Production of conditional, phenotype-restricted hypoimmune progenitor cells

Abstract

The present disclosure relates to recombinant nucleic acid molecules and related methods for production of conditional, phenotype-restricted hypoimmune progenitor cells.

Description

UR 6-24056 / FR: 161118.06801 Production Of Conditional, Phenotype-Restricted Hypoimmune Progenitor CellsCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing date of U.S. Provisional Patent Application No. 63 / 759,640, filed on February 18, 2025, which is hereby incorporated by reference in its entirety.GOVERNMENT INTERESTS

[0002] This invention was made with government support under AG072298 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (161118.06801SeqList.xml; Size: 113,944 bytes; and Date of Creation: February 6, 2026) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0004] This disclosure relates to reagents and methods for producing conditional, phenotype-restricted hypoimmune progenitor cells, related cell preparations, and uses thereof.BACKGROUND

[0005] Pluripotent stem cell or progenitor cell technology provides opportunities in regenerative medicine to generate grafts from donors for transplantation. Yet, particularly when non-autologus (e.g., allogeneic or xenogeneic) cells are used, immune suppression is generally required to avoid rejection of grafts or progenies derived from the transplanted cells. Strategies exist for the engineering of pluripotent stem / progenitor cells so as to permanently remove their immune-recognition molecules, including HLA class 1 and 2 antigens, while inserting checkpoint genes such as CD47 and PDL1, in order to produce lines whose derivatives can be transplanted as allografts without the need for immunosuppression. However, this process has a significant deficiency in that should undesired effects occur from, or phenotypes arise from, the transplanted population, then there is no further recourse for their targeted immunodepletion. There is a need for such recourse or targeted immunodepletion.SUMMARY

[0006] This disclosure addresses the need mentioned above in a number of aspects.1182281269.1UR 6-24056 / FR: 161118.06801

[0007] In one aspect, the disclosure provides a recombinant nucleic acid molecule comprising: (1) a first expression cassette comprising a first promoter, a second promoter, a first coding-sequence, and second coding-sequence, wherein the first coding-sequence and second coding-sequence are operably linked to the first promoter and the second promoter; (2) a second expression cassette joined to the first expression cassette in an opposite expression direction, comprising a third promoter and a third coding-sequence operably linked to the third promoter; (3) a first pair of sites of recombinase-mediated recombination, and (4) a second pair of sites of recombinase-mediated recombination. A segment of the recombinant nucleic acid molecule between the two members of the first pair or between the two members of the second pair is reversable upon exposure of the two members to a recombinase. A first member and a second member of the first pair are in the first expression cassette and the second expression cassette, respectively. A first member and a second member of the second pair are in the first expression cassette and the second expression cassette, respectively. The second member of the first pair is between the two members of the second pair, and the first member of the second pair is between the two members of the first pair. The first promoter and the second promoter regulate, respectively, expressions of genes or cassettes of genes sequentially expressed at two different stages of differentiation within a common lineage.

[0008] In one embodiment, (i) the recombinant nucleic acid molecule is for conditional, stage-and phenotype-specific expression of one or more transgenes in a differentiated cell derived from a pluripotent stem cell and (ii) the first coding-sequence, the second coding-sequence, or the third coding-sequence encodes the transgenes.

[0009] In one embodiment, (i) the recombinant nucleic acid molecule is for conditionally rendering a cell hypo-immune and / or non-immunogenic in a developmentally-restricted and phenotype-specific fashion and (ii) the second coding-sequence encodes for an immune checkpoint protein.

[0010] In one embodiment, the first coding-sequence, the second coding-sequence, or the third coding-sequence encodes for one or more proteins or RNAs.

[0011] In one embodiment, the first promoter or the second promoter or both are tissuespecific, differentiation stage-specific, cell-specific, or ligand inducible.

[0012] In one embodiment, the first promoter regulates gene expression specifically in a progenitor cell.

[0013] In one embodiment, the second promoter regulates gene expression specifically in a differentiated cell derived from the progenitor cell.2182281269.1UR 6-24056 / FR: 161118.06801

[0014] In one embodiment, the progenitor cell is a glial progenitor cell, a neural progenitor cell, an oligodendrocyte progenitor cell, or an astrocyte progenitor cell. In one embodiment, the differentiated cell is an astrocyte, an oligodendrocyte, or a neuron.

[0015] In one embodiment, the progenitor cell is a pancreatic Islet progenitor cell, a pancreatic Islet beta cell, or a pancreatic Islet alpha cell. In one embodiment, the differentiated cell is a pancreatic Islet insulin-producing beta cell or glucagon-secreting alpha cell.

[0016] In one embodiment, the second promoter is embedded within an intron and flanked by a splice donor site and a splice acceptor site.

[0017] In one embodiment, the second promoter is followed by a second splice donor site that is 5’ end to said splice acceptor site.

[0018] In one embodiment, the first promoter is selected from the group consisting of promoters of Neurogenin2, NeuroD family, GPR17, NG2, NGN3, PDX1, and NKX6.1.

[0019] In one embodiment, the second promoter is selected from the group consisting of promoters of: ELAV4, SYN1, MAP2, SOX10, MYRF, MAG, GFAP, AQP4, INS (beta), GCG (alpha), and SST (delta).

[0020] In one embodiment, the third promoter is a constitutive promoter. In one embodiment, the third promoter is selected from the group consisting of promoters of CAG, CBH, spleen focus-forming virus (SFFV), CBG, CMV, EFl alpha, UBC, and PGK.

[0021] In some embodiments, the first coding-sequence encodes a recombinase. In one embodiment, the first coding-sequence encoding the recombinase comprises an intron. In one embodiment, the recombinase is a Cre recombinase or a FLP recombinase.

[0022] In some embodiments, the second coding-sequence encodes for an immune checkpoint protein. In one embodiment, the immune checkpoint protein is selected from the group consisting of CD47, PDL1, HLA-E, CD200, CD36, and CD64. In one embodiment, the second coding-sequence further encodes for a first reporter protein.

[0023] In some embodiments, the third coding-sequence encodes for β2-microglobulin protein. In one embodiment, the third coding-sequence further encodes for a second reporter protein.

[0024] In one embodiment, the first expression cassette comprises a first poly adenylation signal (PAI) 3’ end to the first coding-sequence; or the first expression cassette comprises a second poly adenylation signal (PA2) 3’ end to the second coding-sequence; or the second expression cassette comprises a third poly adenylation signal (PA3) 3’ end to the third coding-sequence.3182281269.1UR 6-24056 / FR: 161118.06801

[0025] In one embodiment, (A) the first member of the first pair is located between the second promoter and the first coding-sequence; (B) the second member of the first pair is located between the PA3 and the third coding sequence; (C) the first member of the second pair is located between the PAI and the second coding-sequence, and / or (D) the second member of the second pair is located between the third coding sequence and the third promoter.

[0026] In a second aspect, the disclosure provides a polynucleotide derived from exposing the recombinant nucleic acid molecule described herein to a recombinase. In one embodiment, the polynucleotide comprises from the 5’ end thereof to the 3’ end thereof: the third promoter; the third coding-sequence; the complement of the second promoter, and the complement of the first promoter. In one embodiment, the polynucleotide further comprises the PA2 and the complement of PA3.

[0027] In a third aspect, the disclosure features a vector comprising the recombinant nucleic acid molecule or polynucleotide described above. In one embodiment, the vector is a viral vector or a plasmid vector. In one embodiment, the vector further comprises one or more elements selected from the group consisting of a DNA nuclear targeting sequence and a barcoding sequence.

[0028] In a fourth aspect, the disclosure provides a composition comprising (i) the recombinant nucleic acid molecule described above, the polynucleotide described above, or the vector described above, and (ii) a pharmaceutically acceptable carrier. In one embodiment, the carrier comprises a liposome, or a nanoparticle, or an exosome.

[0029] In a fifth aspect, the disclosure provides a cell or a progeny thereof. The cell comprises the recombinant nucleic acid molecule described above, the polynucleotide described above, or the vector described above. In one embodiment, the cell is a pluripotent stem cell. In one embodiment, the cell is an embryonic stem cell. In one embodiment, the cell is an induced pluripotent stem cell. In one embodiment, the cell is a human cell.

[0030] In a further aspect, the disclosure provides a method of conditional, stage- and phenotype-specific expression of a transgene in a differentiated cell derived from a pluripotent stem cell. The method comprises obtaining a pluripotent stem cell; introducing into the pluripotent stem cell the recombinant nucleic acid molecule or the vector described above; and culturing the pluripotent stem cell containing the recombinant nucleic acid molecule or vector under conditions suitable for (i) differentiation of the pluripotent stem cell and (ii) expression of the proteins encoded by the recombinant nucleic acid molecule under the control of the first4182281269.1UR 6-24056 / FR: 161118.06801 promoter or the second promoter, thereby to obtain one or more differentiated cells derived from the pluripotent stem cell.

[0031] The disclosure further provides a method of conditionally rendering or obtaining one or more hypo-immune and / or non-immunogenic cells. The method comprises obtaining a starting cell; introducing into the starting cell the recombinant nucleic acid molecule described above or a vector comprising the recombinant nucleic acid molecule, wherein the second coding-sequence encodes for an immune checkpoint protein; and culturing the starting cell containing the recombinant nucleic acid molecule or vector under conditions permitting expression of the proteins encoded by the recombinant nucleic acid molecule under the control of the first promoter or the second promoter thereby to obtain one or more hypo-immune and / or non-immunogenic cells.

[0032] In one embodiment, the starting cell lacks a functional β2-microglobulin protein and / or a functional CIITA protein.

[0033] The details of one or more embodiments of the disclosure are set forth in the description below. Other features, objectives, and advantages of the disclosure will be apparent from the description and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIGs. 1 A, IB, 1C, and ID show expression cassettes for glial specific expression.

[0035] FIG. 2 shows an expression cassette for glial specific immunoavoidance.

[0036] FIG. 3 shows schemes to achieve concurrent expression CD47 in astrocytes and glial progenitor cells and their progeny thereafter. Cell specific promoters were placed in tandem to drive Cre expression through an alternative splice of Minute virus of mice (MVM) intron. A GFAP (ABC ID) promoter was inserted inside the intron and then both inserted immediately after the GPR17 promoter. In glial progenitor cells, the expression occurs from GPR17 promoter. In this case the intron - including the GFAP promoter region - is spliced out. In astrocytes, the GFAP promoter is activated: a site donor (SD) inserted immediately after this promoter to allow for the splicing of the distal tier of the intron. SA: site acceptor. PA: Poly adenylation signal.

[0037] FIG. 4 shows an expression cassette that is inserted in cells in which the HLA complexes I and II are depleted by knockout of native B2M and CIITA respectively. These cells are nonetheless immunogenic since a synthetic B2M is expressed under the CAG promoter to confer immunogenicity to cells in the absence of Cre activation.5182281269.1UR 6-24056 / FR: 161118.06801

[0038] FIG. 5 shows Cre expression in astrocytes of GPCs and targeting lox sequences in the expression cassette.

[0039] FIG. 6 shows that Cre and B2M coding sequences are removed.

[0040] FIG. 7 shows that the CD47 coding sequence is inverted.

[0041] FIG. 8 shows that upon Cre-mediated recombination, CD47 then driven by constitutive promoter (CAG). At the same time, the cells are no longer immunogenic because of (i) loss of B2M and (ii) CD47 protect the cells of interest from the host immune system.

[0042] FIG. 9 shows validation of self-excision and recombination of a full knockin cassette, where 293FT cells knockin clones were transduced with rtTA-3G-expressing lentivirus.

[0043] FIG. 10A shows self-excision and recombination of a knockin cassette at the genomic level.

[0044] FIG. 10B shows 293FT cells knockin clones transduced with rtTA-3G-expresing virus.

[0045] FIG. 11 shows validation of gene expression following recombination.

[0046] FIG. 12 shows validation of protein expression following recombination.

[0047] FIG. 13 shows an all-in-one conditional expression cassette for the generation of hypoimmunogenic progenitor cell lines, further incorporating transcriptional stabilization elements. Depicted is a representative gene-targeting (16.8 KB) construct of the type described herein, configured for insertion into a genomic safe-harbor locus and comprising an all-in-one conditional expression cassette. Constructs that use cell-specific recombination mediated by a recombinase (e.g., Cre) result in the rearrangement and excision of intervening sequences, such that, following recombination, expression is restricted to the CD47 coding sequence. To reduce epigenetic silencing of the integrated transgene following differentiation from pluripotent progenitors, the illustrated construct includes chromatin-stabilizing regulatory elements, such as a ubiquitous chromatin opening element (UCOE). insulator sequences, and a post-transcriptional regulatory element, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Homology arms (HA-L / HA-R) flank the cassette to allow for homology-directed recombination and targeted integration at the selected safe-harbor locus.

[0048] FIG. 14 shows an alternative split-allele configuration distributing a conditional CD47 expression system across two alleles to reduce cassette size. Depicted is alternative construct in which the components of a conditional CD47 expression system are divided between two alleles (allele 1: 11.5Kb and allele 2: 10.3Kb) of the same genomic safe-harbor locus. In this configuration, each allele contributes to the overall expression architecture, so that CD47 expression occurs only after specific Cre expression in glial progenitor cells. Partitioning the 6182281269.1UR 6-24056 / FR: 161118.06801 system across two alleles reduces the size of each targeting cassette relative to an all-in-one configuration, potentially reducing epigenetic shutdown of the transgene. To further reduce epigenetic silencing, both transgenes incorporate transcriptional stabilization elements, including UCOE sequences, insulators, and post-transcriptional regulatory elements such as WPRE, positioned to mitigate trans gene silencing following differentiation. HA-L: Homology arm- Left; HA-R: Homolog}’ arm- Right; T2A: self-cleaving 2A peptide derived from the Thosea asi'gna virus: PuroR: Puromycin resistance gene; bGH: The bovine growth hormone (bGH) polyadenylation signal (polyA); SRF-UCOE: Serum Response Factor-Ubiquitous Chromatin Opening Element; CMV enhancer: enhancer of the human cytomegalovirus (hCMV) immediate-early promoter; Lox: locus of crossover in bacteriophage Pl; tRFP: Turbo Red Fluorescent Protein; WPRE: Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element; cHS4 core: core insulator, a 250-bp DNA sequence derived from the chicken beta-globin locus; Insulator: Insulator sequence from chicken betaglobin locus; CD47: Cluster of Differentiation 47 protein coding sequence; Cre: Cre recombinase protein coding sequence; GPR17 / Pr: G Protein-coupled Receptor 17 promoter sequence; CAG promoter: Cytomegalovirus (CMV) immediate-early enhancer element fused to the Actin the promoter, and first exon, and first intron of the chicken beta- actin gene followed by the splice acceptor from the rabbit beta-Globin gene; hGH: human growth hormone polyA signal; EGFP: Enhanced Fluorescent Protein; SV40 PA: Simian Virus 40 polyadenylation signal; GFAP / Pr: gfaABCID promoter derived from human Glial Fibrillary Acidic Protein gene; SA: Splice Acceptor.DETAILED DESCRIPTION OF THE INVENTION

[0049] This disclosure relates nucleic acids, vectors, compositions, and methods for conditionally rendering immunocompetent cells as immunodeficient, once they have become differentiated to the point of yielding desired progenitor cells and their lineal progeny.

[0050] In some embodiments, immunosuppression can be supplied for some period of time after either the allogeneic or xenogeneic transplantation of human somatic progenitor cells, allowing these cells to migrate to desired sites and differentiating into desired phenotypes. Upon differentiation, these cells conditionally activate checkpoint genes / proteins expressions that prevent or evade innate cell atack, thereby achieving immune-avoidant stability. Those cells that have not yet achieved that degree of differentiation fail to activate the conditional promoters, and remain recognizable by the innate system, and are subject to immune elimination. At that point, comprising a predefined period after transplant, immunosuppression 7182281269.1UR 6-24056 / FR: 161118.06801 is withdrawn, and residual undesired, undifferentiated cells remain visible to the immune system, and are rejected by their now-immunocompetent hosts. Unique to this system is the acquisition of conditional immuneoavoidance by both the progenitor cells and their terminally differentiated derivatives.

[0051] In some embodiments, human pluripotent stem cells (PSCs) - whether embryonic stem cells or induced pluripotent stem cells - are edited to render them deficient in HLA class I and II antigen expression, by CRISPR / Cas-mediated knock-out or any means of stable genetic editing, (such as Zn-finger nucleases) of the beta2 microglobulin and class 2 transactivator genes, respectively. The cells are also edited to include a selection cassette bearing a dual promoter-regulated Cre recombinase, with the two promoters regulating Cre expression comprising the regulatory elements controlling serial stages in lineage progression (promoters 1 and 2); these are placed in tandem, with the 3’ promoter (promoter 2, driving the expression of the mature phenotype) embedded within a Minute virus of mice (MVM) intron, flanked with or next to 5’ splice donor site and / or 3 ' splice acceptor sites. This element in turn regulates the expression of an immune checkpoint protein, which may include CD47, PDL1, or HLA-E, or other competent immune checkpoint proteins evading immune surveillance.

[0052] By way of example, one embodiment of this disclosure comprises the immunoprotection of human glial progenitor cells and their derived oligodendrocytes and astrocytes. Shown in Fig. 3 is an exemplary recombinant nucleic acid to achieve concurrent expression CD47 in astrocytes and glial progenitor cells and their progeny thereafter.

[0053] Specifically, cell specific promoters are placed in tandem to drive Cre expression through an alternative splice of the Minute virus of mice (MVM) intron. In this case, the promoter for glial fibrillary acidic protein GFAP (ABC ID segment) was inserted inside the intron, and then both were inserted 3' to directly follow the GPR17 promoter. In glial progenitor cells, transgene expression then occurs from the GPR17 promoter, since in this case the intron - including the GFAP promoter region - is spliced out. In contrast, in mature GFAP-expressing astrocytes, the GFAP promoter is instead activated, since a site donor (SD) inserted immediately after this promoter allows for the splicing of the distal portion of the intron.

[0054] This strategy necessarily avoids use of promoters for glial genes that might also be expressed in undesired phenotypes, such as in glioma cells or other neuroepithelial-derived neoplasms. Such genes, for instance OLIG2, would not be appropriate drivers of these selection cassettes, whose purpose is to hide desired allogeneic cells from immune surveillance.8182281269.1UR 6-24056 / FR: 161118.06801 As such, promoters used to conditionally restrict immuneoavoidance are optimally utilized only by the desired cell types of interest.

[0055] In that regard, examples of other potential pairs of promoters for progenitors and their differentiated derivatives include, among others: for neural progenitor cells and neurons: Neurogenin2, NeuroD family; ELAV4, SYN1, and MAP2; for oligodendrocyte progenitor cells and oligodendrocytes: GPR17, NG2; SOXIO, MYRF, and MAG; and for astrocyte progenitor cells and astrocytes: GPR17, GFAP, and AQP4.

[0056] In another embodiment of this disclosure, this approach can also be used to render transplantable pancreatic Islet progenitors and their derived pancreatic beta cells conditionally immunoavoidant, using pairs of regulatory' elements that may include the transcriptional promoters for the following genes, among others: for pancreatic neuroendocrine progenitors: NGN3, PDX1, NKX6.1; and for mature Islet cells INS (beta), GCG (alpha), and SST (delta).

[0057] In one embodiment, to allow one to combine Cre and its Lox(s) in the same construct without recombination during bacterial production, a second small and constitutive intron from GAPDH gene was inserted inside CRE; this intron is not spliced out until Cre expression is activated under the regulatory control of the promoter 1 -defined and targeted phenotype.

[0058] The nucleic acids and strategies described herein can be used for the conditional expression of therapeutic transgenes by any suitable different maturational stages of lineally-related cells within a common lineage.Recombinant Nucleic Acid Molecules / Constructs

[0059] The present disclosure provides recombinant nucleic acid molecules or recombinant genetic constructs.

[0060] As used herein, the “recombinant nucleic acid molecule” and “recombinant genetic construct” are used interchangeably, and each refers to a nucleic acid molecule containing a combination of two or more genetic elements not naturally occurring together. The recombinant nucleic acid molecule / genetic construct comprises a non-naturally occurring nucleotide sequence that can be in the form of linear DNA, circular DNA, e.g., placed within a vector (e.g., a bacterial vector, a viral vector), or integrated into a genome. The terms means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature, or which is synthetic, which comprises one or more (e.g., several) control sequences.9182281269.1UR 6-24056 / FR: 161118.06801

[0061] The term “recombinant” means, for example, that a nucleic acid sequence is made by an artificial combination of two otherwise separated segments of sequence, e.g.. by chemical synthesis or by the manipulation of isolated nucleic acids by genetic engineering techniques.

[0062] Recombination refers to a process in which a molecule of nucleic acid is broken and then joined to a different one. The recombination process of the disclosure ty pically involves the artificial and deliberate recombination of disparate nucleic acid molecules, which may be from the same or different organism, so as to create recombinant nucleic acids.

[0063] The recombinant nucleic acid molecule of this disclosure comprises two expression cassettes that are joined in the opposite expression direction. The first expression cassette comprises a first promoter, a second promoter, a first coding-sequence, and second coding-sequence. The first coding-sequence and second coding-sequence are operably linked to the first promoter and the second promoter. The second expression cassette is joined to the first expression cassette in an opposite expression direction, comprising a third promoter and a third coding-sequence operably linked to the third promoter.

[0064] The term “operable linkage” or “operably linked” or the like are defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the production of an mRNA or a polypeptide. “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a nucleic acid sequence is capable of effecting the expression of that sequence when the proper enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence. Thus, the term "operably linked" is intended to encompass any spacing or orientation of the promoter element and the DNA sequence of interest which allows for initiation of transcription of the DNA sequence of interest upon recognition of the promoter element by a transcription complex.

