pL to about 10 pg / pL, about 0.25 pg / pL to about 8 pg / pL, about 0.5 pg / pL to about 5 pg / pL, and / or about 1 pg / pL to about 3 pg / pL per 1 x 106cells. The transfection medium may include isotonic buffers containing magnesium chloride, potassium phosphate, and / or HEPES adjusted to a pH of about 6.8 to about 7.6, about 7.0 to about 7.4, and / or about 7.2 to about 7.3.

[0128] In some embodiments, the differentiation factor gene incorporated within the piggyBac vector backbone comprises SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4. SEQ ID NO. 2 corresponds to Macaca fascicularis SOX10, SEQ ID NO. 3 corresponds to Macaca fascicularis OLIG2, and SEQ ID NO. 4 corresponds to Macaca fascicularis NKX6- 2. These genes represent critical transcriptional regulators of oligodendrocyte lineage progression. SOX10 initiates early glial lineage commitment, OLIG2 regulates the proliferation and specification of oligodendrocyte precursor cells, and / or NKX6-2 promotes terminal maturation and myelination. In some embodiments, the combination of SOX10, OLIG2, and / or NKX6-2 expression may occur sequentially or simultaneously to coordinate the transition from pluripotent to differentiated states.

[0129] The relative timing and expression intensity of each differentiation factor gene may be modulated by promoter strength and induction timing. In an embodiment, SOX10 expression may occur between about day 1 and about day 10 post-transfection, OLIG2 expression between about day 5 and about day 20, and NKX6-2 expression between about day 10 and about day 30. In some examples, the differentiation factor genes may be expressed at ratios ranging from about 1 : 1 : 1 to about 1 :5:3, about 1 :3:2 to about 1 :4:2, or about 1 :2:2 relative to one another, depending on the desired maturation efficiency. TheDocket No. 0138-706.600INTERNATIONAL APPLICATION resulting expression cascade may induce the iPSCs to express oligodendrocyte lineage markers such as PDGFRA, 04, 01, and / or myelin basic protein (MBP).

[0130] In some embodiments, the transfected iPSCs are cultured in a differentiation medium configured to promote oligodendrocyte lineage specification. The medium may include Neurobasal or DMEM / F-12 as a base medium supplemented with N2 and B27, L- glutamine, platelet-derived growth factor (PDGF), neurotrophin-3 (NT3), insulin-like growth factor-1 (IGF-1), triiodothyronine (T3), and dibutyryl cyclic AMP (dbcAMP). Concentrations of PDGF, NT3, and IGF-1 may range from about 1 ng / mL to about 50 ng / mL, about 5 ng / mL to about 40 ng / mL, and / or about 10 ng / mL to about 30 ng / mL. The concentration of T3 may range from about 5 ng / mL to about 100 ng / mL, about 10 ng / mL to about 80 ng / mL, or about 20 ng / mL to about 60 ng / mL, and dbcAMP may range from about 10 pM to about 500 pM, about 25 pM to about 400 pM, or about 50 pM to about 300 pM. The differentiation medium may be replaced daily, every other day, or every third day, for a total duration ranging from about one week to about six weeks, about two weeks to about five weeks, and / or about three weeks to about four weeks.

[0131] In some embodiments, the differentiated nonhuman primate iPSCs exhibit morphological and molecular features of mature oligodendrocytes, including branched processes and expression of myelination markers such as MBP and proteolipid protein 1 (PLP1). The efficiency of differentiation may range from about 30% to about 95%, about 40% to about 90%, and / or about 50% to about 85% of total surviving transfected cells, depending on induction timing and growth factor concentration.

[0132] In certain embodiments, the piggyBac vector backbone comprises SEQ ID NO. 1. SEQ ID NO. 1 provides the backbone sequence for vector assembly and includes one or more terminal inverted repeats and transposase recognition sites. These structural features facilitate site-specific excision and integration of the inserted differentiation factor gene into genomic loci. In some embodiments, the transposase-mediated integration occurs at TTAA or other transposase-compatible motifs, including AT -rich and / or TA-dinucleotide sites, at frequencies ranging from about 30% to about 98%, about 40% to about 95%, and / or about 50% to about 90% per population of transfected cells.