[0065] The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a protein. The boundaries of a coding sequence that encodes one protein are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA. cDNA. synthetic DNA, or a combination thereof.10182281269.1UR 6-24056 / FR: 161118.06801

[0066] The term “control sequences” is defined herein to include all components, which are necessary’ or advantageous for the production of mRNA or a polypeptide, either in vitro or in vivo in a host cell. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, Shine-Delgamo sequence, optimal translation initiation sequences (as described in Kozak, 1991, J. Biol. Chem. 266:19867-19870), a polyadenylation sequence, a pro-peptide sequence, a pre-pro-peptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and a transcriptional stop signal as well as translational start and stop signals. Control sequences may be optimized to their specific purpose. Preferred optimized control sequences used in the present invention are those described in W02006 / 077258.

[0067] The term “promoter” is defined herein as a DNA sequence that binds RNA polymerase and directs the polymerase to the correct downstream transcriptional start site of a nucleic acid sequence encoding a biological compound to initiate transcription. RNA polymerase effectively catalyzes the assembly of messenger RNA complementary’ to the appropriate DNA strand of a coding region. The term “promoter” will also be understood to include the 5'-non-coding region (between promoter and translation start) for translation after transcription into mRNA, cis-acting transcription control elements such as enhancers, and other nucleotide sequences capable of interacting with transcription factors.

[0068] Accordingly, a specific promoter may be strategically placed in an intro (e.g., the GFAP promoter shown in Fig. 3) or a coding sequence may be strategically placed between sites of recombinase-mediated recombination such that the promoter and coding sequence are brought into operable linkage on recombination to direct the expression of the coding sequence in a cell type-specific, tissue-specific, or differentiation stage-specific manner.

[0069] The promoter may be any appropriate promoter sequence suitable for a eukaryotic or prokaryotic host cell, which shows transcriptional activity’, including mutant, truncated, and hybrid promoters. The promoter may be a constitutive or inducible promoter. The promoter may also be cell type-specific, tissue-specific, or differentiation stage-specific. Expression of the recombinase by an inducible promoter, a cell type-specific promoter, a tissue-specific promoter, or a differentiation stage-specific promoter will allow recombination of the sequence located between the site-specific recombination sites to be controlled or tied to the specific cell tissue, or stage, for example including the recombinase encoding sequence.11182281269.1UR 6-24056 / FR: 161118.06801

[0070] By way of example, the first promoter and the second promoter regulate, respectively, expressions of genes or cassettes of genes sequentially expressed at two different stages of differentiation within a common lineage. For example, as shown in the figures, such as Figs.3 -5, the first expression cassette comprises a GPR17 promoter (the first promoter), a GFAP promoter (the second promoter), a sequence encoding a Cre polypeptide (the first coding-sequence), and a sequence encoding a CD47 polypeptide (the second coding-sequence). The first expression cassette shown in Fig. 3 also includes poly adenylation signals PA1 and PA2. The second expression cassette is joined to the first expression cassette in the opposite expression direction, and comprises a CAG promoter (the third promoter) and a sequence encoding a B2M polypeptide or a tRFP polypeptide (the third coding-sequence). The second expression cassette shown in Fig. 3 also include a poly adenylation signal PA3.

[0071] The recombinant nucleic acid molecule further comprises a first pair of sites of recombinase-mediated recombination, and a second pair of sites of recombinase-mediated recombination. In the figures, such as Figs. 3-5, each pair is represented by a pair of triangles of the same kind. More specifically, the first pair includes a first member and a second number shown as light gray (or dark gray) triangles in Fig. 3. The second pair includes a first member and a second number shown as black triangles in Fig. 3.

[0072] The segment of the nucleic acid molecule between the two members of the first pair or between the two members of the second pair is reversable upon exposure of the two members to a recombinase (such as a Cre recombinase). The first member and the second member of the first pair are in the first expression cassette and the second expression cassette, respectively. The first member and the second member of the second pair are in the first expression cassette and the second expression cassette, respectively. The second member of the first pair is between the two members of the second pair. The first member of the second pair is between the two members of the first pair.

[0073] In one embodiment, the nucleic acid molecule is for conditional, stage- and phenotypespecific expression of a transgene in a differentiated cell derived from a pluripotent stem cell. In that case, the first coding-sequence, second coding-sequence, or the third coding-sequence encodes the transgene.

[0074] In one embodiment, the nucleic acid molecule is for conditionally rendering a cell hypo-immune and / or non-immunogenic in a developmentally restricted and phenotype-specific fashion. In that case, the second coding-sequence encodes an immune checkpoint gene.12182281269.1UR 6-24056 / FR: 161118.06801

[0075] In one embodiment, the first coding-sequence, the second coding-sequence, or the third coding-sequence encodes one or more same or different proteins or RNAs.Promoters

[0076] In the nucleic acid molecule described herein, the first promoter or the second promoter or both can be tissue-specific, differentiation stage-specific, cell-specific, or ligand inducible.

[0077] In one embodiment, the first promoter regulates gene expression specifically in a progenitor cell. In that case, the second promoter regulates gene expression specifically in a differentiated cell derived from the progenitor cell.

[0078] In some embodiments, the progenitor cell is a glial progenitor cell, a neural progenitor cell, an oligodendrocyte progenitor cell, or an astrocyte progenitor cell. The corresponding differentiated cell is an astrocyte, an oligodendrocyte, or a neuron. Examples of other suitable cells include those described herein.

[0079] In some embodiments, the progenitor cell is a pancreatic Islet progenitor cell, a pancreatic Islet beta cell, or a pancreatic Islet alpha cell. The corresponding differentiated cell is a pancreatic Islet insulin-producing beta cell or glucagon-secreting alpha cell.

[0080] In one embodiment, the second promoter (e g.. the GFAP promoter in Fig. 3) is embedded within an intron and flanked by or next to a splice donor site and / or a splice acceptor site. In one embodiment, the second promoter is followed by a second splice donor site that is 5' end to said splice acceptor site.

[0081] Shown in the table below are examples of cell lineages and corresponding promoters.Table 1A. Exemplary Lineages Specific PromotersLineages 1stPromoter (progenitor specific) 2ndPromoter (differentiated / mature cell specific)Neural Neurogenin2, NeuroD family ELAV4, SYN1, MAP2 Oligodendrocyte GPR17, NG2 (CSPG4), PDGFRA SOX 10, MYRF, MAGAstrocyte PDGFRA, SOX9 GFAP, AQP4Pancreatic NGN3, PDX1, NKX6.1 INS (beta), GCG (alpha), and SST (delta)neuroendocrine

[0082] Exemplary lineages specific promoters suitable for inclusion in the recombinant nucleic acid molecule / construct described herein are well known in the art and are shown in the table below.

[0083] Promoters useful in this disclosure may be constitutive or regulated or inducible. In some cases, a promoter useful may be regulated with cell signaling mechanisms involved in differentiation, either to be expressed at a higher level in differentiating cells or expressed at a 13182281269.1UR 6-24056 / FR: 161118.06801 lower level in differentiated cells compared to undifferentiated cells. For example, the promoter may be upregulated during gliogenesis such that it is expressed at a higher level in differentiating glial cells than in related progenitors. In some cases, the promoter is activated by various glycogenic regulatory factors.Constitutive Promoters

[0084] In some embodiments, the third promoter is a constitutive promoter. Examples of such a promotor include CAG, CBH, spleen focus-forming virus (SFFV), CBG, CMV, EFlalpha, UBC, and PGK. Exemplary constitutive mammalian promoters suitable for inclusion in the recombinant nucleic acid molecule / construct described herein are well known in the art and are shown in the Table below (Qin et al., “Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter,” PLoS One 5(5):el0611 (2010). which is hereby incorporated by reference in its entirety).Table 2. Exemplary Constitutive Promoter SequencesPromoterNucleotide SequenceNameUBC GGTGCAGCGGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGA GC GC T GC C AC GT CAGAC GAAGGGC GCAGGAGC GT T C C T GAT CCTTCCGCCC GGAC GCT C AGG AC AGC GGC CC GC T GC T C AT AAGAC T CGGC C T TAGAAC C C CAGTAT C AGCAGAAGGACAT T T T AGGAC GGGAC T T GGGT GAC T C TAGGGC AC TGGTTTTCT T TC C AGAGAGC GGAAC AGGC GAGG AAAAGT AGTC C C T T C T C GGC GAT T C TGC GGAGGGAT C T C CGT GGGGC GGT GAAC GC CGAT GA T T AT ATAAGGAC GC GC C GGGT GT GGCAC AGC TAGT T C C GTC GCAGC C GGGAT T T GGGT C GC G GTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGGTGAGTTGCGGGCTGCTGGGCTGGCCGG GGCTTTCGTGGCCGCCGGGCCGCTCGGT GGGAC GGAAGC GT GT GGAGAGAC C GC CAAGGGC T GTAGTCTGGGTCCGC GAGC AAGGT T GC C C T GAAC T GGGGGT T GGGGGGAGC GCACAAAAT GG C GGC T GT T CC C GAGT C T T GAAT GGAAGAC GC T T GT AAGGCGGGC T GT GAGGT C GT T GAAACA AGGT GGGGGGC AT GGT GGGC GGCAAGAAC C C AAGGT C T T GAGGC C T T C GC TAAT GC GGGAAA GC T C T T AT TC GGGT GAGAT GGGC T GGGGC AC CAT C T GGGGAC C C T GAC GT GAAGT T TGT C AC T GAC T GGAGAAC T C GGGT T T GT C GT CT GGT T GC GGGGGC GGC AGT T AT GC GGT GC C GT T GGG CAGTGCACCCGTACCTTTGGGAGCGCGCGCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTG GC T T ATAATGC AGGGT GGGGC CAC C TGC C GGTAGGT GT GCGGTAGGC TTTTCTCCGTCGCAG GAC GCAGGGT T C GGGC C T AGGGT AGGC T C T C C T GAAT C GAC AGGC GC C GGAC C T C T GGT GAG GGGAGGGATAAGT GAGGC GTCAGTTTCTTTGGTCGGTTT TAT GTACCTATCTTCT T AAGT AG CTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTGTTTTGTGAAGTTTTTTA GGCAC C T T TT GAAAT GTAAT C AT T T GGGT CAAT AT GTAATT T T C AGT GT T AGAC TAGT AAA( SEQ ID NO: 1 )PGK T T C T AC C GGGT AGGGGAGGC GCTTTTCC C AAGGC AGT C T GGAGC AT GC GC T T TAGC AGC C C C GC T GGGCACT T GGC GC T AC AC AAGT GGCCTCTGGCCTC GCAC AC AT T C CAC AT C CACC GGTA GGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTC AGGAAGT TCCCCCCCGCCCC GCAGC TC GC GT C GT GCAGGAC GT GAC AAAT GGAAGT AGC AC G T C T C AC TAGT C T C GT GC AGAT GGAC AGC AC C GC T GAGCAAT GGAAGC GGGT AGGC C TT T GGG GCAGC GGC CAAT AGCAGC TTTGCTCCTTCGCTTTCT GGGCT C AGAGGC T GGGAAGGGGT GGG T C C GGGGGC GGGC T CAGGGGC GGGC TC AGGGGC GGGGC GGGC GC C C GAAGGT C C T C CGGAGG CCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTC CGGGCCTTTCGACCT ( SEQ ID NO: 2 )EFla GGCTCCGGTGCCCGTCAGT GGGC AGAGC GCACAT C GC C CAC AGT C C C C GAGAAGT T GGGGGG AGGGGT C GGC AAT T GAAC C GGT GC C TAGAGAAGGT GGC GCGGGGTAAAC T GGGAAAGT GAT G T C GT GT AC TGGC TCCGCCTTTTTC C CGAGGGT GGGGGAGAAC C GT AT AT AAGT GCAGT AGT C GC C GT GAACGT TCTTTTTC GC AAC GGGTTTGCCGC CAGAAC ACAGGTAAGT GC C GT GT GT GGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCT 14182281269.1UR 6-24056 / FR: 161118.06801 GGCT GC AGTAC GT GAT T C T T GAT C C CGAGC T T C GGGT T GGAAGT GGGT GGGAGAGTTGGAGG CCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGG CCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAG CCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAAT GC GGGC CAAGAT C T GC AC AC T GGTATT T C GGT T T T T GGGGC C GC GGGC GGC GAC GGGGC C C G TGCGTCCCAGC GCACAT GT T C GGC GAGGC GGGGC C T GC GAGC GC GGC C AC C GAGAATC GGAC GGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGTCTCGCGCCGCCGTGTATCGCC CCGCCCTGGGC GGC AAGGC T GGC C C GGT C GGCAC C AGT T GC GT GAGC GGAAAGAT GGC C GC T TCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTG AGT C AC C C AC AC AAAGGAAAAGGGC CT T T C C GT C C T C AGCC GT C GC T T CAT GT GAC TC C AC G GAGT AC CGGGCGCCGTC C AGGCAC C TC GAT T AGT T C T C GAGC T T T T GGAGT AC GTCGTCTTT AGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAG TTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCT TGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA ( SEQ ID NO: 3 )CMV TAGT TAT T AAT AGT AAT C AAT TAC GGGGT CAT T AGT T CATAGC C CAT AT AT GGAGT TC C GC G T T AC ATAACT T AC GGT AAAT GGC C C GC C T GGC T GAC C GC CC AAC GAC C C C C GC C CATT GAC G T C AAT AAT GAC GTAT GT T C C C AT AGTAAC GC CAAT AGGGAC T T T C C AT T GAC GT CAAT GGGT GGAGTAT T TAC GGT AAAC T GC C C AC TT GGCAGT AC AT CAAGT GTAT CAT AT GC CAAGT AC GC C C C C TAT T GAC GT CAAT GAC GGT AAAT GGC C C GC C T GGC AT TAT GC C C AGT ACAT GAC C T TA T GGGAC TTTCCTACTT GGC AGTACATC T AC GTAT TAGT CAT C GC TAT TAC CAT GGT GAT GC G GT T T T GGC AGT ACAT CAAT GGGC GT GGAT AGC GGT T T GACT C AC GGGGAT T T C CAAGT C T C C AC C C CAT T GAC GT CAAT GGGAGT T T GT T T T GGC AC CAAAAT C AAC GGGAC T T T C CAAAAT GT C GTAACAACT C C GC C C CAT T GAC GC AAAT GGGC GGTAGGCGT GT AC GGT GGGAGGT CT AT AT AAGCAGAGCTGGTTTAGTGAACCGTCAGATC ( SEQ ID NO: 4 )CAGG AC TAGT TATT AATAGT AAT CAAT TACGGGGT CAT T AGT T CATAGC C CAT AT AT GGAGT T C C G C GT TAC AT AAC T TAC GGT AAAT GGC CC GC C T GGC T GAC C GC C CAAC GAC C C C C GC C CAT T GA C GT CAAT AAT GAC GTAT GT T C C CAT AGTAAC GC CAAT AGGGAC T T T C C AT T GAC GT CAAT GG GT GGAGTATT T AC GGT AAAC T GC C C AC T T GGCAGT AC AT CAAGT GTAT CAT AT GC CAAGT AC GC C C C C TATT GAC GT CAAT GAC GGT AAAT GGC C C GC C T GGC AT T AT GC C C AGTACATGAC C T TAT GGGAC TT T C C T AC T T GGC AGTAC AT C TAC GTAT T AGTC AT C GC TAT TAC CAT GGT C GAG GTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTA T T TAT T TATT T T T T AAT T AT T T T GT GC AGC GAT GGGGGC GGGGGGGGGGGGGGGGC GC GC GC CAGGC GGGGC GGGGC GGGGC GAGGGGC GGGGC GGGGC GAGGC GGAGAGGT GC GGC GGC AGC C AAT C AGAGCGGC GC GC T C C GAAAGT TT C C T T T T AT GGC GAGGC GGC GGC GGC GGC GGC C C TA TAAAAAGCGAAGCGCGCGGCGGGCGGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCG CTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGA GC GGGC GGGAC GGC C C T T C T C C T C C GGGC T GTAAT TAGC GC T T GGT T T AAT GAC GGCT T GT T T C T T T T C T GT GGC T GC GT GAAAGC C TT GAGGGGC T C C GGGAGGGC C C T T T GT GC GGGGGGAG C GGC T C GGGGGGT GC GT GC GT GT GT GT GT GC GT GGGGAGCGC CGCGTGCGGCTCCGCGCTGC CCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAG GGGAGC GC GGC C GGGGGC GGT GC C C CGC GGT GC GGGGGGGGC T GC GAGGGGAACAAAGGC T G C GT GC GGGGT GT GT GC GT GGGGGGGTGAGCAGGGGGT GTGGGCGCGTCGGTC GGGC TGCAAC CCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGT ACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGG C GGGGC GGGGC C GC C T C GGGC C GGGGAGGGC T C GGGGGAGGGGC GC GGC GGC C C C C GGAGC G CCGGCGGCTGTC GAGGC GC GGC GAGCC GC AGC CATTGCCTTT TAT GGT AAT C GT GC GAGAGG GC GC AGGGAC TTCCTTTGTCC CAAATC T GT GC GGAGC C GAAAT C T GGGAGGC GC C GCC GC AC C C C C T C TAGC GGGC GC GGGGC GAAGCGGT GC GGC GC C GGCAGGAAGGAAAT GGGC GGGGAGG GCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGG GGGGAC GGCT GC C T T C GGGGGGGAC GGGGCAGGGC GGGGTT C GGC T T C T GGC GT GT GAC C GG CGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCA ACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTC ( SEQ ID NO: 5 ) SV40 C T GT GGAATGT GT GT C AGT T AGGGT GT GGAAAGT C C C CAGGC T C C C CAGC AGGC AGAAGT AT GC AAAGCATGC AT C T CAAT TAGT CAGC AAC C AGGT GT GGAAAGT CCCCAGGCTCCC CAGC AG GC AGAAGT AT GC AAAGC AT GC AT C T CAAT TAGT C AGCAACC ATAGT CCCGCCCC T AAC T C C G CCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTT T T T T AT T T AT GC AGAGGC C GAGGC CGCCTCTGCCTCT GAGC TAT T C CAGAAGTAGT GAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCT ( SEQ ID NO: 6 )15182281269.1UR 6-24056 / FR: 161118.06801 Inducible Promoters

[0085] In some cases, the promoter may be inducible by adding an exogenous compound. An example of an inducible promoter is seen in the Tet-On system. The Tet-On system includes a protein called reverse tetracycline-controlled transactivator (rtTA), which in turn interacts with a rtTA responsive promoter called TRE. The rtTA is a transcription factor, a protein that binds to DNA and regulates gene expression. The TRE promoter is a DNA sequence positioned upstream of a gene and must be activated to induce expression of that gene. In the absence of tetracycline or doxycycline, the rtTA cannot bind to the TRE promoter to drive gene expression. When tetracycline or doxycycline is added, the antibiotic complexes with rtTA, which allows it to bind to the TRE promoter. The TRE promoter then allows for the recruitment of RNA polymerase to transcribe the gene. The Tet-On system may be used to drive expression of a gene, for example the Tet-On system may be used to control expression of a recombinase enzyme.

[0086] Other examples of inducible promoters that can be used include the promoters described in W02006 / 092396 and W02005 / 100573, which are herein incorporated by reference. Other examples of promoters include a starch-, cellulose-, hemicellulose (such as xylan- and / or xylose-inducible), copper-, oleic acid-inducible promoters described in US Patent 9657309. Additional examples of the use of promoters are described in W02008 / 098933. Other examples of inducible (heterologous) promoters are the alcohol inducible promoter alcA. the tet system using the tetracycline-responsive promoter, the estrogen-responsive promoter (Pachlinger et al. (2005), Appl & Environmental Microbiol 672-678).Immune Checkpoint Proteins

[0087] In the nucleic acid molecule described herein, the second coding-sequence encodes for an immune checkpoint protein. In some embodiments, the immune checkpoint protein is selected from the group consisting of CD47, PDL1, HLA-E, CD200, CD36, and CD64.

[0088] The immune checkpoint protein encoded by the nucleotide sequence of the recombinant genetic construct of the present disclosure can be any protein, or peptide thereof, that is involved in immune system downregulation and / or that promotes immune self-tolerance. In one embodiment, the immune checkpoint protein, or peptide thereof, is one that suppresses the activity of the acquired immune response. In one embodiment, the immune checkpoint protein, or peptide thereof, is one that suppresses the activi ty of the innate immune response.16182281269.1UR 6-24056 / FR: 161118.06801

[0089] In one embodiment, the immune checkpoint protein encoded by the recombinant genetic construct is programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), or functionally active peptides thereof, that bind to the inhibitory programmed cell death protein 1 (PD-1). PD-1 is primarily expressed on mature T cells in peripheral tissues and the tumor microenvironment. It is also expressed on other non-T cell subsets including B cells, professional APCs, and natural killer (NK) cells. PD-1 signaling is mediated through interaction with its ligands PD-L1 (also known as B7-H1 and CD274) and PD-L2 (also known as B7-DC and CD273). Interaction of PD-1 with any of its ligands, z.e., PD-L1 and PD-L2, transmits an inhibitory signal which reduces the proliferation of CD8+T cells at the lymph nodes, thereby suppressing the immune response.

[0090] Suitable nucleotide sequences encoding human PD-L1 and PD-L2 for inclusion in the recombinant genetic construct as described herein are set forth in Table 1 below. Suitable nucleotide sequences also include nucleotide sequences having about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the PD-L1 and PD-L2 coding sequences provided in Table 3 below.Table 3. Suitable PD-L1 and PD-L2 Coding Sequences and Amino Acid Sequences17182281269.1UR 6-24056 / FR: 161118.06801Amino Acid Name Coding Sequence Sequence Homo AT GAGGAT AT TTGCTGTCTT TAT AT T CAT GAC C TAC T GG MRI FAVE I FMT YWH sapiens GBk NM0012677061 NM0141433enan..__ C AT T T GCT GAAC GC C C CATACAAC AAAAT CAAC CAAAGA LLNAPYNKINQRIL CD274 Aiccesson AT T T T GGT T GT GGAT C CAGT CAC C T C TGAAC AT GAAC TG WDPVTSEHELTCQ molecule Number ACAT GT CAGGC T GAGGGC TACC C C AAGGC C GAAGT CATC AEGYPKAEVIWTS S (CD274), T GGAC AAGC AGT GAC CAT CAAGT C C T GAGT GGT AAGACC DHQVLSGKTTTTNS transcript AC CAC CAC CAAT T C CAAGAGAGAGGAGAAGC T T T T CAAT KREEKLFNVTSTLR variant 2, GT GAC C AGC ACAC T GAGAAT CAAC AC AACAAC T AAT GAG INTTTNE IFYCTFR mRNA AT T T T C TAC T GC AC T T T T AGGAGAT T AGAT C C T GAGGAA RLDPEENHTAELVI AAC CAT AC AGC T GAAT T GGT CAT C C C AGAAC TAC C T C TG PELPLAHPPNERTH GCACATCCTC C AAAT GAAAGGAC T C AC T T GGT AAT T C T G LVILGAILLCIGVA GGAGC C AT C T TAT T AT GC C T TGGT GT AGC AC T GAC AT TC LTFIFRLRKGRMMD ATCTTCCGTT TAAGAAAAGGGAGAAT GAT GGAT GT GAAA VKKCGIQDTNSKKQ AAAT GT GGC AT C CAAGAT AC AAAC T C AAAGAAGCAAAGT SDTHLEET ( SEQ GATACACATTTGGAGGAGACGTAA ( SEQ ID NO: 7 ) ID NO: 8 ) Homo AT GAGGAT AT TTGCTGTCTT TAT AT T CAT GAC C TAC T GG MRIFAVFIFMTYWH sapiens C AT T T GCT GAAC GC AT T T AC TGT C AC GGT T C C C AAGGAC LLNAFTVTVPKDLY CD274 C TAT AT GT GGTAGAGT AT GGTAGC AATAT GAC AAT T GAA WEYGSNMT IECKF molecule T GCAAATT C C CAGT AGAAAAACAAT TAGAC C T GGC T GCA PVEKQLDLAAI IVY (CD274), C TAAT T GT C T AT T GGGAAAT GGAGGATAAGAAC AT TATT WEMEDKNIIQFVHG transcript CAAT T T GT GC AT GGAGAGGAAGAC C T GAAGGT T CAGC AT EEDLKVQHS SYRQR variant 1, AGT AGC TAC AGACAGAGGGC CC GGC T GT T GAAGGAC C AG ARLLKDQLSLGNAA mRNA C T C T C C CT GGGAAAT GC T GC AC T T CAGAT CAC AGAT GTG LQITDVKLQDAGVY AAAT T GCAGGAT GC AGGGGT GT AC C GCT GCAT GAT CAGC RCMIS YGGADYKRI TAT GGT GGT GC C GAC TAC AAGC GAAT TAC TGT GAAAGTC TVKVNAPYNKINQR AAT GC C CC AT AC AACAAAAT CAAC CAAAGAAT T T T GGTT ILWDPVTSEHELT GT GGAT CC AGT C AC C T C T GAAC AT GAAC T GAC AT GT C AG CQAEGYPKAEVIWT GCT GAGGGC T AC C C C AAGGC C GAAGT CAT C T GGAC AAGC S SDHQVLSGKTTTT AGT GAC CAT CAAGT C C T GAGTGGT AAGAC CAC CAC CACC NSKREEKLFNVTST AAT T C CAAGAGAGAGGAGAAGC T T T T CAAT GT GAC CAGC LRINTTTNE IFYCT ACAC T GAGAAT CAAC ACAAC AAC T AATGAGAT T T T C T AC FRRLDPEENHTAEL T GC AC T TT T AGGAGAT TAGATC C T GAGGAAAAC CATACA VIPEL PLAHPPNER GCT GAAT TGGTCATCC C AGAAC TACCTCTGGCACATCCT THLVILGAILLCLG C CAAAT GAAAGGAC T C AC T T GGTAAT TC T GGGAGC CATC VALTFIFRLRKGRM T TAT TATGCCTTGGT GTAGC AC T GAC AT T CAT C T T C C GT MDVKKCGIQDTNSK T TAAGAAAAGGGAGAAT GAT GGAT GT GAAAAAAT GT GGC KQSDTHLEET AT C CAAGAT AC7AAC T C7AAGAAGC7AAGT GAT AC AC AT ( SEQ ID NO: 10 )TTGGAGGAGACGTAA ( SEQ ID NO: 9 )18182281269.1UR 6-24056 / FR: 161118.06801 Homo ATGATCTTCCTCCTGCTAATGTTGAGCCTGGAATTGCAG MIFLLLMLSLELQL sapiens C T T C AC CAGATAGC AGC T T TAT T CAC AGT GAC AGT C C CT HQIAALFTVTVPKE programme AAGGAACT GTAC ATAATAGAGCATGGCAGCAAT GT GACC LYIIEHGSNVTLEC d cell death C T GGAATGCAAC T T T GAC AC TGGAAGTC AT GT GAAC C TT NFDTGSHVNLGAIT 1 ligand 2 GGAGC AAT AACAGC C AGT T T GC AAAAGGT GGAAAAT GAT ASLQKVENDTS PHR (PDCD1L NM0252393 XM0052516003..__ ACAT CCCCACACCGT GAAAGAGC C AC TT T GC T GGAGGAG ERATLLEEQLPLGK G2), CAGCTGCCCC TAGGGAAGGC CT C GT T CC AC AT AC C T C AA ASFHI PQVQVRDEG mRNA GT C CAAGT GAGGGAC GAAGGAC AGTACC AAT GC AT AATC QYQCI IIYGVAWDY AT C TAT GGGGT C GC C T GGGACT AC AAGT AC C T GAC T C TG KYLTLKVKASYRKI AAAGT C AAAGC T T C C T AC AGGAAAAT AAACAC T CACATC NTHILKVPETDEVE C TAAAGGT T C CAGAAACAGATGAGGT AGAGC T C AC C T GC LTCQATGYPLAEVS C AGGC T AC AGGT TAT C C T C T GGCAGAAGT AT C C T GGC CA WPNVSVPANTSHSR AAC GT C AGC GT T C C T GC C AACAC C AGCC AC T C C AGGACC T PEGLYQVTSVLRL C C T GAAGGC C T C TAC C AGGT CAC CAGTGTTCTGCGCCTA KPPPGRNFSCVFWN AAGCCACCCCCTGGCAGAAACTTCAGCTGTGTGTTCTGG THVRELTLAS IDLQ AAT AC T CAC GT GAGGGAAC T TAC T T T GGC CAGC AT T GAC SQMEPRTHPTWLLH C T T CAAAGT C AGAT GGAAC C CAGGAC CC AT C CAAC T T GG IFIPFCI IAFIFIA CTGCTTCACATTTTCATCCCCTTCTGCATCATTGCTTTC TVIALRKQLCQKLY AT T T T CAT AGC CAC AGT GAT AGC C C T AAGAAAACAAC TC S SKDTTKRPVTTTK T GT CAAAAGC T GTAT T C T T C AAAAGACAC AACAAAAAGA REVNSAI ( SEQ ID C C T GT C AC C ACAAC AAAGAGGGAAGT GAACAGT GC TATC NO: 12 )TGA ( SEQ ID NO: 11 )Homo ATGATCTTCCTCCTGCTAATGTTGAGCCTGGAATTGCAG MIFLLLMLSLELQL sapiens C T T CAC CAGATAGC AGC T T TAT T CAC AGT GAC AGT C C CT HQIAALFTVTVPKE programme AAGGAACT GTAC AT AAT AGAGC AT GGCAGC AAT GT GACC LYIIEHGSNVTLEC d cell death C T GGAATGCAAC T T T GAC AC TGGAAGTC AT GT GAAC C TT NFDTGSHVNLGAIT 1 ligand 2 GGAGC AAT AACAGC C AGT T T GC AAAAGGT GGAAAAT GAT ASLQKVENDTS PHR (PDCD1L ACAT CCCCACACCGT GAAAGAGC C AC TT T GC T GGAGGAG ERATLLEEQLPLGK G2), CAGCTGCCCC TAGGGAAGGC CT C GT T CC AC AT AC C T C AA ASFHI PQVQVRDEG transcript GT C CAAGT GAGGGAC GAAGGAC AGTACC AAT GC AT AATC QYQCI IIYGVAWDY variant XI, AT C TAT GGGGT C GC C T GGGACT AC AAGT AC C T GAC T C TG KYLTLKVKASYRKI mRNA AAAGT C AAAGC T T C C TAC AGGAAAAT AAAC AC T CACATC NTHILKVPETDEVE C TAAAGGT T C CAGAAACAGATGAGGT AGAGC T C AC C T GC LTCQATGYPLAEVS C AGGC TAC AGGT TAT C C T C T GGCAGAAGT AT C C T GGC CA WPNVSVPANTSHSR AAC GT C AGC GT T C C T GC C AACAC C AGCC AC T C C AGGACC T PEGLYQVTSVLRL C C T GAAGGC C T C TAC C AGGT CAC CAGTGTTCTGCGCCTA KPPPGRNFSCVFWN AAGCCACCCCCTGGCAGAAACTTCAGCTGTGTGTTCTGG THVRELTLAS IDLQ AAT AC T CAC GT GAGGGAAC T TAC T T T GGC CAGC AT T GAC SQMEPRTHPTWLLH C T T CAAAGT C AGAT GGAAC C CAGGAC CC AT C CAAC T T GG IFIPFCI IAFIFIA CTGCTTCACATTTTCATCCCCTTCTGCATCATTGCTTTC TVIALRKQLCQKLY AT T T T CAT AGC CAC AGT GAT AGC C C T AAGAAAACAAC TC S SKDTTKRPVTTTK T GT CAAAAGC T GTAT T C T T C AAAAGACAC AACAAAAAGA REVNSAVNLNLWSW C C T GT C AC C ACAAC AAAGAGGGAAGT GAACAGT GC T GTG E PG ( SEQ ID NO: AATCTGAACCTGTGGTCTTGGGAGCCAGGGTGA ( SEQ 14 )ID NO: 13 )

[0091] Additional suitable human PD-L1 encoding nucleotide sequences that can be incorporated in the recombinant genetic construct described herein are known in the art, see e.g, GenBank Accession Nos. BC113734.1, BC113736.1, BC074984.2, and BC069381.1, which are hereby incorporated by reference in their entirety.

[0092] Additional suitable human PDL-2 encoding nucleotides sequences that can be incorporated in the recombinant genetic construct described herein are known in the art, see e.g., GenBank Accession Nos. BC113680.1, BC 113678.1, and BC074766.2, which are hereby incorporated by reference in their entirety.19182281269.1UR 6-24056 / FR: 161118.06801

[0093] In another embodiment, the immune checkpoint protein or peptide encoded by the recombinant genetic construct of the present disclosure is the cell surface antigen, cluster of differentiation 47 (CD47; integrin associated protein (IAP)). The phagocytic acini ty of macrophages is regulated by activating ("eat") and inhibitory ("do not eat") signals. Under normal physiologic conditions, the ubiquitously expressed CD47 suppresses phagocytosis by¬ binding to signal regulatory protein alpha (SIRPa) on macrophages. SIRPa, also known as Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 / brain Ig-like molecule with tyrosine-based activation motif / cl uster of differentiation antigen-like family member A (SHPS-l / BIT / CD172a), is another membrane protein of the immunoglobulin superfamily that is particularly abundant in the myeloid-lineage hematopoietic cells such as macrophages and dendritic cells. The ligation of SIRPa on phagocytes by CD47 expressed on a neighboring cell results in phosphorylation of SIRPa cytoplasmic immunoreceptor tyrosine-based inhibition (ITIM) motifs, leading to the recruitment of SHP-1 and SHP-2 phosphatases. One resulting downstream effect is the prevention of myosin-IIA accumulation at the phagocytic synapse and consequently inhibition of phagocytosis. Thus, CD47-SIRPa interaction functions as a negative immune checkpoint to send a “don’t eat me” signal to ensure that healthy autologous cells are not inappropriately phagocytosed (Lui et al., “Is CD47 an Innate Immune Checkpoint for Tumor Evasion?” J. Hematol. Oncol. 10:12 (2017), which is hereby incorporated byreference in its entirety ).

[0094] Suitable nucleotide sequences encoding human CD47 for inclusion in the recombinant genetic construct as described herein are set forth in Table 4 below. Suitable nucleotide sequences also include nucleotide sequences having about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the CD47 coding sequences provided in Table 4 below.Table 4. Exemplary CD47 Coding Sequences and Amino Acid Sequences20182281269.1UR 6-24056 / FR: 161118.06801Amino Acid Sequences Name Coding SequenceGBkenanHomo ATGTGGCCCCTGGTAGCGGCGCTGTTGCTGGGCTCGGCG MWPLVAALLLGSA NM_001777.3 NM_198793.2 Aiccesson..__sapiens T GC T GC GGAT CAGC T CAGC TAC TAT T TAATAAAAC AAAA CCGSAQLLFNKTK NumberCD47 T C T GT AGAAT T C AC GT T T T GTAAT GACAC T GT C GT CATT SVEFTFCNDTWI molecule C CAT GC TT T GT T AC TAAT AT GGAGGC AC AAAAC AC TACT PCFVTNMEAQNTT (CD47), GAAGT ATAC GTAAAGT GGAAAT T T AAAGGAAGAGATATT EVYVKWKFKGRDI T AC AC C TT T GAT GGAGC T C T AAACAAGT C CAC T GT C C CC YTFDGALNKSTVP transcriptAC T GAC TT T AGT AGT GCAAAAAT T GAAGT C T C ACAAT TA TDFSSAKIEVSQL variant 1,C TAAAAGGAGAT GC C T C T T T GAAGAT GGATAAGAGT GAT LKGDASLKMDKSD mRNAGCT GT C TCACAC ACAGGAAACTACAC TT GTGAAGTAACA AVSHTGNYTCEVT GAAT T AAC C AGAGAAGGT GAAAC GAT CAT C GAGC T AAAA ELTREGETIIELK T AT C GT GT T GT T T C AT GGT T TT C T C CAAAT GAAAATATT YRWSWFS PNENI CTTATTGTTATTTTCCCAATTTTTGCTATACTCCTGTTC L IVIFPIFAILLF T GGGGACAGT T T GGT AT T AAAACAC T TAAAT AT AGAT CC WGQFGIKTLKYRS GGT GGT AT GGAT GAGAAAAC AAT T GC TT T AC T T GT T GCT GGMDEKT IALLVA GGAC T AGT GAT CACTGTCATTGTCATTGTT GGAGC CATT GLVITVIVIVGAI CTTTTCGTCCCAGGT GAAT AT T C AT T AAAGAAT GC T AC T LFVPGEYSLKNAT GGC C T T GGT T TAAT T GT GAC TT C T AC AGGGAT AT T AATA GLGLIVTSTGILI T TAC T T CAC T AC TAT GT GT T TAGT AC AGC GAT T GGAT TA LLHYYVFSTAIGL ACCTCCTTCGTCATTGCCATATT GGT TAT T C AGGT GATA TSFVIAILVIQVI GCCTATATCCTCGCTGTGGTTGGACTGAGTCTCTGTATT AYILAWGLSLCI GC GGC GTGT ATAC C AAT GCATGGC CCTCTTCT GAT T T CA AACIPMHGPLL IS GGT T T GAGT AT C T T AGC T C T AGCACAAT T AC T T GGAC TA GLS ILALAQLLGL GT T TAT AT GAAAT TTGTGGCTTC C AATC AGAAGAC TATA VYMKFVASNQKTI C AAC C T CC T AGGAAAGC T GT AGAGGAAC C C C T TAAT GCA QPPRKAVEE PINA T T C AAAGAAT CAAAAGGAAT GAT GAATGAT GAATAA FKESKGMMNDE( SEQ ID NO: 15 ) ( SEQ ID NO:16 ) MWPLVAALLLGSA ATGTGGCCCCTGGTAGCGGCGCTGTTGCTGGGCTCGGCG HomoT GC T GC GGAT CAGC T CAGC TAC TAT T TAAT AAAAC AAAA CCGSAQLLFNKTK sapiensT C T GT AGAAT T C AC GT T T T GTAAT GACAC T GT C GT CATT SVEFTFCNDTWI CD47C CAT GC TT T GT T AC TAAT AT GGAGGC AC AAAAC AC TACT PCFVTNMEAQNTT moleculeGAAGT ATAC GTAAAGT GGAAAT T T AAAGGAAGAGATATT EVYVKWKFKGRDI (CD47),T AC AC C TT T GAT GGAGC T C T AAACAAGT C CAC T GT C C CC YTFDGALNKSTVP transcriptAC T GAC TT TAGT AGT GCAAAAAT T GAAGT C T CAC AAT TA TDFSSAKIEVSQL variant 2,C T AAAAGGAGAT GC C T C T T T GAAGAT GGATAAGAGT GAT LKGDASLKMDKSD mRNAGCT GT C TCACAC ACAGGAAACTACAC TT GTGAAGTAACA AVSHTGNYTCEVT GAAT T AAC C AGAGAAGGT GAAAC GAT CAT C GAGC T AAAA ELTREGETIIELK T AT C GT GT T GT T T CAT GGT T TT C T C CAAAT GAAAATATT YRWSWFS PNENI CTTATTGTTATTTTCCCAATTTTTGCTATACTCCTGTTC L IVIFPIFAILLF T GGGGACAGT T T GGT AT T AAAACAC T TAAAT AT AGAT CC WGQFGIKTLKYRS GGT GGT AT GGAT GAGAAAAC AAT T GC TT T AC T T GT T GCT GGMDEKT IALLVA GGAC TAGT GAT CACTGTCATTGTCATTGTT GGAGC CATT GLVITVIVIVGAI CTTTTCGTCCCAGGT GAAT AT T C AT T AAAGAAT GC T AC T LFVPGEYSLKNAT GGC C T T GGT T TAAT T GT GAC TT C TAC AGGGAT AT T AATA GLGLIVTSTGILI T TAC T T CAC TAC TAT GT GT T TAGT AC AGC GAT T GGAT TA LLHYYVFSTAIGL ACCTCCTTCGTCATTGCCATATT GGT TAT T C AGGT GATA TSFVIAILVIQVI GCCTATATCCTCGCTGTGGTTGGACTGAGTCTCTGTATT AYILAWGLSLCI GC GGC GTGT ATAC C AAT GCATGGC CCTCTTCT GAT T T CA AACIPMHGPLL IS GGT T T GAGT AT C T T AGC T C T AGCACAAT T AC T T GGAC TA GLS ILALAQLLGL GT T TAT AT GAAAT TTGTGGCTTC C AATC AGAAGAC TATA VYMKFVASNQKTI CAACCTCCTAGGAATAACTGA ( SEQ ID NO: 17 ) QPPRNN ( SEQ ID NO: 18 )21182281269.1UR 6-24056 / FR: 161118.06801Amino Acid Sequences Name Coding SequenceGBkenanHomo ATGTGGCCCCTGGTAGCGGCGCTGTTGCTGGGCTCGGCG MWPLVAALLLGSA LN680437.1 KJ904432.1 Aiccesson..sapiens T GC T GC GGAT CAGC T CAGC TAC TAT T TAATAAAAC AAAA CCGSAQLLFNKTK NumbermRNA for T C T GT AGAAT T C AC GT T T T GTAAT GACAC T GT C GT CATT SVEFTFCNDTWI C CAT GC TT T GT T AC TAAT AT GGAGGC AC AAAAC AC TACT PCFVTNMEAQNTT CD47GAAGT ATAC GTAAAGT GGAAAT T T AAAGGAAGAGATATT EVYVKWKFKGRDI T AC AC C TT T GAT GGAGC T C T AAACAAGT C CAC T GT C C CC YTFDGALNKSTVP AC T GAC TT T AGT AGT GCAAAAAT T GAAGT C T C ACAAT TA TDFSSAKIEVSQL C TAAAAGGAGAT GC C T C T T T GAAGAT GGATAAGAGT GAT LKGDASLKMDKSD GCT GT C TCACAC ACAGGAAACTACAC TT GTGAAGTAACA AVSHTGNYTCEVT GAAT T AAC C AGAGAAGGT GAAAC GAT CAT C GAGC T AAAA ELTREGETTIIELK T AT C GT GT T GT T T C AT GGT T TT C T C CAAAT GAAAATATT YRWSWFS PNENI CTTATTGTTATTTTCCCAATTTTTGCTATACTCCTGTTC L IVIFPIFAILLF T GGGGACAGT T T GGT AT T AAAACAC T TAAAT AT AGAT CC WGQFGIKTLKYRS GGT GGT AT GGAT GAGAAAAC AAT T GC TT T AC T T GT T GCT GGMDEKT IALLVA GGAC T AGT GAT CACTGTCATTGTCATTGTT GGAGC CATT GLVITVIVIVGAI CTTTTCGTCCCAGGT GAAT AT T C AT T AAAGAAT GC T AC T LFVPGEYSLKNAT GGC C T T GGT T TAAT T GT GAC TT C T AC AGGGAT AT T AATA GLGLIVTSTGILI T TAC T T CAC T AC TAT GT GT T TAGT AC AGC GAT T GGAT TA LLHYYVFSTAIGL ACCTCCTTCGTCATTGCCATATT GGT TAT T C AGGT GATA TSFVIAILVIQVI GCCTATATCCTCGCTGTGGTTGGACTGAGTCTCTGTATT AYILAWGLSLCI GC GGC GTGT ATAC C AAT GCATGGC CCTCTTCT GAT T T CA AACIPMHGPLL IS GGT T T GAGT AT C T T AGC T C T AGCACAAT T AC T T GGAC TA GLS ILALAQLLGL GTTTATATGAAATTTGTGGAATAA ( SEQ ID NO: VYMKFVE ( SEQ 19 ) ID NO: 20 ) ATGTGGCCCCTGGTAGCGGCGCTGTTGCTGGGCTCGGCG MWPLVAALLLGSA SyntheticT GC T GC GGAT CAGC T CAGC TAC TAT T TAATAAAAC AAAA CCGSAQLLFNKTK constructT C T GT AGAAT T C AC GT T T T GTAAT GACAC T GT C GT CATT SVEFTFCNDTWI HomoPCFVTNMEAQNTT C CAT GC TT T GT T AC TAAT AT GGAGGC AC AAAAC AC TACT sapiensGAAGT ATAC GTAAAGT GGAAAT T T AAAGGAAGAGATATT EVYVKWKFKGRDI cloneT AC AC C TT T GAT GGAGC T C TAAAC AAGT C CAC T GT C C CC YTFDGALNKSTVP ccsbBroadAC T GAC TT TAGT AGT GCAAAAAT T GAAGT C T C ACAAT TA TDFSSAKIEVSQL En_13826C TAAAAGGAGAT GC C T C T T T GAAGAT GGATAAGAGT GAT LKGDASLKMDKSD CD47GCT GT C TCACAC ACAGGAAACTACAC TT GTGAAGTAACA AVSHTGNYTCEVT gene,GAAT T AAC C AGAGAAGGT GAAAC GAT CAT C GAGC T AAAA ELTREGETTIIELK encodesT AT C GT GT T GT T T CAT GGT T TT C T C CAAAT GAAAATATT YRWSWFS PNENI completeCTTATTGTTATTTTCCCAATTTTTGCTATACTCCTGTTC L IVIFPIFAILLF proteinT GGGGACAGT T T GGT AT T AAAACAC T TAAAT AT AGAT CC WGQFGIKTLKYRS GGT GGT AT GGAT GAGAAAAC AAT T GC TT T AC T T GT T GCT GGMDEKT IALLVA GGAC TAGT GAT CACTGTCATTGTCATTGTT GGAGC CATT GLVITVIVIVGAI CTTTTCGTCCCAGGT GAAT AT T C AT T AAAGAAT GC T AC T LFVPGEYSLKNAT GGC C T T GGT T TAAT T GT GAC TT C TAC AGGGAT AT T AATA GLGLIVTSTGILI T TAC T T CAC TAC TAT GT GT T TAGT AC AGC GAT T GGAT TA LLHYYVFSTAIGL ACCTCCTTCGTCATTGCCATATT GGT TAT T C AGGT GATA TSFVIAILVIQVI GCCTATATCCTCGCTGTGGTTGGACTGAGTCTCTGTATT AYILAWGLSLCI GC GGC GTGT ATAC C AAT GCATGGC CCTCTTCT GAT T T CA AACIPMHGPLL IS GGT T T GAGT AT C T T AGC T C T AGCACAAT T AC T T GGAC TA GLS ILALAQLLGL GT T TAT AT GAAAT TTGTGGCTTC C AATC AGAAGAC TATA VYMKFVASNQKTI CAACCTCCTGGAATAACTG ( SEQ ID NO: 21 ) QPPGIT ( SEQ ID NO: 22 )