[0133] In some embodiments, stable integration of the vector backbone containing SEQ ID NO. 1 results in one to about ten, about two to about eight, or about three to about six integrated copies per cell. The integrated construct may maintain expression of the differentiation factor genes over extended passaging, for example across about 5 to about 30Docket No. 0138-706.600INTERNATIONAL APPLICATION passages, about 10 to about 25 passages, and / or about 12 to about 20 passages, confirming long-term genomic stability suitable for in vitro and in vivo modeling of oligodendrocyte development in nonhuman primate systems.

[0134] The disclosed vectors and methods provide multiple technical benefits that enhance the efficiency, reproducibility, and stability of induced differentiation of nonhuman primate induced pluripotent stem cells (iPSCs) into oligodendrocytes. By employing a piggyBac vector backbone, the invention enables stable genomic integration of one or more differentiation factor genes under physiological conditions without the insertional mutagenesis risks commonly associated with viral systems. The piggyBac transposase mediates precise excision and reintegration events at TTAA and other AT-rich target motifs, ensuring predictable, high-fidelity genomic incorporation.

[0135] In some embodiments, the use of Macaca fascicularis-specific differentiation factor genes, including SOX10, OLIG2, and / or NKX6-2, improves lineage-specific induction by leveraging species-optimized codon usage and conserved promoter compatibility. This approach enhances transcriptional activity and translation efficiency by about 10% to about 80%, about 20% to about 70%, and / or about 30% to about 60% relative to human homolog sequences. The simultaneous or sequential expression of these genes produces a synergistic activation of the glial lineage program, promoting coordinated oligodendrocyte precursor specification and terminal maturation characterized by myelin basic protein (MBP) expression and process arborization.

[0136] Another advantage of the described piggyBac-based vector system is its compatibility with nonviral cloning methods such as Gibson Assembly and restriction enzyme digestion and ligation. These methods allow flexible and efficient construction of expression cassettes in vitro, supporting fragment sizes from about 1 kilobase to about 20 kilobases, about 2 kilobases to about 15 kilobases, or about 3 kilobases to about 10 kilobases. The ability to interchange promoters, selectable markers, and / or coding sequences provides modularity and reduces development time for optimizing transcriptional control elements.

[0137] The inclusion of an inducible promoter, such as a tetracycline-responsive element, provides precise temporal control of differentiation factor gene activation. This enables staged induction of SOX10, OLIG2, and NKX6-2 expression, allowing users to mimic natural developmental timing. Controlled induction reduces premature differentiation and maintains high viability during early expansion phases. In some examples, cell survival ratesDocket No. 0138-706.600INTERNATIONAL APPLICATION increase by about 15% to about 70%, about 25% to about 60%, and / or about 30% to about 50% relative to uninduced or continuously expressing systems.

[0138] Electroporation-based delivery provides an additional benefit by enabling nonviral, transient transfection with minimal genomic disruption. The optimized electroporation parameters described herein yield transfection efficiencies of about 40% to about 90%, about 50% to about 85%, and / or about 60% to about 80%, depending on voltage and DNA concentration. These parameters support reproducible transfection outcomes across independent iPSC lines and multiple passages.

[0139] Furthermore, the combination of species-specific transcription factors and the piggyBac genomic integration platform produces mature oligodendrocyte populations that exhibit stable gene expression for extended culture durations. The integrated constructs maintain function and stability for about 10 to about 50 days, about 15 to about 40 days, and / or about 20 to about 35 days post-transfection, enabling extended analyses of myelination, neural conductivity, and glial network formation. In some embodiments, the system therefore offers advantages over viral transduction or transient expression methods, providing a nonviral, integration-stable, and modular approach to inducing oligodendrocyte differentiation in nonhuman primate models. This configuration is especially beneficial for translational neuroscience applications, regenerative medicine studies, and preclinical safety and efficacy testing where long-term gene expression, lineage fidelity, and / or genomic stability are performance criteria.