[0095] In another embodiment, the immune checkpoint protein encoded by the recombinant genetic construct is CD200. CD200 (also known as OX-2 membrane glycoprotein) is a 45 kDa 22182281269.1UR 6-24056 / FR: 161118.06801 transmembrane immune checkpoint protein. The CD200 receptor (CD200R) is expressed on cells of the monocyte / macrophage lineage and subsets of B and T cells. Signaling by CD200 prevents normal activation of CD200R bearing myeloid cells, eventuating an immunosuppressive cascade that includes the induction of regulatory T cells (Tregs) (Gaiser et al., “Merke Cell Carcinoma Expresses the Immunoregulatory Ligand CD200 and Induces Immunosuppressive Macrophages and Regulatory’ T Cells,” Oncoimmunology 7(5):el426517 (2018), which is hereby incorporated by reference in its entirety). For example, CD200 signaling inhibits classic macrophage activation (Ml polarization) and supports an immunosuppressive M2 polarized state that secrets high levels of IL-10, thereby inducing Tregs. Thus, cell expression of CD200 via the recombinant genetic construct as described herein, will impart protection to the cell from macrophage and T-cell mediated responses.

[0096] Suitable nucleotide sequences encoding human CD200 for inclusion in the recombinant genetic construct as described herein are set forth in Table 5 below. Suitable nucleotide sequences also include nucleotide sequences having about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%. or 100% sequence identity to the CD200 coding sequences provided in Table 5 below.Table 5. Exemplary CD200 Coding Sequences and Amino Acid Sequences23182281269.1UR 6-24056 / FR: 161118.06801Amino Acid Name Coding SequenceSequence GBkenanHomo AT GGAGAGGC T GGT GAT C AGGAT GCCCTTCTCT CAT C TGT C MERLVIRMPFSHL NM_001004196.3 Aiccesson..sapiens T AC C T ACAGC C T GGT T T GGGTC AT GGCAGCAGT GGT GCT GT STYSLVWVMAAVV NumberCD200 o GCAC AGCACAAGT GCAAGT GGT GAC C CAGGAT GAAAGAGAG LCTAQVQWTQDE molecule C AGC T GTAC ACAC C T GC T T C CT TAAAAT GC T C T C T GC AAAA REQLYTPASLKCS (CD200), 1 T GC C C AGGAAGC C C T C AT T GTGAC AT GGC AGAAAAAGAAAG LQNAQEALIVTWQ transcript C T GTAAGC C C AGAAAACAT GGT CAC C TT C AGC GAGAACC AT KKKAVS PENMVTF variant 1, GGGGT GGT GAT C CAGC C T GC CT AT AAGGAC AAGAT AAAC AT SENHGWIQPAYK mRNA T AC C CAGC T GGGAC T C CAAAAC T C AACC AT CACCTTCTGGA DKINITQLGLQNS AT AT C ACC C T GGAGGAT GAAGGGT GT TAC AT GT GT C T CT T C T ITFWNITLEDEG AAT AC C TT T GGT T T T GGGAAGAT C T C AGGAAC GGC C T GC C T CYMCLFNTFGFGK C AC C GT CT AT GT AC AGC C CATAGT AT CC C T T CAC T AC AAAT ISGTACLTVYVQP TCTCTGAAGACCACCTAAATATCACTTGCTCTGCCACTGCC IVSLHYKFSEDHL CGCCCAGCCCCCATGGTCTTCTGGAAGGTCCCTCGGTCAGG NITCSATARPAPM GAT T GAAAAT AGTAC AGT GACT C T GT CT CAC C C AAAT GGGA VFWKVPRSGIENS C CAC GT CT GT TAC C AGCAT C CT C CAT AT C AAAGAC C C TAAG TVTLSHPNGTTSV AAT C AGGT GGGGAAGGAGGT GAT C T GCC AGGT GC T GC AC C T TS ILHIKDPKNQV GGGGAC TGT GAC C GAC T T TAAGCAAACC GT C AACAAAGGC T GKEVICQVLHLGT ATTGGTTTTCAGTTCCGCTATTGCTAAGCATTGTTTCCCTG VTDFKQTVNKGYW GTAATTCTTCTCGTCCTAATCTCAATCTTACTGTACTGGAA FSVPLLLS IVSLV ACGTCACCGGAATCAGGACCGAGAGCCCTAA ( SEQ ID ILLVL IS ILLYWK NO: 23 ) RHRNQDREP ( SEQ ID NO: 24 )Homo AT GGAGAGGC T GAC T C T GAC CAGGAC AAT TGGGGGCCCTCT MERLTLTRT IGGP sapiens C C T TAC AGC T AC AC T C C T AGGAAAGACC AC CAT CAAT GAT T LLTATLLGKTT IN CD200 ACCAGGTGATCAGGATGCCCTTCTCTCATCTGTCTACCTAC DYQVIRMPFSHLS molecule AGC C T GGT T T GGGT CAT GGCAGCAGT GGT GC T GT GCACAGC T YS LVWVMAAWL (CD200), ACAAGT GCAAGT GGT GAC C CAGGAT GAAAGAGAGC AGCT GT CTAQVQWTQDER transcript AC AC AC C T GC T T C C T T AAAAT GC T C T C T GC AAAAT GC C C AG EQLYT PASLKCSL variant 2, GAAGC C CT CAT T GT GACAT GGC AGAAAAAGAAAGC T GTAAG QNAQEALIVTWQK mRNA C C C AGAAAACAT GGT CAC C T TC AGC GAGAAC CAT GGGGT GG KKAVS PENMVTFS T GAT CCAGCCTGCC T ATAAGGACAAGAT AAAC AT T AC CC AG ENHGWIQPAYKD C T GGGACT C CAAAAC T C AAC CAT C AC CT T C T GGAATATC AC KINITQLGLQNST C C T GGAGGAT GAAGGGT GT T AC AT GT GT C T C T T CAAT AC C T ITFWNITLEDEGC T T GGT T TT GGGAAGAT C T CAGGAAC GGC C T GC C T CAC CGT C YMCLFNTFGFGKI TAT GT ACAGC C C AT AGTAT C CC T T CACT AC AAAT T C T CT GA SGTACLTVYVQPI AGAC C ACC TAAATAT C AC T T GC T C T GCC AC T GC C C GC CC AG VSLHYKFSEDHLN CCCCCATGGTCTTCTGGAAGGTCCCTCGGTCAGGGATTGAA ITCSATARPAPMV AAT AGT AC AGT GAC TCTGTCTCACC C AAAT GGGAC CACGT C FWKVPRSGIENST T GT TAC CAGC AT C C T C CATATC AAAGAC C C T AAGAAT CAGG VTLSHPNGTTSVT T GGGGAAGGAGGT GAT C T GC CAGGT GCT GCAC C T GGGGAC T S ILHIKDPKNQVG GT GAC C GAC T T T AAGC AAAC CGT C AACAAAGGC TAT T GGT T KEVICQVLHLGTV TTCAGTTCCGCTATTGCTAAGCATTGTTTCCCTGGTAATTC TDFKQTVNKGYWF TTCTCGTCCTAATCTCAATCTTACTGTACTGGAAACGTCAC SVPLLLS IVSLVI CGGAATCAGGACCGAGAGCCCTAA ( SEQ ID NO: 25 ) LLVLIS ILLYWKR HRNQDREP( SEQ ID NO:26 )24182281269.1UR 6-24056 / FR: 161118.06801Amino Add Name Coding SequenceSequence GBkenanHomo ATGAAGGGTGTTACATGTGTCTCTTCAATACCTTTGGTTTT MKGVTCVSS IPLV NM_001318826.1 NM_001318828.1 Aiccesson..__sapiens GGGAAGAT C T CAGGAAC GGC CT GC C T CAC C GT C TAT GCC CA LGRSQERPASPSM NumberCD200 T AGT AT CC C T T C AC T ACAAATT C T C T GAAGAC C AC C T AAAT PIVSLHYKFSEDH molecule ATCACTTGCTCTGCCACTGCCCGCCCAGCCCCCATGGTCTT LNITCSATARPAP (CD200), C T GGAAGGT C C C T C GGT C AGGGAT T GAAAAT AGTACAGT GA MVFWKVPRSGIEN transcript C T C T GT CT CAC C CAAAT GGGAC CAC GTC T GT T AC C AGCAT C STVTLSHPNGTTS variant 3, C T C CAT AT CAAAGAC C C T AAGAAT CAGGT GGGGAAGGAGGT VTS ILHIKDPKNQ mRNA GAT C T GCC AGGT GC T GCAC C TGGGGACT GT GAC C GAC TT TA VGKEVICQVLHLG AGC AAACC GT CAAC AAAGGC TAT T GGTT T T C AGT T C C GC TA TVTDFKQTVNKGY TTGCTAAGCATTGTTTCCCTGGTAATTCTTCTCGTCCTAAT WFSVPLLLS IVSL C T C AAT CT T AC T GT AC T GGAAAC GT CAC C GGAAT C AGGAC C VILLVLIS ILLYW GAGAGCCCTAA ( SEQ ID NO: 27 ) KRHRNQDRE P ( SEQ ID NO: 28 )Homo AT GGT C AC C T T C AGC GAGAACC AT GGGGT GGT GAT C C AGC C MPFSHLSTYSLVW sapiens T GC C T ATAAGGACAAGAT AAAC AT TACC C AGC T GGGACT C C VMAAWLCTAQVQ CD200 AAAAC T CAAC CAT C AC C T T C TGGAAT AT C AC C C T GGAGGAT WTQDEREQLYTP molecule GAAGGGTGT T AC AT GT GT C T CT T C AAT AC CTTTGGTTTTGG ASLKCSLQNAQEA (CD200), GAAGAT CT CAGGAAC GGC C T GC C T CACC GT C TAT GTACAGC L IVTWQKKKAVS P transcript C CAT AGTAT C C C T T CAC T AC AAAT T C TC T GAAGAC CACC TA ENMVTFSENHGW variant 4, AATATCACTTGCTCTGCCACTGCCCGCCCAGCCCCCATGGT IQPAYKDKINITQ mRNA C T T C T GGAAGGT C C C T C GGT CAGGGATT GAAAAT AGT AC AG LGLQNSTITFWNI TGACTCTGTCTCACCCAAATGGGACCACGTCTGTTACCAGC TLEDEGCYMCLFN AT C C T C CAT AT C AAAGAC C C TAAGAATC AGGT GGGGAAGGA TFGFGKISGTACL GGT GAT CT GC CAGGT GC T GC AC C T GGGGAC T GT GAC C GAC T TVYVQPIVSLHYK T TAAGC AAAC C GT C AACAAAGGC TATTGGTTTT CAGT TC C G FSEDHLNITCSAT CTATTGCTAAGCATTGTTTCCCTGGTAATTCTTCTCGTCCT ARPAPMVFWKVPR AAT C T C AAT C T T AC T GTAC T GGAAAC GT CAC C GGAAT CAGG SGIENSTVTLSHP ACC GAGAGCCCTAA ( SEQ ID NO: 2 9 ) NGTTSVTS ILHIK DPKNQVGKEVICQ VLHLGTVTDFKQT VNKGYWFSVPLLL S IVSLVILLVL IS ILLYWKRHRNQDR E P ( SEQ IDNO: 30 )

[0097] In another embodiment, the immune checkpoint protein encoded by the recombinant genetic construct is CTLA-4. In the immune recognition process, two signals are required for T lymphocyte expansion and differentiation: the T-cell receptor (TCR) binding to the HLA molecule-peptide complex and an antigen-independent costimulatory signal provided by the B7 (CD80 and Cd86) / CD28 interaction. The cytotoxic T-lymphocyte antigen (CTLA-4) is a homologous molecule of CD28 that is a competitive antagonist for B7. CTLA-4 has a greater affinity and avidity for B7 than does CD28, and its translocation to the cell surface after T-cell activation results in B7 sequestration and transduction of a negative signal, responsible for T-cell inactivation (Perez-Garcia et al., “CTLA-4 Polymorphisms and Clinical Outcome after 25182281269.1UR 6-24056 / FR: 161118.06801 Allogeneic StemCell Transplantation from HLA-Identical Sibling Donors,’’ Blood 110(l):461-7 (2007). which is hereby incorporated by reference in its entirety). Thus, cell expression of CTLA-4 via the recombinant genetic construct as described herein, will impart protection to the cell from cytotoxic T-cell mediated lysis.GBkenan

[0098] The CTLA-4 AF414120.1 gene is translated into 2 isoforms: a full-length protein (flCLTA-4) and a Aiccesson.soluble counterpart (sCTLA-4), which lacks exon 3 (responsible for coding the transmembrane domain) due to alternative splicing. flCTLA-4 down-regulates T-cell responses by inducing cell-cycle arrest and blocking cytokine production. Thus, in some embodiments, the immune checkpoint protein encoded by the recombinant genetic construct is full length CTLA-4 (flCTLA-4).

[0099] Suitable nucleotide sequences encoding human CTLA-4 for inclusion in the recombinant genetic construct as described herein are set forth in Table 6 below. Suitable nucleotide sequences also include nucleotide sequences having about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the CTLA-4 coding sequences provided in Table 6 below.Table 6. Exemplary CTLA-4 Coding Sequences and Amino Acid Sequences Amino Acid Sequence Name Coding SequenceHomo AT GGC T T GC C T T GGAT T TO AGO GGC ACAAGGC T C AGO MACLGFQRHKAQ sapiens TGAACCTGGCTACCAGGACCTGGCCCTGCACTCTCCT LNLATRTWPCTLGT T TT TTCT TC TC TT CATC CCTGTC T TC TGCAAAGCALFFLLFIPVFCK CTLA4 AT GCAC GT GGC C C AGC C TGC T GT GGT AC T GGC CAGC A AMHVAQPAWLA (CTLA4) GC C GAGGCAT C GC CAGC TT T GT GTGT GAGT AT GC AT C S SRGIASFVCEY mRNA, T C C AGGC AAAGC C AC T GAGGT C C GGGT GAC AGT GCT T AS PGKATEVRVT complete cds C GGCAGGC T GAC AGC CAGGT GAC TGAAGT CTGTGCGG VLRQADSQVTEV CAACC T ACAT GAT GGGGAAT GAGTT GAC C T T C C T AGA CAATYMMGNELT T GATT C C AT C T GC AC GGGC AC C T CC AGT GGAAAT CAA FLDDS ICTGTSS GT GAAC C T C AC TAT C CAAGGAC T GAGGGC CAT GGAC A GNQVNLTIQGLR C GGGAC T C T AC AT C T GC AAGGT GGAGC T CAT GTACC C AMDTGLYICKVE AC C GC CAT AC T AC C T GGGC AT AGGC AAC GGAAC C CAG LMYPPPYYLGIG AT T TAT GTAAT T GAT C C AGAAC C GT GC C C AGAT T CT G NGTQIYVIDPEP ACTTCCTCCTCTGGATCCTTGCAGCAGTTAGTTCGGG C PDSDFLLWILA GTTGTTTTTTTATAGCTTTCTCCTCACAGCTGTTTCT AVSSGLFFYS FL T T GAGCAAAAT GC TAAAGAAAAGAAGC C C T C T TACAA LTAVSLSKMLKK C AGGGGT C TAT GT GAAAAT GC C C CC AAC AGAGC C AGA RS PLTTGVYVKM AT GTGAAAAGC AAT T T C AGC C T T AT T T T AT T C C C AT C PPTE PECEKQFQ AATTGA ( SEQ ID NO: 31 ) PYFI PIN ( SEQ ID NO: 32 )26182281269.1UR 6-24056 / FR: 161118.06801Amino Acid Sequence Name Coding SequenceHomo AT GGC T T GC C T T GGAT T TO AGO GGC ACAAGGC T C AGO MACLGFQRHKAQ sapiens GBkenan TGAACCTGGCTACCAGGACCTGGCCCTGCACTCTCCT LNLATRTWPCTL NM_005214.5 NM_001037631.3..__GT T TT TTCT TC TC TT CATC CCTGTC T TC TGCAAAGCAcytotoxic T- Aiccesson LFFLLFIPVFCK AT GCAC GT GGC C C AGC C TGC T GT GGT AC T GGC CAGC A AMHVAQPAWLAlymphocyte Nbmeru GC C GAGGCAT C GC CAGC TT T GT GTGT GAGT AT GC AT C S SRGIASFVCEY associated T C C AGGC AAAGC C AC T GAGGT C C GGGT GAC AGT GCT T AS PGKATEVRVT protein 4 C GGCAGGC T GAC AGC CAGGT GAC TGAAGT CTGTGCGG VLRQADSQVTEV (CTLA4), CAACC T ACAT GAT GGGGAAT GAGTT GAC C T T C C T AGA CAATYMMGNELT T GATT C C AT C T GC AC GGGC AC C T CC AGT GGAAAT CAA FLDDS ICTGTSStranscript GT GAAC C T C AC TAT C CAAGGAC T GAGGGC CAT GGAC A GNQVNLTIQGIR variant 1, C GGGAC T C T AC AT C T GC AAGGT GGAGC T CAT GTACC C AMDTGLYICKVE mRNA AC C GC CAT AC T AC C T GGGC AT AGGC AAC GGAAC C CAG LMYPPPYYLGIG AT T TAT GTAAT T GAT C C AGAAC C GT GC C C AGAT T CT G NGTQIYVIDPEP ACTTCCTCCTCTGGATCCTTGCAGCAGTTAGTTCGGG C PDSDFLLWILA GTTGTTTTTTTATAGCTTTCTCCTCACAGCTGTTTCT AVSSGLFFYS FL T T GAGCAAAAT GC TAAAGAAAAGAAGC C C T C T TACAA LTAVSLSKMLKK C AGGGGT C TAT GT GAAAAT GC C C CC AAC AGAGC C AGA RS PLTTGVYVKM AT GTGAAAAGC AAT T T C AGC C T T AT T T T AT T C C C AT C PPTE PECEKQFQ AATTGA ( SEQ ID NO: 33 ) PYFI PIN ( SEQ ID NO: 34 ) Homo AT GGC T T GC C T T GGAT T TC AGC GGC ACAAGGC T C AGC MACLGFQRHKAQ sapiens TGAACCTGGCTACCAGGACCTGGCCCTGCACTCTCCT LNLATRTWPCTL GTTTTTTCTTCTCTTCATCCCTOTCTTCTGCAAAGCA LFFLLFIPVFCKcytotoxic T- AT GCAC GT GGC C CAGC C TGC T GT GGT AC T GGC CAGC A AMHVAQPAWLA lymphocyte GC C GAGGCAT C GC CAGC TT T GT GTGT GAGT AT GC AT C S SRGIASFVCEY associated T C C AGGC AAAGC C AC T GAGGT C C GGGT GAC AGT GCT T AS PGKATEVRVT protein 4 C GGCAGGC T GAC AGC CAGGT GAC TGAAGT CTGTGCGG VLRQADSQVTEV (CTLA4), CAACC T ACAT GAT GGGGAAT GAGTT GAC C T T C C T AGA CAATYMMGNELT T GATT C C AT C T GC AC GGGC AC C T CC AGT GGAAAT CAA FLDDS ICTGTSStranscript GT GAAC C T C AC TAT C CAAGGAC T GAGGGC CAT GGAC A GNQVNLTIQGIR variant 2, C GGGAC T C T AC AT C T GC AAGGT GGAGC T CAT GTACC C AMDTGLYICKVE mRNA AC C GC CAT AC T AC C T GGGC AT AGGC AAC GGAAC C CAG LMYPPPYYLGIG AT T TAT GTAAT TGC T AAAGAAAAGAAGC CCTCTTACA NGTQIYVIAKEK AC AGGGGT C TAT GT GAAAAT GC C CC C AAC AGAGC CAG KPSYNRGLCENA AATGTGA ( SEQ ID NO: 35 ) PNRARM ( SEQ ID NO: 36 )

[0100] In another embodiment, the immune checkpoint protein encoded by the recombinant genetic construct is HLA-E (major histocompatibility complex, class I, E). Natural killer (NK) cells detect infected cells (mainly infected by viruses), foreign cells, or malignant cells in which expression of MHC molecules has decreased, is altered, abolished, or absent. NK cells distinguish normal host cells through the killer cell immunoglobulin-like receptor (KIR) and CD94-NKG2A inhibitory receptors which recognize the MHC class I expressed on the surface of normal host cells. In particular, CD94-NKG2A recognizes HLA-E on the surface of NK cells and CD8+T cells. The binding of these receptors inhibits lysis 27182281269.1UR 6-24056 / FR: 161118.06801 and cytokine secretion by NK cells. KIRs are also expressed on CD8+T cells and APCs. Thus, cell expression ofHLA-E via the recombinant genetic construct as described herein, will impart protection to the cell from NK cell lysis.