[0140] FIG. 10 illustrates an embodiment of a vector 700 comprising a nucleic acid sequence 710 that includes a piggyBac vector backbone 712. In some embodiments, the piggyBac vector backbone 712 comprises OTPL007-pbvector 1002. The OTPL007-pbvector may serve as a transposable backbone configured to support the stable integration of one or more differentiation factor genes into the host genome. The backbone may include a pair of terminal inverted repeats and one or more transposase recognition sites configured to interact with a piggyBac transposase, as well as one or more promoter elements and selectable markers. The vector backbone may correspond to SEQ ID NO. 1 and have a length ranging from about 6,500 base pairs to about 11,000 base pairs, about 7,000 base pairs to about 10,000 base pairs, about 7,200 base pairs to about 9,800 base pairs, about 7,500 base pairs to about 9,500 base pairs, and / or about 8,000 base pairs to about 9,200 base pairs, depending on incorporated regulatory sequences and cloning sites.Docket No. 0138-706.600INTERNATIONAL APPLICATION

[0141] In some embodiments, the vector 700 is introduced into nonhuman primate iPSCs 908 by electroporation 906. The electroporation process may include one or more voltage pulses to permeabilize the cell membrane and allow entry of the plasmid DNA. The voltage may range from about 200 V / cm to about 2,500 V / cm, about 400 V / cm to about 2,000 V / cm, about 600 V / cm to about 1,800 V / cm, about 800 V / cm to about 1,600 V / cm, about 1,000 V / cm to about 1,400 V / cm, or about 1,200 V / cm to about 1,350 V / cm. Pulse duration may range from about 0.1 milliseconds to about 50 milliseconds, about 1 millisecond to about 25 milliseconds, about 2 milliseconds to about 20 milliseconds, about 4 milliseconds to about 15 milliseconds, and / or about 5 milliseconds to about 10 milliseconds, with the number of pulses ranging from about 1 to about 10, about 2 to about 8, about 3 to about 6, or about 2 to about 4 pulses. The total pulse interval may range from about 0.01 seconds to about 1.0 second, about 0.05 seconds to about 0.5 seconds, and / or about 0.1 seconds to about 0.25 seconds.

[0142] The electroporation buffer may include isotonic solutions containing magnesium chloride, potassium phosphate, calcium chloride, and / or HEPES at concentrations ranging from about 1 mM to about 100 mM, about 5 mM to about 75 mM, and / or about 10 mM to about 50 mM, with pH adjusted to between about 6.5 and about 8.0, about 6.8 and about 7.6, about 7.0 and about 7.4, and / or about 7.2 and about 7.3. The DNA concentration may range from about 0.01 pg / pL to about 20 pg / pL, about 0.05 pg / pL to about 12 pg / pL, about 0.1 pg / pL to about 10 pg / pL, about 0.25 pg / pL to about 8 pg / pL, about 0.5 pg / pL to about 5 pg / pL, and / or about 1 pg / pL to about 3 pg / pL per approximately 1 * 106to about 1 x 107cells. Post-electroporation, the cells may be incubated in a recovery medium for about 1 hour to about 48 hours, about 2 hours to about 36 hours, and / or about 4 hours to about 24 hours before reseeding.

[0143] Following electroporation, the transfected iPSCs 908 may be cultured in a feeder- free differentiation workflow configured to generate oligodendrocyte lineage cells 1040. The workflow may use extracellular matrix coatings such as laminin, vitronectin, Matrigel, Cultrex, or Geltrex, with coating concentrations ranging from about 1 pg / cm2to about 100 pg / cm2, about 5 pg / cm2to about 75 pg / cm2, or about 10 pg / cm2to about 50 pg / cm2. The basal medium may include formulations such as E8, E6, DMEM / F-12, and / or Neurobasal medium supplemented with N2, B27, and / or one or more essential amino acids. Concentrations of L- glutamine may range from about 0.1 mM to about 10 mM, about 0.5 mM to about 5 mM, about 1 mM to about 4 mM, and / or about 2 mM to about 3 mM.Docket No. 0138-706.600INTERNATIONAL APPLICATION

[0144] The culture may be maintained at an oxygen concentration ranging from about 1% to about 21%, about 3% to about 18%, about 5% to about 15%, about 7% to about 12%, or about 8% to about 10% O2, with a carbon dioxide concentration of about 2% to about 10%, about 3% to about 8%, and / or about 4% to about 6%. The temperature may be maintained between about 35°C to about 39°C, about 36°C to about 38°C, and / or about 37°C ± 0.5°C. The medium may further contain growth factors including platelet-derived growth factor (PDGF), neurotrophin-3 (NT3), insulin-like growth factor-1 (IGF-1), and / or triiodothyronine (T3). Concentrations of these factors may range from about 0.1 ng / mL to about 100 ng / mL, about 1 ng / mL to about 75 ng / mL, about 5 ng / mL to about 50 ng / mL, about 8 ng / mL to about 40 ng / mL, and / or about 10 ng / mL to about 30 ng / mL. The differentiation medium may be replaced every 12 hours to every 96 hours, every 18 hours to every 72 hours, every 24 hours to every 48 hours, and / or once every 36 hours depending on cell density and pH stability.