[0101] Like other HLA class I proteins, HLA-E is a heterodimer consisting of a heavy chain (a chain) and light chain (02 microglobulin). In one embodiment, the recombinant genetic construct may comprise a nucleotide sequence encoding the HLA-E (a chain E) and a nucleotide sequence encoding the 02 microglobulin chain. Alternatively, the recombinant genetic construct may comprise a fusion construct, i. e., a nucleotide sequence encoding a single chain fusion protein that comprises at least a portion of the 02 microglobulin covalently linked to at least a portion of HLA-E. In other embodiments, the HLA-E / 02M fusion protein is sy02M-HLA-E, where syB2M (synthetic B2M) is expressed as complex with HLA-E.

[0102] Exemplary nucleotide sequences encoding human HLA-E (alpha chain) are provided in Table 7 below. Suitable nucleotide sequences also include nucleotides sequence having about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the HLA-E coding sequences provided in Table 7 below.Table 7. Exemplary HLA-E Coding Sequences and Amino Acid Sequences28182281269.1UR 6-24056 / FR: 161118.06801Amino Acid Sequence Coding SequenceNameGBkenanHuman M20022.1 Aiccesson. ATGGTAGATGGAACCCTCCTTTTACTCTCCTCGGAGGCCC MVDGTLLLLSSEA HLA-E Number TGGCCCTTACCCAGACCTGGGCGGGCTCCCACTCCTTGAA LALTQTWAGSHSL Class I GTATTTCCACACTTCCGTGTCCCGGCCCGGCCGCGGGGAG KYFHTSVSRPGRG mRNA CCCCGCTTCATCTCTGTGGGCTACGTGGACGACACCCAGT E PRFISVGYVDDT T C GT GC GC T T C GAC AAC GAC GC C GC GAGT C C GAGGAT GGT QFVRFDNDAAS PR GO C GO GGGC GO C GT GGAT GGAGC AGGAGGGGT C AGAGTAT MVPRAPWMEQEGS T GGGAC CGGGAGAC AC GGAGC GC CAGGGACAC C GC AC AGA EYWDRETRSARDT T T T T C C GAGT GAAC C T GC GGAC GCT GC GC GGC T AC TACAA AQIFRVNLRTLRG T C AGAGCGAGGC C GGGT C T CACACC C T GC AGT GGAT GCAT YYNQSEAGSHTLQ GGC T GC GAGC T GGGGC C C GAC AGGC GCTTCCTCCGCGGGT WMHGCELGPDRRF AT GAAC AGT T C GC C T AC GAC GGC AAGGAT TAT C T C AC CC T LRGYEQFAYDGKD GAAT GAGGAC C T GC GC T C C T GGACC GC GGT GGACAC GGC G YLTLNEDLRSWTA GCT C AGAT C T C C GAGC AAAAGT C AAAT GAT GC C T C T GAGG VDTAAQISEQKSN C GGAGC AC C AGAGAGC C T AC C T GGAAGAC AC AT GC GT GGA DASEAEHQRAYLE GT GGC T CC AC AAAT AC C T GGAGAAGGGGAAGGAGAC GCT G DTCVEWLHKYLEK C T T C AC CT GGAGC C C C C AAAGAC AC AC GT GAC T CAC C AC C GKETLLHLE PPKT CCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCT HVTHHPISDHEAT GGGCTTCTACCCTGC GGAGAT CAC AC T GAC C T GGC AGCAG LRCWALGFYPAEI GAT GGGGAGGGC CAT AC C C AGGACAC GGAGC T C GT GGAGA TLTWQQDGEGHTQ C C AGGC CT GC AGGGGAT GGAAC C TT C C AGAAGT GGGC AGC DTELVETRPAGDG TGTGGTGGTGCCTTCT GGAGAGGAGC AGAGAT ACAC GTGC TFQKWAAVWPSG CAT GT GCAGC AT GAGGGGC TAC C CGAGC CCGTCACCCTGA EEQRYTCHVQHEG GAT GGAAGC C GGC T T C C C AGC C C AC CAT C C C CAT C GT GGG L PE PVTLRWKPAS CATCATTGCTGGCCTGGTTCTCCTTGGATCTGTGGTCTCT QPT IPIVGI IAGL GGAGC T GT GGT TGCTGCTGT GAT AT GGAGGAAGAAGAGC T VLLGSWSGAWA CAGGT GGAAAAGGAGGGAGC TAC TC TAAGGC T GAGT GGAG AVIWRKKSSGGKG C GAC AGTGC C CAGGGGT C T GAGT CT C ACAGC T T GT AA GSYSKAEWSDSAQ( SEQ ID NO: 37 ) GSESHSL ( SEQ ID NO: 38 )29182281269.1UR 6-24056 / FR: 161118.06801Amino Acid Sequence Coding SequenceNameGBkenanHuman AJ293263.1 Aiccesson. ATGGTAGATGGAACCCTCCTTTTACTCCTCTCGGAGGCCC MVDGTLLLLLSEA MHC Number TGGCCCTTACCCAGACCTGGGCGGGCTCCCACTCCTTGAA LALTQTWAGSHSL Class I GTATTTCCACACTTCCGTGTCCCGGCCCGGCCGCGGGGAG KYFHTSVSRPGRG antigen, CCCCGCTTCATCTCTGTGGGCTACGTGGACGACACCCAGT EPRFISVGYVDDT HLA- T C GT GC GC T T C GAC AAC GAC GC C GC GAGT C C GAGGAT GGT QFVRFDNDAAS PR E*01033 GO C GO GGGC GO C GT GGAT GGAGCAGGAGGGGT C AGAGTAT MVPRAPWMEQEGS allele T GGGAC CGGGAGAC AC GGAGCGC C AGGGAC AC C GC AC AGA EYWDRETRSARDT T T T T C C GAGT GAAT C T GC GGAC GC T GCGC GGC T AC TACAA AQIFRVNLRTLRG T CAGAGCGAGGC CGGGTCTCACACCCTGCAGT GGAT GCAT YYNQSEAGSHTLQ GGC T GC GAGC T GGGGC C C GACGGGC GCTTCCTCCGCGGGT WMHGCELGPDGRF AT GAAC AGT T C GC C T AC GAC GGCAAGGAT TAT C T C AC CC T LRGYEQFAYDGKD GAAT GAGGAC C T GC GC T C C T GGAC C GCGGT GGACAC GGC G YLTLNEDLRSWTA GC T C AGAT C T C C GAGC AAAAGT CAAATGAT GC T T C T GAGG VDTAAQISEQKSN C GGAGC AC C AGAGAGC C T AC CT GGAAGAC AC AT GC GT GGA DASEAEHQRAYLE GT GGC T CC AC AAAT AC C T GGAGAAGGGGAAGGAGAC GCT G DTCVEWLHKYLEK C T T C AC CT GGAGC C C C CAAAGACACACGT GAC T CAC C AC C GKETLLHLEPPKT CCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCT HVTHHPISDHEAT GGGCTTCTACCCTGC GGAGATC AC AC TGAC C T GGC AGCAG LRCWALGFYPAEI GAT GGGGAGGGC CAT AC C CAGGAC AC GGAGC T C GT GGAGA TLTWQQDGEGHTQ C CAGGC CT GC AGGGGAT GGAAC C T T C CAGAAGT GGGC AGC DTELVETRPAGDG T GT GGT GGT GC C T T C T GGAGAGGAGC AGAGAT ACAC GTGC T F QKWAAVWP S G CAT GT GCAGC AT GAGGGGC T AC C C GAGC CCGTCACCCTGA EEQRYTCHVQHEG GAT GGAAGC C GGC T T C C C AGCC CAC C AT C C C CAT C GT GGG LPEPVTLRWKPAS CATCATTGCTGGCCTGGTTCTCCTTGGATCTGTGGTCTCT QPTIPIVGIIAGL GGAGC T GT GGT T GC T GC T GT GAT AT GGAGGAAGAAGAGC T VLLGSWSGAWA C AGGT GGAAAAGGAGGGAGC TAC T C T AAGGC T GAGT GGAG AVIWRKKS SGGKG C GAC AGTGC C CAGGGGT C T GAGT C T CAC AGC T T GT AA GSYSKAEWSDSAQ( SEQ ID NO: 39 ) GSESHSL ( SEQ ID NO: 40 )30182281269.1UR 6-24056 / FR: 161118.06801Amino Acid Sequence Coding SequenceNameGBkenanHuman AJ293264.1 Accession ATGGTAGATGGAACCCTCCTTTTACTCCTCTCGGAGGCCC MVDGTLLLLLSEA MHC Number TGGCCCTTACCCAGACCTGGGCGGGCTCCCACTCCTTGAA LALTQTWAGSHSL Class I GTATTTCCACACTTCCGTGTCCCGGCCCGGCCGCGGGGAG KYFHTSVSRPGRG antigen, CCCCGCTTCATCTCTGTGGGCTACGTGGACGACACCCAGT EPRFISVGYVDDT HLA- T C GT GC GC T T C GAC AAC GAC GC C GC GAGT C C GAGGAT GGT QFVRFDNDAAS PR E*0101 GO C GO GGGC GO C GT GGAT GGAGCAGGAGGGGT C AGAGTAT MVPRAPWMEQEGS allele T GGGAC CGGGAGAC AC GGAGCGC C AGGGAC AC C GC AC AGA EYWDRETRSARDT T T T T C C GAGT GAAC C T GC GGAC GC T GCGC GGC T AC TACAA AQIFRVNLRTLRG T CAGAGCGAGGC CGGGTCTCACACCCTGCAGT GGAT GCAT YYNQSEAGSHTLQ GGC T GC GAGC T GGGGC C C GACAGGC GCTTCCTCCGCGGGT WMHGCELGPDRRF AT GAAC AGT T C GC C T AC GAC GGCAAGGAT TAT C T C AC CC T LRGYEQFAYDGKD GAAT GAGGAC C T GC GC T C C T GGAC C GCGGT GGACAC GGC G YLTLNEDLRSWTA GC T C AGAT C T C C GAGC AAAAGT CAAATGAT GC C T C T GAGG VDTAAQISEQKSN C GGAGC AC C AGAGAGC C T AC CT GGAAGAC AC AT GC GT GGA DASEAEHQRAYLE GT GGC T CC AC AAAT AC C T GGAGAAGGGGAAGGAGAC GCT G DTCVEWLHKYLEK C T T C AC CT GGAGC C C C CAAAGACACACGT GAC T CAC C AC C GKETLLHLEPPKT CCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCT HVTHHPISDHEAT GGGCTTCTACCCTGC GGAGATC AC AC TGAC C T GGC AGCAG LRCWALGFYPAEI GAT GGGGAGGGC CAT AC C CAGGAC AC GGAGC T C GT GGAGA TLTWQQDGEGHTQ C CAGGC CT GC AGGGGAT GGAAC C T T C CAGAAGT GGGC AGC DTELVETRPAGDG T GT GGT GGT GC C T T C T GGAGAGGAGC AGAGAT ACAC GTGC T F QKWAAVWP S G CAT GT GCAGC AT GAGGGGC T AC C C GAGC CCGTCACCCTGA EEQRYTCHVQHEG GAT GGAAGC C GGC T T C C C AGCC CAC C AT C C C CAT C GT GGG LPEPVTLRWKPAS CATCATTGCTGGCCTGGTTCTCCTTGGATCTGTGGTCTCT QPTIPIVGIIAGL GGAGC T GT GGT T GC T GC T GT GAT AT GGAGGAAGAAGAGC T VLLGSWSGAWA C AGGT GGAAAAGGAGGGAGC TAC T C T AAGGC T GAGT GGAG AVIWRKKS SGGKG C GAC AGTGC C CAGGGGT C T GAGT C T CAC AGC T T GT AA GSYSKAEWSDSAQ( SEQ ID NO: 41 ) GSESHSL ( SEQ ID NO: 42 )02M protein

[0103] In the recombinant nucleic acid molecule / genetic construct described herein, the third coding-sequence encodes for p2-microglobulin protein. Exemplary nucleotide sequences encoding for human [EM are provided in Table 8 below. Suitable nucleotide sequences also include nucleotide sequences having about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the [EM coding sequences provided in Table 8 below.Table 8. Suitable fM Coding Sequence and Amino Acid Sequences31182281269.1UR 6-24056 / FR: 161118.06801Amino Acid Sequences Name Coding SequenceGBkenanHomo CR457066.1 BC064910.1 BC032589.1 NM_004048.3 Accession ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCT MSRSVALAVLALL sapiens Number CTCTTTCTGGCCTGGAGGCTATCCAGCGTACTCCAAAGAT SLSGLEAIQRTPK beta-2- T CAGGT TT AC T C AC GT CAT C CAGC AGAGAAT GGAAAGTC A IQVYSRHPAENGK microglobu AATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCG SNFLNCYVSGFHP lin (B2M), AC AT T GAAGT T GAC T T AC T GAAGAAT GGAGAGAGAAT T GA SDIEVDLLKNGER mRNA AAAAGT GGAGCAT T C AGAC T TGT C T T TC AGC AAGGAC TGG IEKVEHSDLSFSK TCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTG DWSFYLLYYTEFT AAAAAGAT GAGT AT GC C T GC CGT GT GAAC CAT GT GAC TT T PTEKDEYACRVNH GT C AC AGC C C AAGAT AGT TAAGT GGGAT C GAGACAT GTAA VTLSQPKIVKWDR(SEQ ID NO: 43 ) DM (SEQ ID NO: 44 ) Homo ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCT MSRSVALAVLALL sapiens full CTCTTTCTGGCCTGGAGGCTATCCAGCGTACTCCAAAGAT SLSGLEAIQRTPK open T CAGGT TT AC T C AC GT CAT C CAGC AGAGAAT GGAAAGTC A IQVYSRHPAENGK reading AATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCG SNFLNCYVSGFHP frame AC AT T GAAGT T GAC T T AC T GAAGAAT GGAGAGAGAAT T GA SDIEVDLLKNGER cDNA AAAAGT GGAGCAT T C AGAC T TGT C T T TC AGC AAGGAC TGG IEKVEHSDLSFSK clone TCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTG DWSFYLLYYTEFT RZPDo834 AAAAAGAT GAGT AT GC C T GC CGT GT GAAC CAT GT GAC TT T PTEKDEYACRVNH B107D for GT C AC AGC C C AAGAT AGT TAAGT GGGAT C GAGACAT T TAA VTLSQPKIVKWDR gene B2M (SEQ ID NO: 45 ) DI (SEQ ID NO: 46 ) Homo ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCT MSRSVALAVLALL sapiens CTCTTTCTGGCCTGGAGGCTATCCAGCGTACTCCAAAGAT SLSGLEAIQRTPK beta-2- T CAGGT TT AC T C AC GT CAT C CAGC AGAGAAT GGAAAGTC A IQVYSRHPAENGK microglobu AATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCG SNFLNCYVSGFHP lin, mRNA AC AT T GAAGT T GAC T T AC T GAAGAAT GGAGAGAGAAT T GA SDIEVDLLKNGER AAAAGT GGAGCAT T C AGAC T TGT C T T TC AGC AAGGAC TGG IEKVEHSDLSFSK TCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTG DWSFYLLYYTEFT AAAAAGAT GAGT AT GC C T GC CGT GT GAAC CAT GT GAC TT T PTEKDEYACRVNH GT C AC AGC C C AAGAT AGT TAAGT GGGAT C GAGACAT GTAA VTLSQPKIVKWDR(SEQ ID NO: 47 ) DM (SEQ ID NO: 48 ) Homo ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCT MSRSVALAVLALL sapiens CTCTTTCTGGCCTGGAGGCTATCCAGCGTACTCCAAAGAT SLSGLEAIQRTPK beta-2- T CAGGT TT AC T C AC GT CAT C CAGC AGAGAAT GGAAAGTC A IQVYSRHPAENGK microglobu AATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCG SNFLNCYVSGFHP lin, mRNA AC AT T GAAGT T GAC T T AC T GAAGAAT GGAGAGAGAAT T GA SDIEVDLLKNGER AAAAGT GGAGCAT T C AGAC T TGT C T T TC AGC AAGGAC TGG IEKVEHSDLSFSK TCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTG DWSFYLLYYTEFT AAAAAGAT GAGT AT GC C T GC CGT GT GAAC CAT GT GAC TT T PTEKDEYACRVNH GT C AC AGC C C AAGAT AGT TAAGT GGGAT C GAGACAT GTAA VTLSQPKIVKWDR(SEQ ID NO: 49 ) DM (SEQ ID NO: 50 )32182281269.1UR 6-24056 / FR: 161118.06801 Recombinases and Sites of Recombinase-Mediated Recombination

[0104] The recombinant nucleic acid molecule and related methods disclosed herein utilize site-specific recombination to rearrange (e.g., flip or reverse the orientation of) or remove certain elements of the recombinant nucleic acid molecule in cells depending on types of the cells and differentiation stages of the cells as well as the promoters that are operative in the cells.

[0105] “Site-specific recombination’’, also known as conservative site-specific recombination, is a type of recombination in which nucleic acid strand exchange takes place between segments possessing only a limited degree of sequence homology. Site-specific recombinase enzymes perform rearrangements of nucleic acid segments by recognizing and binding to short DNA sequences (sites), at which they cleave the DNA backbone, exchange the tw o DNA helices involved and rejoin the DNA strands. In some site-specific recombination systems having just a recombinase enzyme together with the recombination sites is enough to perform all these reactions, in some other systems a number of accessory proteins and accessory sites may also needed.

[0106] The term “recombinase-mediated recombination” means recombination between two recognition sites of identical or near identical sequences (also called repeats) catalyzed by a recombinase.

[0107] The term “repeat” means a fragment of DNA that is repeated at least twice in the recombinant DNA introduced into a host cell and which can facilitate the loss or flip of the section between the repeats (z.e. a coding sequence or a control sequence that is inserted between two repeats, by homologous recombination. A repeat can be an inverted repeat, a direct repeat, or a homologous repeat. In a preferred aspect, the two repeats are identical in sequence. However, two repeats may have a sequence identity to each other of at least 70%, i.e., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%. at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%. or at least 99%.

[0108] The term “repeat sequences” means a set of two sequences, one of which is positioned 5' to a polynucleotide of interest and the other is positioned 3' to the polynucleotide sequence of interest, such that the repeat sequences undergo homologous recombination to remove or flip the polynucleotide. A repeat sequence can be at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 15033182281269.1UR 6-24056 / FR: 161118.06801 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides.

[0109] In the recombinant nucleic acid molecule described herein, the first coding-sequence encodes for a sequence specific recombinase. Thissequence-specific recombinase recognizes a pair of specific DNA sequences (also called “sites of recombinase-mediated recombination.” “site-specific recombination sites,” “recombination recognition sites,” or “recombinase recognition sites” in this disclosure) and mediated recombination between the pair. The site-specific recombination sites and recombinase can be selected such that the recombinase may target the site-specific recombination sites leading to recombination of sequence locate between the recombination sites.

[0110] The terms “recombinase” or “site-specific recombinase” or the like refers to enzymes or recombinases that recognize and bind to a short nucleic acid site or “site-specific recombinase site” and catalyze the recombination of nucleic acid in relation to these sites. Examples of these enzymes include recombinases, transposases, and integrases.

[0111] The “site-specific recombinase site.” “sites of recombinase-mediated recombination,” “site-specific recombination sites,” “recombination recognition sites,” or “recombinase recognition sites ”or the like refers to short nucleic acid sites or sequences, i.e., which are recognized by a sequence- or site-specific recombinase and which become the crossover regions during a site-specific recombination event. Examples of sequence-specific recombinase target sites include, but are not limited to, lox sites, att sites, dif sites and fit sites.

[0112] The term “lox site” as used herein refers to a nucleotide sequence at which the product of the ere gene of bacteriophage Pl, the Cre recombinase, can catalyze a site-specific recombination event. A variety of lox sites are known in the art, including the naturally occurring loxP, loxB, loxL and loxR, as well as a number of mutant or variant, lox sites, such as lox66, lox71, loxP511, loxP514, lox486, lox4117, loxC2, loxP2, loxP3 and lox P23.

[0113] The term “frt site” as used herein refers to a nucleotide sequence at which the product of the FLP gene of the yeast 2 micron plasmid, FLP recombinase, can cataly ze sitespecific recombination.

[0114] The site-specific recombination sites may be such that recombination following recombinase expression gives rise to a single site-specific recombination site at a locus which may or may not be recognized by the recombinase. In particular, the lox sites may be lox66 and lox 71 (Albert, H.. Dale, E. C., Lee, E., & Ow, D. W. (1995). Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant Journal, 7(4). 649- 34182281269.1UR 6-24056 / FR: 161118.06801 659). In a specific embodiment, the lox66 and lox71 site-specific recombination sites may be such that recombination following recombinase expression gives rise to a lox72 mutant sitespecific recombination site at the locus which is not recognized by the recombinase.

[0115] The recombination recognition sites can be any recombination recognition sites useful in the methods of the present disclosure. In one aspect, the recombination recognition sites are selected from the group consisting of a B2 system from Zygosaccharomyces bailii, B3 system from Zygosaccharomyces bisporus. beta-recombinase-six system from a 25 Bacillus subtilis plasmid, Bxbl from phage Bxbl, Cre-lox system of bacteriophase Pl, Dre from Bacteriophage D6, FLP-FRT of Saccharomyces cerevisiae, Delta-gamma-es system from bacterial transposon TnlOOO, Gin-gix system from bacteriophase Mu, HK022 from phage HK022, KD system from Kluyveromyces drosophilarum, Mx9 phage transformation system, Streptomyces phage 1C31, R-RS system of Zygosaccharomyces rouxii, Tn3 fromE. coli, Vika recombinase from Vibrio coralliilyticus, and Xis-att system of temperate lactococcal bacteriophage TP901-1; and combinations thereof. It is understood herein that the enumerated recombination recognition sites encompass homologs and variants thereof. In one embodiment, one of the recombination recognition sites is a B2 system from Zygosaccharomyces bailii. In another embodiment, one of the recombination recognition sites is a B3 system from Zygosaccharomyces bisporus. In another embodiment, one of the recombination recognition sites is a beta-recombinase-six system from a 25 Bacillus subtilis plasmid. In another embodiment, one of the recombination recognition sites is a Bxbl from phage Bxbl. In another embodiment, one of the recombination recognition sites is a Cre-lox system of bacteriophase Pl. In another embodiment, one of the recombination recognition sites is a Dre from Bacteriophage D6. In another embodiment, one of the recombination recognition sites is a FLP-FRT of Saccharomyces cerevisiae. In another embodiment, one of the recombination recognition sites is a Delta-gamma-es system from bacterial transposon TnlOOO. In another embodiment, one of the recombination recognition sites is a Gin-gix system from bacteriophase Mu. In another embodiment, one of the recombination recognition sites is a HK022 from phage HK022. In another embodiment, one of the recombination recognition sites is a KD system from Kluyveromyces drosophilarum. In another embodiment, one of the recombination recognition sites is a Mx9 phage transformation system. In another embodiment, one of the recombination recognition sites is a Streptomyces phage 1C31. In another embodiment, one of the recombination recognition sites is a R-RS system of Zygosaccharomyces rouxii. In another embodiment, one of the recombination recognition sites is a Tn3 from E. coli. In another 35182281269.1UR 6-24056 / FR: 161118.06801 embodiment, one of the recombination recognition sites is a Vika recombinase from Vibrio coralliilyticus. In another embodiment, one of the recombination recognition sites is a Xis-att system of temperate lactococcal bacteriophage TP901-1.

[0116] In one aspect, the flippase recognition sites of the FLP-FRT system are selected from the group consisting of F, F3, F10, F13, F14, F15, Fa, and F3a, and combinations thereof. It is understood herein that the enumerated flippase recognition sites encompass homologs and variants thereof. In one embodiment, the flippase recognition site is F. In another embodiment, the flippase recognition site is F3. In another embodiment, the flippase recognition site is F10. In another embodiment, the flippase recognition site is F13. In another embodiment, the flippase recognition site is F14. In another embodiment, the flippase recognition site is F15. In another embodiment, the flippase recognition site is Fa. In another embodiment, the flippase recognition site is F3a. See Turan et al., 2010, J. Mol. Biol. 402: 52-69, which is incorporated herein in its entirety.

[0117] In another aspect, the TP901-1 sites of the Xis-att sy stem are selected from the group consisting of attB and attP. and combinations thereof. It is understood herein that the enumerated TP901-1 sites encompass homologs and variants thereof. In one embodiment, the TP901-1 site is attB. In another embodiment, the TP901-1 site is attP.In another aspect, the Lox sites of the Cre-lox system are selected from the group consisting of LoxP, Lox71, Lox66, Lox511. Lox5171, Lox2272, M2, M3, M7, and MH, and combinations thereof. It is understood herein that the enumerated Lox sites encompass homologs and variants thereof. In one embodiment, the Lox site is LoxP. In another embodiment, the Lox site is Lox71. In another embodiment, the Lox site is Lox66. In another embodiment, the Lox site is Lox511. In another embodiment, the Lox site is Lox5171. In another embodiment, the Lox site is M2. In another embodiment, the Lox site is M3. In another embodiment, the Lox site is M7. In another embodiment, the Lox site is MH

[0118] The recombinase can be any recombinase useful in the methods of the present disclosure. In one embodiment, the recombinase is selected from the group consisting of a Bxbl recombinase, a Cre recombinase, a CinH recombinase, a Flp flippase, a HK022 integrase, a ParA recombinase, a Tnl721 recombinase, a Tn5053 recombinase, a TP901-l integrase, an U153 recombinase, a λ integrase, and a <|> C31 recombinase. It is understood herein that the enumerated recombinases encompass homologs and variants thereof. In one embodiment, the recombinase is a Bxbl recombinase. In another embodiment, the recombinase is a Cre recombinase. In another embodiment, the recombinase is a CinH recombinase. In another 36182281269.1UR 6-24056 / FR: 161118.06801 embodiment, the recombinase is a Flp flippase. In another embodiment, the recombinase is a HK022 integrase. In another embodiment, the recombinase is a ParA recombinase. In another embodiment, the recombinase is a Tnl721 recombinase. In another embodiment, the recombinase is a Tn5053 recombinase. In another embodiment, the recombinase is a TP901-1 integrase. In another embodiment, the recombinase is a U153 recombinase. In another embodiment, the recombinase is a integrase. In another embodiment, the recombinase is a (|)C31 recombinase.

[0119] In one embodiment, the first coding-sequence encoding the recombinase comprises an intron.

[0120] In one embodiment, the recombinase is a Cre recombinase or a FLP recombinase.[Sequence 2 / Construct 2]

[0121] In one example, the recombinant nucleic acid molecule comprises the sequence of Sequence 2 / Construct 2 shown in the example section below. Sequence 2 / Construct 2 and the elements therein are shown in the opposite orientation of the construct illustrated in Fig. 3. More specifically, the second expression cassette (which includes the elements of < G4G-Pfomofer>< LOXP>< B2M-Optimized>< T2A><tRFP>< LOX2272><gG7f POLY(A) SIGNAL>) is at the 5’ part of Sequence 2 / Construct 2 shown below, while in Fig. 3 the second expression cassette is shown at the 3’ part. By the same token, while the first expression cassette is shown at the 5’ part of the construct illustrated in Fig. 3, the reverse complement of the first expression cassette as well as elements therein (including: < HGH POLY(A) SIGNAL ><egfp>< P2A>< CD47>< LOXP>< SV4Q poly(a) signal>< Cre-C’TERMINAL>< GAPDH-INTRON>< Cre-N,TERMINAL>< LOX2272><fflvm introriXGFAP PROMOTERxmvm intronxGWAl PROMOTERS is at the 3’ part of Sequence 2 / Construct 2 shown below.

[0122] In this particular example, the first pair sites of recombinase-mediated recombination (corresponding to the light gray or dark gray triangles in Fig. 3) are a Lox2272 pair as shown below. The second pair sites of recombinase-mediated recombination (corresponding to the black triangles in Fig. 3) are a LoxP pair.Lox2272 pair:5 ' ATAACTTCGTATAAAGTATCCTATACGAAGTTAT3 ' (SEQ ID NO: 51 ) 5' ATAACTTCGTATAGGATACTTTATACGAAGTTAT3 ' (SEQ ID NO: 52 ) LoxP pair:5 ' ATAACTTCGTATAGCATACATTATACGAAGTTAT3 ' (SEQ ID NO: 53 ) 5' ATAACTTCGTATAATGTATGCTATACGAAGTTAT3 ' (SEQ ID NO: 54 )37182281269.1UR 6-24056 / FR: 161118.06801

[0123] In some embodiments, (A) the first member of the first pair is located between the second promoter and the first coding-sequence (e.g, between the GFAP promoter and the Cre-coding sequence as in Fig. 3); (B) the second member of the first pair is located between the PA3 and the third coding sequence (e.g., between the PA3 and the tRFP-coding sequence as in Fig. 3); (C) the first member of the second pair is located between the PAI and the second coding-sequence (e.g., between the PAI and the CD47 coding sequence as in Fig. 3); and / or (D) the second member of the second pair is located between the third coding sequence and the third promoter (e.g., between the GAG promoter and the B2M-coding sequence as in Fig. 3).

[0124] Within the scope of this disclosure is a polynucleotide derived from exposing the recombinant nucleic acid molecule or constructs described above to a recombinase. In one embodiment, this polynucleotide comprises from the 5’ end thereof to the 3’ end thereof: the third promoter; the third coding-sequence; the complement of the second promoter, and the complement of the first promoter. In one embodiment, the polynucleotide further comprise the PA2 and the complement of PA3, for example between the third coding-sequence and the complement of the second promoter. A non-limiting example of the polynucleotide is shown in Fig. 8.Reporter / marker proteins

[0125] In some embodiments, the recombinant nucleic acid molecule / genetic construct further encodes at least one reporter or marker protein. Non-limiting examples of such reporter / marker protein include fluorescent proteins, purification tags, and epitope tags.

[0126] In some examples, the reporter / marker protein may be a fluorescent protein. Non limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry. mRFPl, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein.38182281269.1UR 6-24056 / FR: 161118.06801

[0127] In one embodiment, the second coding-sequence further encodes for a first reporter protein. In one embodiment, the third coding-sequence further encodes for a second reporter protein.

[0128] In some embodiments, the recombinant nucleic acid molecule / genetic construct further comprises a selection marker. Suitable selection markers for mammalian cells are known in the art, and include for example, thymidine kinase, dihydrofolate reductase (together with methotrexate as a DHFR amplifier), aminoglycoside phosphotransferase, hygromycin B phosphotransferase, asparagine synthetase, adenosine deaminase, metallothionein, and antibiotic resistant genes, e.g., the puromycin resistance gene or the neomycin resistance gene.

[0129] In some examples, the selection marker protein may be a purification tag and / or an epitope tag. Exemplary tags include, but are not limited to, glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity’ purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, SI, T7, V5, VSV-G, 6xHis, biotin carboxyl carrier protein (BCCP), and calmodulin.Other Elements

[0130] The recombinant nucleic acid molecule described herein may include one or more additional elements, control sequences, or coding sequences.

[0131] The control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3 '-terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence, which is functional in the cell, may be used in the recombinant nucleic acid molecule described herein.

[0132] In one embodiment, the first expression cassette comprises a first poly adenylation signal (PAI) 3’ end to the first coding-sequence. In one embodiment, the first expression cassette comprises a second poly adenylation signal (PA2) 3’ end to the second coding-sequence. In one embodiment, the second expression cassette comprises a third poly adenylation signal (PA3) 3’ end to the third coding-sequence.Vectors

[0133] In one aspect, the present disclosure provides a vector comprising the nucleic acid molecule or polynucleotide described above. In some embodiments, the vector is a viral vector or a plasmid vector.39182281269.1UR 6-24056 / FR: 161118.06801

[0134] In some embodiments, the recombinant nucleic acid molecule or polynucleotide of the present disclosure is incorporated into a delivery vector. Suitable delivery vectors include, without limitation, plasmid vectors, viral vectors, including without limitation, vaccina vectors, lentiviral vector (integration competent or integration-defective lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, vectors for baculovirus expression, transposon based vectors or any other vector suitable for introduction of the nucleic acid molecule / recombinant genetic construct described herein into a cell by any means to facilitate the gene / cell selective expression of the recombinant construct.

[0135] In some embodiments, the vector further comprises one or more elements selected from the group consisting of a DNA nuclear targeting sequence and a barcoding sequence.

[0136] A nucleic acid molecule or polynucleotide or vector of the present disclosure can be introduced into any type of target or host cell. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g, mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be delivered or transferred into a host cell by physical, chemical, or biological means.

[0137] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0138] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U. S. Pat. Nos.5,350,674 and 5,585,362.

[0139] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome e.g., an artificial membrane vesicle).40182281269.1UR 6-24056 / FR: 161118.06801

[0140] As disclosed herein, the nucleic acid molecule or polynucleotide described above can be used for treating a disorder in a subject. In some embodiments, a polynucleotide encoding a therapeutic agent (RNA or protein) can be inserted into, or encoded by, vectors such as plasmids or viral vectors. Preferably, the polynucleotide is inserted into, or encoded by, viral vectors. A variety of viral-derived vectors can be used for transfection and integration into a mammalian cell genome. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses (AAV), herpes viruses, and lentiviruses. In some embodiments, the protein may be encoded by a retroviral vector, such as a lentiviral vector (See, e.g, U. S. Pat. Nos. 5,399,346; 5,124,263; 4,650,764 and 4,980,289; the content of each of which is incorporated herein by reference in its entirety). In some specific embodiments, the viral vectors are AAV vectors.Lentiviral vectors

[0141] Lentiviruses, such as HIV, are ‘‘slow viruses.” Vectors derived from lentiviruses can be expressed long-term in the host cells after a few administrations to the patients, i. e., via ex vivo transduced stem cells or progenitor cells. For most diseases and disorders, including genetic diseases, cancer, and neurological disease, long-term expression is crucial to successful treatment. Regarding safety with lentiviral vectors, a number of strategies for eliminating the ability' of lentiviral vectors to replicate have now been known in the art. See i.e., US 20210401868 and 20210403517, each of which is incorporated herein by reference in its entirety’. For example, the deletion of promoter and enhancer elements from the U3 region of the long terminal repeat (LTR) are thought to have no LTR-directed transcription. The resulting vectors are called “self-inactivating” (SIN).

[0142] Lentiviral vectors are particularly suitable to achieving long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as CNS cells. They also have the added advantage of low immunogenicity. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (i.e., WOOl / 96584 and W001 / 29058; and U. S. Pat. No. 6,326,193). Several vector promoter sequences are available for expression of the transgenes. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any 41182281269.1UR 6-24056 / FR: 161118.06801 polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is EFla. However, other constitutive promoter sequences can also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as. but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Inducible promoters include, but are not limited to a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0143] The present disclosure provides a recombinant lentivirus capable of infecting dividing and non-dividing cells, such oligodendrocytes or oligodendrocyte progenitor cells. The virus is useful for the in vivo and ex vivo transfer and expression of nucleic acid sequences. Lentiviral vectors of the present disclosure may be lentiviral transfer plasmids or infectious lentiviral particles. Construction of lentiviral vectors, helper constructs, envelope constructs, etc., for use in lentiviral transfer systems has been described in, e.g., US 20210401868 and 20210403517, each of which is incorporated herein by reference in its entirety.Adenoviruses

[0144] Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid molecule to a variety of cell types m vivo, and have been used extensively in gene therapy protocols, including for targeting genes to neural cells and glial cells. Various replication defective adenovirus and minimum adenovirus vectors have been described for nucleic acid therapeutics (See, e.g., PCT Patent Publication Nos. WO199426914, WO 199502697, WO199428152. WO199412649, WO199502697 and WO199622378; the content of each of which is incorporated by reference in their entirety). Such adenoviral vectors may also be used to deliver therapeutic molecules of the present disclosure to cells.Adeno-Associated Virus

[0145] The adeno-associated virus is a widely used gene therapy vector due to its clinical safety record, non-pathogenic nature, ability to infect non-dividing cells (like neurons), and ability to provide long-term gene expression after a single administration. Currently, many human and non-human primate AAV serotypes have been identified. AAV vectors have demonstrated safety in hundreds of clinical trials worldwide, and clinical efficacy has been42182281269.1UR 6-24056 / FR: 161118.06801 shown in trials of hemophilia B, spinal muscular atrophy, alpha 1 antitrypsin, and Leber congenital amaurosis.

[0146] Because of their safety, nonpathogenic nature, and ability to infect many cells including neurons, AAVs such as AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, and AAV9 are commonly used gene therapy vectors for CNS applications. However, after direct CNS infusion, these serotypes exhibit a dominant neuronal tropism and expression in oligodendrocytes is low, especially when gene expression is driven by a constitutive promoter, which restricts their potential for use in treating white matter diseases. AAV1 / 2, AAV2, and AAV8 have been shown transduce oligodendrocytes. Reliance on cell-specific promoters for expression specificity allows for the possibility of nonselective cellular uptake and leaky transgene expression through cryptic promoter activity in non-oligodendrocyte lineage cells.

[0147] The approach described herein to alleviate these issues includes using AAV serotypes with high tropism for oligodendrocytes or glial progenitor cells, such as oligodendrocyte progenitor cells. Recently, using DNA shuffling and directed evolution, a chimeric AAV capsid with strong selectivity for oligodendrocytes, AAV / OligOOl, has been described (Powell et al., 2016, Gene Ther 23:807-814). Subsequently, AAV / OligOOl was shown to transduce neonatal oligodendrocytes in a mouse model of Canavan disease (Francis et al., 2021. Mol Ther Methods Clin Dev 20:520-534). Other approaches such as random mutagenesis and peptide library insertion can be used to generate capsid libraries that can be screened for tropism and selectivity for oligodendrocytes or glial progenitor cells.

[0148] As discussed above, the terms “adeno-associated virus” and / orC'AAV” refer to parvoviruses with a linear single-stranded DNA genome and variants thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. Parvoviruses, including AAV, are useful as gene therapy vectors as they can penetrate a cell and introduce a nucleic acid (e.g., transgene) into the nucleus. In some embodiments, the introduced nucleic acid (e.g., rAAV vector genome) forms circular concatemers that persist as episomes in the nucleus of transduced cells. In some embodiments, a transgene is inserted in specific sites in the host cell genome. Site-specific integration, as opposed to random integration, is believed to likely result in a predictable long-term expression profile. The insertion site of AAV into the human genome is referred to as AAV S 1. Once introduced into a cell, RNAs or polypeptides encoded by the nucleic acid can be expressed by the cell. Because AAV is not associated with any pathogenic disease in humans, a nucleic acid43182281269.1UR 6-24056 / FR: 161118.06801 delivered by AAV can be used to express a therapeutic RNA or polypeptide for the treatment of a disease, disorder and / or condition in a human subject.

[0149] Multiple serotypes of AAV exist in nature with at least fifteen wild type serotypes having been identified from humans thus far (i.e., AAV 1 -AAV 15). Naturally occurring and variant serotypes are distinguished by having a protein capsid that is serologically distinct from other AAV serotypes. Examples include AAV1, AAV2, AAV, AAV3 (including AAV3A and AAV3B), AAV4. AAV5, AAV6, AAV7. AAV8, AAV9. AAV10, AAV12, AAVrhlO, AAVrh74 (see WO 2016 / 210170), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, and recombinantly produced variants (e.g., capsid variants with insertions, deletions and substitutions, etc.), such as variants referred to as AAV2i8, NP4, NP22. NP66, DJ, DJ / 8, DJ / 9, LK.3, RHM4-1, among many others. “Primate AAV’’ refers to AAV that infect primates, “non-primate AAV” refers to AAV that infect non-primate mammals, “bovine AAV” refers to AAV that infect bovine mammals, and so on.

[0150] Serotype distinctiveness is determined on the basis of the lack of crossreactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences and antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). However, some naturally occurring AAV or manufactured AAV mutants (e.g., recombinant AAV) may not exhibit serological difference with any of the currently known serotypes. These viruses may then be considered a subgroup of the corresponding type, or more simply a variant AAV. Thus, as used herein, the term “serotype” refers to both serologically distinct viruses, as well as viruses that are not serologically distinct but that may be within a subgroup or a variant of a given serotype.

[0151] A comprehensive list and alignment of amino acid sequences of capsids of known AAV serotypes is provided by Marsic et al. (2014) Molecular Therapy 22(11): 1900-1909. Genomic sequences of various seroty pes of AAV, as w ell as sequences of the native ITRs, rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), NC_001863 (AAV3B), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), NC_001862 (AAV6), AF513851 (AAV7), AF513852 (AAV8), and NC 006261 (AAV8); the disclosures of which are incorporated by reference herein. See also,44182281269.1UR 6-24056 / FR: 161118.06801 e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology' 33:375-383; international patent publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; WO 2013 / 063379; WO 2014 / 194132; WO 2015 / 121501, and U. S. Patent No. 6,156,303 and U. S. Patent No. 7,906,111.