[0145] In some embodiments, the differentiated iPSCs are identified as oligodendrocyte lineage cells 1040 based on the expression of 04 1020 and myelin basic protein (MBP) 1030. 04 expression may be detected as early as about 5 days to about 15 days, about 6 days to about 20 days, or about 8 days to about 25 days post-transfection. The proportion of 04- positive cells may range from about 10% to about 95%, about 20% to about 90%, about 30% to about 85%, about 40% to about 80%, and / or about 50% to about 75% depending on induction timing and factor expression levels. MBP expression may be observed between about 10 days and about 45 days, about 15 days and about 40 days, about 18 days and about 35 days, and / or about 20 days and about 30 days of differentiation, indicating formation of mature myelin-producing oligodendrocytes.

[0146] Marker expression may be quantified by immunofluorescence microscopy, flow cytometry, or quantitative PCR. Expression intensity of 04 and MBP may increase between about 1.5-fold and about 20-fold, about 2-fold and about 15-fold, about 3-fold and about 12- fold, or about 4-fold and about 10-fold relative to undifferentiated control iPSCs. In some examples, mature oligodendrocyte morphology characterized by branched processes and compacted myelin structures may be observed after about 2 weeks to about 8 weeks, about 3 weeks to about 7 weeks, about 4 weeks to about 6 weeks, and / or about 25 days to about 45 days of induction.

[0147] The feeder-free system eliminates dependence on mouse embryonic fibroblasts (MEFs) and other feeder cells, reducing exposure to undefined xenogenic factors that can inhibit neural lineage commitment. As a result, the workflow achieves reproducibleDocket No. 0138-706.600INTERNATIONAL APPLICATION oligodendrocyte differentiation efficiencies ranging from about 60% to about 98%, about 70% to about 95%, about 75% to about 90%, and / or about 80% to about 85% across independently derived cultures. The OTPL007-pbvector 1002 architecture provides modular flexibility for additional use cases. In some embodiments, the backbone may be adapted to express alternative differentiation factors or regulatory elements for glial, astrocytic, and / or neuronal specification while maintaining similar electroporation, culture, and induction conditions. This allows scalable and reproducible generation of lineage-defined cell populations across experimental replicates and NHP species.

[0148] EXAMPLES

[0149] Embodiment 1. A vector for differentiating nonhuman primate induced pluripotent stem cells (iPSCs) from Macaca fascicularis into oligodendrocytes, wherein the vector comprises a nucleic acid sequence comprising a sequence encoding: a piggyBac vector backbone comprising: one or more terminal inverted repeats; one or more transposase recognition sites configured to interact with a piggyBac transposase; and a differentiation factor gene integrated within the piggyBac vector backbone and configured to induce oligodendrocyte lineage differentiation in one or more transfected iPSCs.

[0150] Embodiment 2. The vector of embodiment 1, wherein the piggyBac vector backbone further comprises a second sequence encoding a selectable marker and a third sequence encoding a promoter; wherein the selectable marker is configured to confer resistance for selection of one or more transfected iPSCs.

[0151] Embodiment 3. The vector of embodiment 1, wherein the differentiation factor gene is configured to be inserted into the piggyBac vector backbone in vitro using Gibson Assembly prior to transposase-mediated genomic integration.

[0152] Embodiment 4. The vector of embodiment 1, wherein the differentiation factor gene is configured to be inserted into the piggyBac vector backbone in vitro using restriction enzyme digestion and ligation prior to transposase-mediated genomic integration.

[0153] Embodiment 5. The vector of embodiment 1, wherein the vector is configured to be electroporated into the nonhuman primate iPSCs.

[0154] Embodiment 6. The vector of embodiment 1, wherein differentiating comprises transfecting.

[0155] Embodiment 7. The vector of embodiment 1, wherein the differentiation factor gene is SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4.Docket No. 0138-706.600INTERNATIONAL APPLICATION

[0156] Embodiment 8. The vector of embodiment 1, wherein the differentiation factor gene comprises Macaca fascicularis SOXIO, Macaca fascicularis OLIG2, and / or Macaca fascicularis NKX6-2.

[0157] Embodiment 9. The vector of embodiment 1, wherein the nonhuman primate iPSCs are differentiated into oligodendrocytes.

[0158] Embodiment 10. The vector of embodiment 1, wherein the piggyBac vector backbone comprises SEQ ID NO. 1.

[0159] Embodiment 11. The vector of embodiment 1, wherein the piggyBac vector backbone comprises OTPL007-pbvector.

[0160] Embodiment 12. The vector of embodiment 1, wherein the vector is introduced into the iPSCs by electroporation and used in a feeder-free differentiation workflow to generate oligodendrocyte lineage cells identified by expression of 04 and myelin basic protein (MBP)

[0161] Embodiment 13. A vector for differentiating nonhuman primate induced pluripotent stem cells (iPSCs) toward an oligodendrocyte lineage, wherein the vector comprises a nucleic acid sequence comprising a sequence encoding: a piggyBac vector backbone; and a differentiation factor gene configured to promote oligodendrocyte lineage development and maturation.

[0162] Embodiment 14. The vector of embodiment 13, wherein the piggyBac vector backbone further comprises a second sequence encoding a selectable marker and a third sequence encoding a promoter.

[0163] Embodiment 15. The vector of embodiment 13, wherein the differentiation factor gene is configured to be inserted into the piggyBac vector backbone using Gibson Assembly.

[0164] Embodiment 16. The vector of embodiment 13, wherein the differentiation factor gene is configured to be inserted into the piggyBac vector backbone using restriction enzymes.

[0165] Embodiment 17. The vector of embodiment 13, wherein the vector is configured to be electroporated into the nonhuman primate iPSCs.

[0166] Embodiment 18. The vector of embodiment 13, wherein differentiating comprises transfecting.

[0167] Embodiment 19. The vector of embodiment 13, wherein the differentiation factor gene is SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4.Docket No. 0138-706.600INTERNATIONAL APPLICATION

[0168] Embodiment 20. The vector of embodiment 13, wherein the differentiation factor gene comprises Macaca fascicularis SOXIO, Macaca fascicularis OLIG2, and / or Macaca fascicularis NKX6-2 of SEQ ID NO. 4.

[0169] Embodiment 21. The vector of embodiment 13, wherein the nonhuman primate iPSCs are differentiated into oligodendrocytes.

[0170] Embodiment 22. The vector of embodiment 13, wherein the piggyBac vector backbone comprises SEQ ID NO. 1.

[0171] Embodiment 23. The vector of embodiment 13, wherein the piggyBac vector backbone comprises OTPL007-pbvector.

[0172] References in the specification to “one embodiment,” “an embodiment,” “an illustrative embodiment,” “some embodiments,” etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may or may not necessarily include that particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to effect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.

[0173] As used in the description and claims, the singular form “a”, “an” and “the” include both singular and plural references unless the context clearly dictates otherwise. For example, the term “cell” may include, and is contemplated to include, a plurality of cells. At times, the claims and disclosure may include terms such as “a plurality,” “one or more,” or “at least one;” however, the absence of such terms is not intended to mean, and should not be interpreted to mean, that a plurality is not conceived.

[0174] The term “about” or “approximately,” when used before a numerical designation or range (e.g., to define a length or pressure), indicates approximations which may vary by ( + ) or ( - ) 5%, 1% or 0.1%. All numerical ranges provided herein are inclusive of the stated start and end numbers. The term “substantially” indicates mostly (i.e., greater than 50%) or essentially all of a device, substance, or composition.