[0152] As discussed herein, a ‘'recombinant adeno-associated virus” or '‘rAAV” is distinguished from a wild-type AAV by replacement of all or part of the endogenous viral genome with a non-native sequence. Incorporation of a non-native sequence within the virus defines the viral vector as a “recombinant” vector, and hence a “rAAV vector.” An rAAV vector can include a heterologous polynucleotide encoding a desired RNA or protein or polypeptide (e.g, an RNA molecule disclosed herein). A recombinant vector sequence may be encapsidated or packaged into an AAV capsid and referred to as an “rAAV vector,” an “rAAV vector particle.” “rAAV viral particle” or simply a “rAAV.”

[0153] The present disclosure provides for an rAAV vector comprising a polynucleotide sequence not of AAV origin (e.g., a polynucleotide heterologous to AAV). The heterologous polynucleotide may be flanked by at least one, and sometimes by two, AAV terminal repeat sequences (e.g, inverted terminal repeats). The heterologous polynucleotide flanked by ITRs. also referred to herein as a “vector genome.” typically encodes an RNA or a polypeptide of interest, or a gene of interest, such as a target for therapeutic treatment. Delivery or administration of an rAAV vector to a subject (e.g. a patient) provides encoded RNAs / proteins / peptides to the subject. Thus, an rAAV vector can be used to transfer / deliver a heterologous polynucleotide for expression for, e.g, treating a variety of diseases, disorders and conditions.

[0154] rAAV vector genomes generally retain 145 base ITRs in cis to the heterologous nucleic acid sequence that replaced the viral rep and cap genes. Such ITRs are useful to produce a recombinant AAV vector; however, modified AAV ITRs and non-AAV terminal repeats including partially or completely synthetic sequences can also sen e this purpose. ITRs form hairpin structures and function to, for example, serve as primers for host-cell-mediated synthesis of the complementary DNA strand after infection. ITRs also play a role in viral packaging, integration, etc. ITRs are the only AAV viral elements which are required in cis for AAV genome replication and packaging into rAAV vectors. An rAAV vector genome 45182281269.1UR 6-24056 / FR: 161118.06801 optionally comprises two ITRs which are generally at the 5’ and 3’ ends of the vector genome comprising a heterologous sequence (e.g., a transgene encoding a gene of interest, or a nucleic acid sequence of interest including, but not limited to, an antisense, and siRNA, a CRISPR molecule, among many others). A 5’ and a 3’ ITR may both comprise the same sequence, or each may comprise a different sequence. An AAV ITR may be from any AAV including by not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or any other AAV.

[0155] An rAAV vector of the disclosure may comprise an ITR from an AAV serotype (e.g., wild-type AAV2, a fragment or variant thereof) that differs from the serotype of the capsid (e.g., AAV8, OligOOl). Such an rAAV vector comprising at least one ITR from one serotype, but comprising a capsid from a different serotype, may be referred to as a hybrid viral vector (see U. S. Patent No. 7.172,893). An AAV ITR may include the entire wild type ITR sequence, or be a variant, fragment, or modification thereof, but will retain functionality.

[0156] In some embodiments, an rAAV vector genome is linear, single-stranded and flanked by AAV ITRs. Prior to transcription and translation of the heterologous gene, a single stranded DNA genome of approximately 4700 nucleotides must be converted to a doublestranded form by DNA polymerases (e.g, DNA polymerases within the transduced cell) using the free 3’-OH of one of the self-priming ITRs to initiate second-strand synthesis. In some embodiments, full length-single stranded vector genomes (i.e., sense and anti-sense) anneal to generate a full length-double stranded vector genome. This may occur when multiple rAAV vectors carrying genomes of opposite polarity (i.e., sense or anti-sense) simultaneously transduce the same cell. Regardless of how they are produced, once double-stranded vector genomes are formed, the cell can transcribe and translate the double-stranded DNA and express the heterologous gene.

[0157] The efficiency of transgene expression from an rAAV vector can be hindered by the need to convert a single stranded rAAV genome (ssAAV) into double-stranded DNA prior to expression. This step can be circumvented by using a self-complementary AAV genome (scAAV) that can package an inverted repeat genome that can fold into doublestranded DNA without the need for DNA synthesis or base-pairing between multiple vector genomes. See, e.g., U. S. Patent No. 8,784,799; McCarty, (2008) Molec. Therapy 16(10):1648-1656; and McCarty et al., (2001) Gene Therapy 8:1248-1254; McCarty et al., (2003) Gene Therapy 10:2112-2118.

[0158] A viral capsid of an rAAV vector may be from a wild type AAV or a variant AAV such as AAV1. AAV2, AAV3, AAV3A. AAV3B, AAV4, AAV5. AAV6, AAV7,46182281269.1UR 6-24056 / FR: 161118.06801 AAV8, AAV9, AAV10, AAVrhlO, AAVrh74 (see W02016 / 210170), AAV12, AAV2i8, AAV1.1. AAV2.5, AAV6.1. AAV6.3.1, AAV9.45, RHM4-1 (SEQ ID NO:5 of WO 2015 / 013313), RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, RHM15-6, AAV hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9,45, AAV218, AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAV avian AAV, bovine AAV, canine AAV. equine AAV, primate AAV. non-primate AAV, snake AAV, goat AAV, shrimp AAV, ovine AAV and variants thereof (see, e.g., Fields etal., VIROLOGY, volume 2, chapter 69 (4thed., Lippincott-Raven Publishers). Capsids may be derived from a number of AAV serotypes disclosed in U. S. Patent No. 7,906,111; Gao et al. (2004) J. Virol. 78:6381; Morris et al. (2004) Virol. 33:375; WO 2013 / 063379; WO 2014 / 194132; and include true type AAV (AAV-TT) variants disclosed in WO 2015 / 121501, and RHM4-1. RHM15-1 through RHM15-6, and variants thereof, disclosed in WO 2015 / 013313. A full complement of AAV cap proteins includes VP1, VP2, and VP3. The ORF comprising nucleotide sequences encoding AAV VP capsid proteins may comprise less than a full complement AAV Cap proteins or the full complement of AAV cap proteins may be provided.

[0159] In some embodiments, an rAAV vector comprising a capsid protein encoded by a nucleotide sequence derived from more than one AAV serotype (e.g., wild type AAV serotypes, variant AAV serotypes) is referred to as a “chimeric vector” or “chimeric capsid” (See U. S. Patent No. 6,491,907, the entire disclosure of which is incorporated herein by reference). In some embodiments, a chimeric capsid protein is encoded by a nucleic acid sequence derived from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more AAV serotypes. In some embodiments, a recombinant AAV vector includes a capsid sequence derived from e.g., AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, AAVrhlO, AAV2i8, or variant thereof, resulting in a chimeric capsid protein comprising a combination of amino acids from any of the foregoing AAV serotypes (see, Rabinowitz et al. (2002) J. Virology 76(2):791-801). Alternatively, a chimeric capsid can comprise a mixture of a VP1 from one serotype, a VP2 from a different serotype, a VP3 from yet a different serotype, and a combination thereof. For example, a chimeric virus capsid may include an AAV1 cap protein or subunit and at least one AAV2 cap protein or subunit. A chimeric capsid can, for example, include an AAV capsid with one or more B19 cap subunits, e.g., an AAV cap protein or subunit can be replaced by a B19 cap protein or subunit. For example, in one embodiment, a VP3 subunit of an AAV capsid can be replaced by a VP247182281269.1UR 6-24056 / FR: 161118.06801 subunit of Bl 9. In some embodiments, a chimeric capsid is an OligOOl capsid as described in WO2021221995 and WO2014052789, which are incorporated herein by reference.

[0160] In some embodiments, chimeric vectors have been engineered to exhibit altered tropism or tropism for a particular tissue or cell type. The term “tropism” refers to preferential entry of the virus into certain cells (e.g., oligodendrocytes) or tissue types and / or preferential interaction with the cell surface that facilitates entry into certain cell or tissue types. AAV tropism is generally determined by the specific interaction between distinct viral capsid proteins and their cognate cellular receptors (Lykken etal. (2018) J. Neurodev. Disord. 10: 16). Preferably, once a virus or viral vector has entered a cell, sequences (e.g., heterologous sequences such as a transgene) carried by the vector genome (e.g, an rAAV vector genome) are expressed.

[0161] A “tropism profile” refers to a pattern of transduction of one or more target cells in various tissues and / or organs. For example, a chimeric AAV capsid may have a tropism profile characterized by efficient transduction of oligodendrocytes or oligodendrocyte progenitor cells with only low transduction of neurons, astrocytes and other CNS cells. See WO2014 / 052789, incorporated herein by reference. Such a chimeric capsid may be considered “specific for oligodendrocytes or oligodendrocyte progenitor cells” exhibiting tropism for oligodendrocytes or oligodendrocyte progenitor cells, and referred to herein as “oligotropism,” if when administered directly into the CNS, preferentially transduces oligodendrocytes or oligodendrocyte progenitor cells over neurons, astrocytes and other CNS cell types. In some embodiments, at least about 80% of cells that are transduced by a capsid specific for oligodendrocytes or oligodendrocyte progenitor cells are oligodendrocytes or oligodendrocyte progenitor cells, e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the transduced cells are oligodendrocytes or oligodendrocyte progenitor cells.

[0162] In some embodiments, an rAAV vector is useful for treating or preventing a “disorder associated with oligodendrocyte dysfunction.” As used herein, the term “associated with oligodendrocyte dysfunction” refers to a disease, disorder or condition in which oligodendrocytes are damaged, lost or function improperly compared to otherwise identical normal oligodendrocytes. The term includes diseases, disorders and conditions in which oligodendrocytes are directly affected as well as diseases, disorders or conditions in which oligodendrocytes become dysfunctional secondary to damage to other cells. In some embodiments, a disorder associated with oligodendrocyte dysfunction is demyelination.48182281269.1UR 6-24056 / FR: 161118.06801 Cells

[0163] As described in more detail herein, the recombinant nucleic acid molecules / constructs or vector described herein can be introduced into the genome of host cells of interest to effectuate the expression of the one or more immune checkpoint proteins and / or one or more agents (peptides or RNAs) of interest. In some embodiments, the one or more agents that confer immunogenicity to cells containing the recombinant nucleic acid molecules / recombinant genetic constructs in the absence of Cre activation.

[0164] The term “host cell” means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide encoding a protein. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0165] Accordingly, the present disclosure provides a cell or a progeny thereof. The cell comprises the recombinant nucleic acid molecule / construct described above, the polynucleotide described above, or the vector described above. In some embodiments, the cell is a pluripotent stem cell. In one embodiment, the cell is an embryonic stem cell. In one embodiment, the cell is an induced pluripotent stem cell. In one embodiment, the cell is a human cell.

[0166] Another aspect of the disclosure relates to a preparation of the cells or progenies described above. The preparation may be a preparation of cells from any organism. In some embodiments, the preparation is a preparation of mammalian cells, e.g., a preparation of rodent cells (i.e., mouse or rat cells), rabbit cells, guinea pig cells, feline cells, canine cells, porcine cells, equine cells, bovine cell, ovine cells, monkey cells, or human cells. In one embodiment, the preparation is a preparation of human cells. Suitable cells as described herein include primary or immortalized embry onic cells, fetal cells, or adult cells, at any stage of their lineage, e.g., totipotent, pluripotent, multipotent, or differentiated cells.

[0167] In some embodiments, the preparation is a preparation of pluripotent stem cells. Pluripotent stem cells can give rise to any cell of the three germ layers (i.e., endoderm, mesoderm and ectoderm). In one embodiment, the preparation of cells is a preparation of induced pluripotent stem cells (iPSCs). In another embodiment, the preparation of cells comprising the recombinant genetic construct is a preparation of pluripotent embryonic stem cells.49182281269.1UR 6-24056 / FR: 161118.06801

[0168] In another embodiment, the preparation of one or more cells may be a preparation of multipotent stem cells. Multipotent stem cells can develop into a limited number of cells in a particular lineage. Examples of multipotent stem cells include progenitor cells, e.g., neural progenitor cells which give rise to cells of the central nervous system such as neurons, astrocytes and oligodendrocytes. Progenitor cells are an immature or undifferentiated cell population having the potential to mature and differentiate into a more specialized, differentiated cell type. A progenitor cell can also proliferate to make more progenitor cells that are similarly immature or undifferentiated. Suitable preparations of progenitor cells include, without limitation, preparations of neural progenitor cells, neuronal progenitor cells, glial progenitor cells, oligodendrocyte-biased progenitor cells, and astrocyte-biased progenitor cells. Other suitable progenitor cell populations include, without limitation, bone marrow progenitor cells, cardiac progenitor cells, endothelial progenitor cells, epithelial progenitor cells, hematopoietic progenitor cells, hepatic progenitor cells, osteoprogenitor cells, muscle progenitor cells, pancreatic progenitor cells, pulmonary progenitor cells, renal progenitor cells, vascular progenitor cells, retinal progenitor cells.

[0169] The preparation of cells comprising the recombinant nucleic acid molecule or a polynucleotide derived from exposing the recombinant nucleic acid molecule to a recombinase as described herein can also be a preparation of terminally differentiated cells. In one embodiment, the preparation of one or more cells may be a preparation of terminally differentiated neurons, oligodendrocytes, or astrocytes. In another embodiment, the preparation of one or more cells is a preparation of adipocytes, chondrocytes, endothelial cells, epithelial cells (keratinocytes, melanocytes), bone cells (osteoblasts, osteoclasts), liver cells (cholangiocytes, hepatocytes), muscle cells (cardiomyocytes, skeletal muscle cells, smooth muscle cells), retinal cells (ganglion cells, muller cells, photoreceptor cells), retinal pigment epithelial cells, renal cells (podocytes, proximal tubule cells, collecting duct cells, distal tubule cells), adrenal cells (cortical adrenal cells, medullary adrenal cells), pancreatic or pancreatic islet cells (alpha cells, beta cells, delta cells, epsilon cells, pancreatic polypeptide producing cells, exocrine cells); lung cells, bone marrow cells (early B-cell development, early T-cell development, macrophages, monocytes), urothelial cells, fibroblasts, parathyroid cells, thyroid cells, hypothalamic cells, pituitary cells, salivary gland cells, ovarian cells, and testicular cells.

[0170] Additional exemplary cell types comprising the recombinant nucleic acid molecule described herein or progenies include, without limitation, placental cells, keratinocytes, basal epidermal cells, urinary epithelial cells, salivary gland cells, mucous cells,50182281269.1UR 6-24056 / FR: 161118.06801 serous cells, von Ebner's gland cells, mammary gland cells, lacrimal gland cells, eccrine sweat gland cells, apocrine sweat gland cells. MpH gland cells, sebaceous gland cells. Bowman's gland cells, Brunner's gland cells, seminal vesicle cells, prostate gland cells, bulbourethral gland cells, Bartholin's gland cells, Littre gland cells, uterine endometrial cells, goblet cells of the respiratory or digestive tracts, mucous cells of the stomach, zymogenic cells of the gastric gland, oxyntic cells of the gastric gland, insulin-producing P cells, glucagon-producing α cells, somatostatin-producing δ cells, pancreatic polypeptide-producing cells, pancreatic ductal cells, Paneth cells of the small intestine, type II pneumocytes of the lung, Clara cells of the lung, anterior pituitary cells, intermediate pituitary cells, posterior pituitary cells, hormone secreting cells of the gut or respiratory tract, gonad cells, juxtaglomerular cells of the kidney, macula densa cells of the kidney, peri polar cells of the kidney, mesangial cells of the kidney, brush border cells of the intestine, striated ducted cells of exocrine glands, gall bladder epithelial cells, brush border cells of the proximal tubule of the kidney, distal tubule cells of the kidney, conciliated cells of the ductulus efferens, epididymal principal cells, epididymal basal cells, hepatocytes, fat cells, type I pneumocytes, pancreatic duct cells, nonstriated duct cells of the sweat gland, nonstriated duct cells of the salivary gland, nonstriated duct cells of the mammary gland, parietal cells of the kidney glomerulus, podocytes of the kidney glomerulus, cells of the thin segment of the loop of Henle, collecting duct cells, duct cells of the seminal vesicle, duct cells of the prostate gland, vascular endothelial cells, synovial cells, serosal cells, squamous cells lining the perilymphatic space of the ear. cells lining the endolymphatic space of the ear, choroid plexus cells, squamous cells of the pia-arachnoid, ciliary epithelial cells of the eye, comeal endothelial cells, ciliated cells having propulsive function, ameloblasts, planum semilunatum cells of the vestibular apparatus of the ear, interdental cells of the organ of Corti, fibroblasts, pericytes of blood capillaries, nucleus pulposus cells of the intervertebral disc, cementoblasts, cementocytes, odontoblasts, odontocytes, chondrocytes, osteocytes, osteoprogenitor cells, hyalocytes of the vitreous body of the eye, stellate cells of the perilymphatic space of the ear, skeletal muscle cells, heart muscle cells, smooth muscle cells, myoepithelial cells, platelets, megakaryocytes, monocytes, connective tissue macrophages, Langerhan's cells, osteoclasts, dendritic cells, microglial cells, neutrophils, eosinophils, basophils, mast cells, plasma cells, helper T cells, suppressor T cells, killer T cells, killer cells, rod cells, cone cells, inner hair cells of the organ of Corti, outer hair cells of the organ of Corti, type I hair cells, cells of the vestibular apparatus of the ear, type II cells of the vestibular apparatus of the ear, type II taste bud cells, olfactory neurons, basal cells of olfactory 51182281269.1UR 6-24056 / FR: 161118.06801 epithelium, type I carotid body cells, type II carotid body cells, Merkel cells, primary sensory neurons, cholinergic neurons of the autonomic nervous system, adrenergic neurons of the autonomic nervous system, peptidergic neurons of the autonomic nervous system, inner pillar cells of the organ of Corti, outer pillar cells of the organ of Corti, inner phalangeal cells of the organ of Corti, outer phalangeal cells of the organ of Corti, border cells, Hensen cells, supporting cells of the vestibular apparatus, supporting cells of the taste bud, supporting cells of the olfactory epithelium, Schwann cells, satellite cells, enteric glial cells, neurons of the central nervous system, astrocytes of the central nervous system, oligodendrocytes of the central nervous system, anterior lens epithelial cells, lens fiber cells, melanocytes, retinal pigmented epithelial cells, iris pigment epithelial cells, oogonium, oocytes, spermatocytes, spermatogonium, ovarian cells, Sertoli cells, and thymus epithelial cells.

[0171] In accordance with this aspect of the disclosure, the recombinant nucleic acid molecule is integrated into the chromosome of the one or more cells in the preparation. The term “integrated,” when used in the context of the recombinant nucleic acid molecule of the present disclosure means that the recombinant nucleic acid molecule is inserted into the genome or the genomic sequence of the one or more cells in the preparation. When integrated, the integrated recombinant nucleic acid molecule is replicated and passed along to daughter cells of a dividing cell in the same manner as the original genome of the cell. In accordance with the design of the recombinant nucleic acid molecule, the genomic integration of the molecule is targeted to a desired gene of interest to achieve the cell selective expression of the one or more immune checkpoint proteins.Methods and uses for modifying cells

[0172] The present disclosure further provides a method of conditional, stage- and phenotype-specific expression of a transgene in a differentiated cell derived from a pluripotent stem cell. The method comprises (a) obtaining a pluripotent stem cell; (b) introducing into the pluripotent stem cell the recombinant nucleic acid molecule / construct described above or the vector described above; and (c) culturing the pluripotent stem cell containing the recombinant nucleic acid molecule / construct or vector under conditions suitable for (i) differentiation of the pluripotent stem cell and (ii) expression of the proteins encoded by the recombinant nucleic acid molecule / construct under the control of the first promoter or the second promoter thereby to obtain one or more differentiated cells derived from the pluripotent stem cell.

[0173] Also provided is a method of conditionally rendering or obtaining one or more hypo-immune and / or non-immunogenic cells. The method comprises (a) obtaining a starting 52182281269.1UR 6-24056 / FR: 161118.06801 cell; (b) introducing into the starting cell the recombinant nucleic acid molecule / construct described above or a vector comprising the recombinant nucleic acid molecule / construct, wherein the second coding-sequence encodes an immune checkpoint protein; (c) culturing the starting cell containing the nucleic acid molecule or vector under conditions permitting expression of the proteins encoded by the nucleic acid molecule under the control of the first promoter or the second promoter thereby to obtain one or more hypo-immune and / or non-immunogenic cells.

[0174] In some embodiments, the starting cell lacks a functional β2-microglobulin protein and / or a functional CIITA protein.

[0175] Cells that can be modified in accordance with this aspect of the disclosure include cells from any organism. In some embodiments, the preparation is a preparation of mammalian cells, e.g., a preparation of rodent cells (z. e., mouse or rat cells), rabbit cells, guinea pig cells, feline cells, canine cells, porcine cells, equine cells, bovine cell, ovine cells, monkey cells, or human cells. Suitable cells include primary or immortalized embryonic cells, fetal cells, or adult cells, at any stage of their lineage, e.g., totipotent, pluripotent, multipotent, or differentiated cells.

[0176] In some embodiments, modifying the cells of interest involves introducing into the cell a sequence-specific nuclease that cleaves a target locus. Once the target gene is cleaved by a sequence-specific nuclease, the method further involves introducing into the target locus, for example, by way of homologous recombination, any of the recombinant nucleic acid molecule described herein.