[0175] As used herein, the term “comprising” or “comprises” is intended to mean that the devices, systems, and methods include the recited elements, and may additionally include any other elements. “Consisting essentially of’ shall mean that the devices, systems, and methods include the recited elements and exclude other elements of essential significance toDocket No. 0138-706.600INTERNATIONAL APPLICATION the combination for the stated purpose. Thus, a system or method consisting essentially of the elements as defined herein would not exclude other materials, features, or steps that do not materially affect the basic and novel characteristic(s) of the claimed disclosure. “Consisting of’ shall mean that the devices, systems, and methods include the recited elements and exclude anything more than a trivial or inconsequential element or step. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0176] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. Other embodiments may be utilized and derived therefrom, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is in fact disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

Docket No. 0138-706.600INTERNATIONAL APPLICATIONCLAIMSWHAT IS CLAIMED IS:

1. A vector for differentiating nonhuman primate induced pluripotent stem cells (iPSCs) from Macaca fascicularis into oligodendrocytes, wherein the vector comprises a nucleic acid sequence comprising a sequence encoding: a piggyBac vector backbone comprising: one or more terminal inverted repeats; one or more transposase recognition sites configured to interact with a piggyBac transposase; and a differentiation factor gene integrated within the piggyBac vector backbone and configured to induce oligodendrocyte lineage differentiation in one or more transfected iPSCs.

2. The vector of claim 1, wherein the piggyBac vector backbone further comprises a second sequence encoding a selectable marker and a third sequence encoding a promoter; wherein the selectable marker is configured to confer resistance for selection of one or more transfected iPSCs.

3. The vector of claim 1, wherein the differentiation factor gene is configured to be inserted into the piggyBac vector backbone in vitro using Gibson Assembly prior to transposase-mediated genomic integration.

4. The vector of claim 1, wherein the differentiation factor gene is configured to be inserted into the piggyBac vector backbone in vitro using restriction enzyme digestion and ligation prior to transposase-mediated genomic integration.

5. The vector of claim 1, wherein the vector is configured to be electroporated into the nonhuman primate iPSCs.

6. The vector of claim 1, wherein differentiating comprises transfecting.

7. The vector of claim 1, wherein the differentiation factor gene is SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4.Docket No. 0138-706.600INTERNATIONAL APPLICATION8. The vector of claim 1, wherein the differentiation factor gene comprises Macaca fascicularis SOXIO, Macaca fascicularis OLIG2, and / or Macaca fascicularis NKX6- 2.

9. The vector of claim 1, wherein the nonhuman primate iPSCs are differentiated into oligodendrocytes.

10. The vector of claim 1, wherein the piggyBac vector backbone comprises SEQ ID NO. 1.

11. The vector of claim 1, wherein the piggyBac vector backbone comprises OTPL007- pbvector.

12. The vector of claim 1, wherein the vector is introduced into the iPSCs by electroporation and used in a feeder-free differentiation workflow to generate oligodendrocyte lineage cells identified by expression of 04 and myelin basic protein (MBP)13. A vector for differentiating nonhuman primate induced pluripotent stem cells (iPSCs) toward an oligodendrocyte lineage, wherein the vector comprises a nucleic acid sequence comprising a sequence encoding: a piggyBac vector backbone; and a differentiation factor gene configured to promote oligodendrocyte lineage development and maturation.

14. The vector of claim 13, wherein the piggyBac vector backbone further comprises a second sequence encoding a selectable marker and a third sequence encoding a promoter.

15. The vector of claim 13, wherein the differentiation factor gene is configured to be inserted into the piggyBac vector backbone using Gibson Assembly.

16. The vector of claim 13, wherein the differentiation factor gene is configured to be inserted into the piggyBac vector backbone using restriction enzymes.

17. The vector of claim 13, wherein the vector is configured to be electroporated into the nonhuman primate iPSCs.

18. The vector of claim 13, wherein differentiating comprises transfecting.

19. The vector of claim 13, wherein the differentiation factor gene is SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4.Docket No. 0138-706.600 INTERNATIONAL APPLICATION20. The vector of claim 13, wherein the differentiation factor gene comprises Macaca fascicularis SOX10, Macaca fascicularis OLIG2, and / or Macaca fascicularis NKX6- 2 of SEQ ID NO. 4.

21. The vector of claim 13, wherein the nonhuman primate iPSCs are differentiated into oligodendrocytes.

22. The vector of claim 13, wherein the piggyBac vector backbone comprises SEQ ID NO. 1.

23. The vector of claim 13, wherein the piggyBac vector backbone comprises OTPL007- pbvector.

Images