[0177] Suitable sequence specific nucleases for cleaving the target gene to introduce the recombinant genetic construct include, without limitation, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and an RNA-guided nucleases. In some embodiments, the sequence-specific nuclease is introduced into the cell as a protein, mRNA, or cDNA.

[0178] Zinc finger nucleases are a class of engineered DNA binding proteins that facilitate targeted editing of DNA by introducing double strand DNA breaks in a sequence specific manner. Each ZFN comprises two functional domains, i.e., a DNA-binding domain comprised of a chain of two-finger modules, each recognizing a unique hexamer sequence of DNA, and a DNA-cleaving domain comprised of the nuclease domain of Fok I. ZFNs suitable for targeted cleavage of the target genes described herein to facilitate insertion of the recombinant genetic construct are known in the art. see e.g, U. S. Patent No. 8.106,255 to 53182281269.1UR 6-24056 / FR: 161118.06801 Carroll et al., U. S. Patent No. 9,428,756 to Cai et al., U. S. Patent Publication No. 20110281306 to Soo and Joo; U. S. Patent Publication No. 20050130304 to Cox et al., which are hereby incorporated by reference in their entirety.

[0179] In another embodiment transcription activator-like effector nuclease (TALEN)-mediated DNA editing is utilized to introduce the recombinant nucleic acid molecule described herein into a target locus of interest. A functional TALEN consists of a DNA binding domain, which is derived from transcription activator-like effector (TALE) proteins, and a nuclease catalytic domain from a DNA nuclease, Fokl. The DNA binding domain of TALE features an array of 33-34 amino acid repeats. Each repeat is conserved, with the exception of the repeat variable di-residues (RVDs) at amino acid positions 12 and 13, which determine which nucleotide of the targeted DNA sequence each repeat recognizes. Methods of customizing TALE proteins to bind to a target site using canonical or non-canonical RVDs within the repeat units are known in the art and suitable for use in accordance with the present disclosure (see, e.g., U. S. Pat. Nos. 8,586,526 to Philip et al. and 9,458,205 to Philip et al., which are hereby incorporated by reference in their entirety). Likewise, methods of using TALEN for gene editing that are suitable for use in accordance with the present disclosure are also known in the art, see e.g., U. S. Patent No. 9,393,257 to Osbom et al., which is hereby incorporated by reference in its entirety.

[0180] In another embodiment, the sequence specific nuclease used to introduce the recombinant genetic construct described herein into a target gene of interest is an RNA-guided nuclease in the form of Cas9. Cas9 is a CRISPR-associated protein containing two nuclease domains, that, when complexed with CRISPR RNA (cRNA) and trans-activating rRNA, can achieve site-specific DNA recognition and double strand cleavage. CRISPR-Cas9 systems and methods for nucleic acid editing that are suitable for use in accordance with the present disclosure are well known in the art, see, e.g., Jinek, M., et al. “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity,” Science 337:816-821 (2012); Doench et al., “Rational Design of Highly Active sgRNAs for CRISPR-mediated Gene Inactivation,” Nature Biotechnol. 32(12): 1262-7 (2014) U. S. Patent No. 9,970,001 to Miller; U. S. Patent Publication No. 20180282762 to Gori et al., and U. S. Patent Publication No.20160201089 to Gersbach et al., which are hereby incorporated by reference in their entirety.Compositions

[0181] In a further aspect, the present disclosure provides a composition comprising (i) the recombinant nucleic acid molecule / construct described above, or the polynucleotide 54182281269.1UR 6-24056 / FR: 161118.06801 described above, or the vector described above, or the cell described above, and (ii) a pharmaceutically acceptable carrier. In some embodiments, the carrier comprises a liposome, or a nanoparticle or an exosome.

[0182] In one embodiment of the present application, the delivery vehicle is a nanoparticle. A variety' of nanoparticle delivery vehicles are known in the art and are suitable for delivery’ of the genetic constructs of the present application (see, e.g., van Vlerken et al., ■'Multi-functional Polymeric Nanoparticles for Tumour-Targeted Drug Delivery / ’ Expert Opin. Drug Deliv. 3(2):205-216 (2006), which is hereby incorporated by reference in its entirety). Suitable nanoparticles include, without limitation, poly(beta-amino esters) (Sawicki et al., “Nanoparticle Delivery of Suicide DNA for Epithelial Ovarian Cancer Cell Therapy,” Adv. Exp. Med. Biol. 622:209-219 (2008), which is hereby incorporated by reference in its entirety), polyethylenimine-alt-poly(ethylene glycol) copolymers (Park et al., “Degradable Polyethylenimine-alt-Poly(ethylene glycol) Copolymers As Novel Gene Carriers,” J. Control Release 105(3): 367-80 (2005) and Park et al., “Intratumoral Administration of Anti-KITENIN shRNA-Loaded PEI-alt-PEG Nanoparticles Suppressed Colon Carcinoma Established Subcutaneously in Mice,” J Nanosci. Nanotechnology 10(5):3280-3 (2010), which are hereby incorporated by reference in their entirety), poly(d,l-lactide-coglycolide) (Chan et al., “Antisense Oligonucleotides: From Design to Therapeutic Application,” Clin. Exp. Pharm. Physiol. 33: 533-540 (2006), which is hereby incorporated by reference in its entirety), and liposome- entrapped siRNA nanoparticles (Kenny et al., “Novel Multifunctional Nanoparticle Mediates siRNA Tumor Delivery, Visualization and Therapeutic Tumor Reduction In vivo,” J. Control Release 149(2): 111-116 (2011), which is hereby incorporated by reference in its entirety). Other nanoparticle vehicles suitable for use in the present application include microcapsule nanotube devices disclosed in U. S. Patent Publication No. 2010 / 0215724 to Prakash et al., which is hereby incorporated by reference in its entirety.

[0183] In another embodiment, the recombinant nucleic acid or genetic construct is contained in a liposome delivery vehicle. The term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.55182281269.1UR 6-24056 / FR: 161118.06801

[0184] Several advantages of liposomes include: their biocompatibility and biodegradability, incorporation of a wide range of water and lipid soluble drugs; and they afford protection to encapsulated molecules from metabolism and degradation. Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0185] Liposomes are useful for the transfer and delivery’ of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

[0186] Methods for preparing liposomes include those disclosed in Bangham et al., “Diffusion of Univalent Ions Across the Lamellae of Swollen Phospholipids,” J. Mol. Biol.13:238-52 (1965); U. S. Patent No. 5,653,996 to Hsu; U. S. Patent No. 5,643,599 to Lee et al.,- U. S. PatentNo. 5,885,613 to Holland et al.,- U. S. Patent No. 5,631,237 to Dzaueta / .; and U. S. Patent No. 5.059,421 to Loughrey et al., which are hereby incorporated by reference in their entirety.

[0187] As disclosed herein, in another embodiment, the genetic construct, expression cassette, or expression vector can be administered in association with a glial progenitor cell-targeted fusogen or a glial progenitor cell-selective surface-binding moiety. For example, the genetic construct, expression cassette, or expression vector can be in or associated with a fusosome.

[0188] As used herein, "fusogen" refers to an agent or molecule that creates an interaction between two membrane enclosed lumens. In embodiments, the fusogen facilitates fusion of the membranes. In other embodiments, the fusogen creates a connection, e.g.. a pore, between two lumens (e.g., a lumen of a liposome and a cytoplasm of a target cell, or a lumen of a viral vector and a cytoplasm of a target cell). In some embodiments, the fusogen comprises a protein or a complex of two or more proteins having a targeting domain or binding moiety'. In some examples, the targeting domain or binding moiety’ specifically targets or binds to a molecule on glial progenitor cell or a glial progenitor cell. Examples of the molecule include, but not limited to, CD140a, NG2 / CSPG4, A2B5 gangliosides, 04 sulfatides, or CD133. A targeting domain or binding moiety’ can be a receptor ligand, a peptide / polypeptide, an antibody, or an antigen-binding portion thereof that specifically binds to a molecule or marker on a glial progenitor cell or a glial progenitor cell. Non-limiting examples of human and non- 56182281269.1UR 6-24056 / FR: 161118.06801 human fusogens are described in, e.g., US 20210198698 and US 20210137839, which are incorporated by reference in their entireties.

[0189] As used herein, "fusosome" refers to a bilayer of amphipathic lipids enclosing a lumen or cavity and a fusogen that interacts with the amphipathic lipid bilayer. In some embodiments, the fusosome comprises a nucleic acid. In some embodiments, the fusosome is a membrane enclosed preparation. In some embodiments, the fusosome is derived from a source cell.

[0190] Fusosomes can take various forms. For example, in some embodiments, a fusosome described herein is derived from a source cell. A fusosome may be or comprise, e.g., an extracellular vesicle, a microvesicle, a nanovesicle, an exosome, a microparticle, or any combination thereof. In some embodiments, a fusosome is released naturally from a source cell, and in some embodiments, the source cell is treated to enhance formation of fusosomes. In some embodiments, the fusosome is between about 10-10,000 nm in diameter, e.g., about 30-100 nm in diameter. In some embodiments, the fusosome comprises one or more synthetic lipids.

[0191] In some embodiments, the fusosome is or comprises a virus, e.g.. a retrovirus, e.g., a lentivirus. For instance, in some embodiments, the fusosome's bilayer of amphipathic lipids is or comprises the viral envelope. The viral envelope may comprise a fusogen, e.g., a fusogen that is endogenous to the virus or a pseudotyped fusogen. In some embodiments, the fusosome's lumen or cavity comprises a viral nucleic acid, e.g., a retroviral nucleic acid, e.g., a lentiviral nucleic acid. The viral nucleic acid may be a viral genome. In some embodiments, the fusosome further comprises one or more viral non-structural proteins, e.g., in its cavity or lumen.

[0192] Fusosomes may have various structures or properties that facilitate delivery’ of a payload to a target cell. For instance, in some embodiments, the fusosome and the source cell together comprise nucleic acid(s) sufficient to make a particle that can fuse with a target cell. In embodiments, these nucleic acid(s) encode proteins having one or more of (e.g., all of) the following activities: gag polyprotein activity, polymerase activity, integrase activity, protease activity, and fusogen activity.

[0193] Compositions comprising cells can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid 57182281269.1UR 6-24056 / FR: 161118.06801 compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

[0194] Sterile injectable solutions can be prepared by incorporating the therapeutic active agent described herein in a suitable amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g, methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”. 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0195] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the genetically modified beta-like cells or their progenitors or descendants.

[0196] The compositions can be isotonic, i. e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.

[0197] Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the therapeutic active agent (e.g, the cells as described herein). This will present no problem to 58182281269.1UR 6-24056 / FR: 161118.06801 those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

[0198] One consideration concerning the therapeutic use of the cells as described herein is the quantity of cells necessary to achieve an optimal effect. The quantity of cells to be administered will vary for the subject being treated. In one embodiment, between 104to 108, between 105to 107, or between 106and 107cells are administered to a human subject. In preferred embodiments, at least about 1×107, 2×107, 3×107, 4×107, and 5×107cells are administered to a human subject. The precise determination of what would be considered an effective dose may be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.

[0199] The present disclosure provides a pharmaceutical composition, or medicament, for preventing or treating an inherited or acquired disorder. In some embodiments, a pharmaceutical composition comprises one or more of the above-described protein molecules, recombinant nucleic acid molecule / genetic construct, polynucleotide, expression cassette, expression vector (e.g., viral vector genome, expression vector, rAAV vector), and host cell.

[0200] The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, adjuvant, diluent, excipient and / or other medicinal agents. A pharmaceutically acceptable carrier, adjuvant, diluent, excipient or other medicinal agent is one that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing undesirable biological effects which outweigh the advantageous biological effects of the material. Any suitable pharmaceutically acceptable carrier or excipient can be used in the preparation of a pharmaceutical composition according to the invention (See e.g., Remington The Science and Practice of Pharmacy, Adeboye Adejare (Editor) Academic Press, November 2020).

[0201] A pharmaceutical composition is typically sterile, pyrogen-free and stable under the conditions of manufacture and storage. A pharmaceutical composition may be formulated as a solution (e.g., water, saline, dextrose solution, buffered solution, or other pharmaceutically sterile fluid), microemulsion, liposome, or other ordered structure suitable to accommodate a high product (e.g, viral vector particles, microparticles or nanoparticles) concentration.

[0202] In some embodiments, a pharmaceutical composition comprising the abovedescribed protein, recombinant nucleic acid molecule / genetic construct, polynucleotide,59182281269.1UR 6-24056 / FR: 161118.06801 expression cassette, expression vector, vector genome, host cell, or rAAV vector of the disclosure is formulated in water or a buffered saline solution. A carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of a coating such as lecithin, by maintenance of a required particle size, in the case of dispersion, and by the use of surfactants. In some embodiments, it may be preferable to include isotonic agents, for example, a sugar, a polyalcohol such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged adsorption of an injectable composition can be brought about by including, in the composition, an agent which delays absorption, e.g., a monostearate salt and gelatin. In some embodiments, a nucleic acid, vector and / or host cell of the disclosure may be administered in a controlled release formulation, for example, in a composition which includes a slow-release polymer or other carrier that protects the product against rapid release, including an implant and microencapsulated delivery' system.

[0203] In some embodiments, a pharmaceutical composition of the disclosure is a parenteral pharmaceutical composition, including a composition suitable for intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intraarticular, intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV) and / or intracistemal magna (ICM) administration. In some embodiments, a pharmaceutical composition of this disclosure is formulated for administration by ICV injection. In some embodiments, a vector (e.g., a viral vector such as AAV) may be formulated in 350 mM NaCl and 5% D-sorbitol in PBS.Therapeutic Uses

[0204] The nucleic acids, genetic constructs, expression cassettes, expression vectors, and cells described herein may be used for gene or cell therapy treatment and / or prevention of a disease, disorder or condition. In particular, they can be used for treating or preventing a disease, disorder or condition associated with loss or deficiency or dysfunction of glial cells / myelin. of neurons, or of pancreatic cells.Cell Therapy

[0205] Within scope of this disclosure is a host cell comprising the recombinant nucleic acid molecule / genetic construct, cassette, or expression vector described above, or a progeny cell of the host cell. The host cell can be a stem cell or a progenitor cell. Examples of stem 60182281269.1UR 6-24056 / FR: 161118.06801 cells include embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells and others. In some embodiments, the host cell is a glial progenitor cell, such as an oligodendrocyte or astrocyte progenitor cell. In some embodiments, the host cell is a neural progenitor cell, such as neuronal progenitor cell. In some embodiments, the host cell is a pancreatic islet progenitor cell including a beta or alpha progenitor cell. The host cell or a progeny thereof can be used as a therapeutic cell or agent for treating the disorders or conditions described herein.

[0206] Suitable methods of introducing or transplanting cells (such as the abovedescribed host cells or progenies thereof) into a target tissue or target site such as the striatum, forebrain, brain stem, and / or cerebellum or one in other tissues of a subject are well known to those of skill in the art and include, but are not limited to, injection, deposition, and grafting as described herein.

[0207] The cellular transplants can be optionally injected as dissociated cells but can also be provided by local placement of non-dissociated cells. In either case, cellular transplants optionally comprise an acceptable solution. Such acceptable solutions include solutions that avoid undesirable biological activities and contamination. Suitable solutions include an appropriate amount of pharmaceutically acceptable salt to render the formulation isotonic. Examples of pharmaceutically acceptable solutions include, but are not limited to, saline, Ringer’s solution, dextrose solution, and culture media. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.

[0208] The injection of the dissociated cellular transplant can be a streaming injection made across the entry path, the exit path, or both the entry and exit paths of the injection device (e.g, a cannula, a needle, or a tube). Automation can be used to provide a uniform entry and exit speed and an injection speed and volume.

[0209] The number of progenitor cells administered to the subject can range from about 102-1010at each administration (e.g., injection site), depending on the size and species of the recipient, and the volume of tissue requiring cell replacement. Single administration (e.g., injection) doses can span ranges of 103- 105, 104- 107, and 105- 108cells, or any amount in total for a transplant recipient patient.

[0210] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to 61182281269.1UR 6-24056 / FR: 161118.06801 the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0211] Since the CNS is an immunologically privileged site, administered cells, including xenogeneic, can survive and, optionally, no immunosuppressant drugs or a typical regimen of immunosuppressant agents are used in the treatment methods. However, optionally, an immunosuppressant agent may also be administered to the subject. Immunosuppressant agents and their dosing regimens are known to one of skill in the art and include such agents as Azathioprine. Azathioprine Sodium, Cyclosporine, Daltroban, Gusperimus Trihydrochloride, Sirolimus, and Tacrolimus. Dosages ranges and duration of the regimen can be varied with the disorder being treated; the extent of rejection; the activity of the specific immunosuppressant employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the specific immunosuppressant employed; the duration and frequency of the treatment; and drugs used in combination. One of skill in the art can determine acceptable dosages for and duration of immunosuppression. The dosage regimen can be adjusted by the individual physician in the event of any contraindications or change in the subject’s status.

[0212] Another aspect of the present disclosure relates to a method of administering a preparation of cells comprising the recombinant nucleic acid molecule as described herein to a subject or a patient in need thereof.

[0213] As used herein, a “subject” or a “patient” suitable for administering a preparation of cells described herein encompasses any animal, preferably a mammal. Suitable subjects include, without limitation, domesticated and undomesticated animals such as rodents (mouse or rat), cats, dogs, rabbits, horses, sheep, pigs, and monkeys. In one embodiment the subject is a human subject. Suitable human subjects include, without limitation, infants, children, adults, and elderly subjects.

[0214] In one embodiment, the subject is in need of a terminally differentiated cell type. For example, the subject has a condition mediated by the loss of or dysfunction of a differentiated cell population. Thus, a cell preparation described herein is administered to such subject in an amount sufficient to restore normal levels and / or function of the differentiated cell population in the selected subject, thereby treating the condition. In some embodiments,62182281269.1UR 6-24056 / FR: 161118.06801 the cell preparation that is administered to the subject is a preparation of the differentiated cell population that is lost or dysfunctional in the subject. In another embodiment, the cell preparation that is administered to the subject is a preparation of precursor or progenitor cells of the differentiated cell population. In accordance with this embodiment, the precursor or progenitor cells comprising the recombinant nucleic acid molecule or genetic construct mature or differentiate into the desired differentiated cell population after administration to the subject in need thereof.

[0215] In carrying out the methods of the present disclosure, "‘treating” or ‘'treatment” includes inhibiting, preventing, ameliorating or delaying onset of a particular condition. Treating and treatment also encompasses any improvement in one or more symptoms of the condition or disorder. Treating and treatment encompasses any modification to the condition or course of disease progression as compared to the condition or disease in the absence of therapeutic intervention.

[0216] In some embodiments, the administering is effective to reduce at least one symptom of a disease or condition that is associated with the loss or dysfunction of the differentiated cell type. In another embodiment, the administering is effective to mediate an improvement in the disease or condition that is associated with the loss or dysfunction of the differentiated cell type. In another embodiment, the administering is effective to prolong survival in the subject as compared to expected survival if no administering were carried out.

[0217] In accordance with this aspect of the present disclosure, the preparation of one or more cells comprising the recombinant nucleic acid molecule may be autologous / autogeneic (“self’) to the recipient subject. In another embodiment, the preparation of cells are non-autologous (“non-self,” e.g., allogeneic, syngeneic, or xenogeneic) to the recipient subject.

[0218] In carrying out the methods of the present disclosure, the administering may be carried out in the absence of immunosuppression, in the presence of immunosuppression or a modified course of immunosuppression therapy. For example, in one embodiment, the administering may be followed up with an initial course of immunosuppression therapy, but the administration of long-term immunosuppression therapy is not required.Therapies for Glial Cell-related Disorders

[0219] In some embodiments, this disclosure provides methods for treating glial cell-related disorders by administering or transplanting to a subject glial progenitor cells comprising the recombinant nucleic acid molecule / genetic construct, cassette, or expression vector described above.63182281269.1UR 6-24056 / FR: 161118.06801

[0220] In one embodiment, the glial progenitor cells are transplanted bilaterally into multiple sites of the subject as described U. S. Patent No. 7,524,491 to Goldman, Windrem et al., “Neonatal Chimerization With Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse,” Cell Stem Cell 2:553-565 (2008), Han et al., “Forebrain Engraftment by Human Glial Progenitor Cells Enhances Synaptic Plasticity and Learning Adult Mice," Cell Stem Cell 12:342-353 (2013). and Wang et al., “Human iPSCs-Derived Oligodendrocyte Progenitor Cells Can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination,” Cell Stem Cell 12:252-264 (2013), which are hereby incorporated by reference in their entirety. Methods for transplanting nerve tissues and cells into host brains are described by NEURAL GRAFTING IN THE MAMMALIAN CNS, Ch. 3-8 (Bjorklund and Stenevi eds., Elsevier. Amsterdam 1985); U.S. Patent No. 5,082,670 to Gage et al.; and U.S. Patent No. 6,497,872 to Weiss et al., which are hereby incorporated by reference in their entirety. Typical procedures include intraparenchymal, intracallosal, intrathecal, intracerebral, intranasal, and intravenous transplantation as well as direct infusion into brain ventricles.

[0221] Intraparenchymal transplantation is achieved by injection or deposition of tissue within the host brain so as to be apposed to the brain parenchyma at the time of transplantation. The two main procedures for intraparenchymal transplantation are: (1) injecting the donor cells within the host brain parenchyma or (2) preparing a cavity by surgical means to expose the host brain parenchyma and then depositing the graft into the cavity (NEURAL GRAFTING IN THE MAMMALIAN CNS, Ch. 3 (Bjorklund and Stenevi eds., Elsevier, Amsterdam 1985), which is hereby incorporated by reference in its entirety)- Both methods provide parenchymal apposition between the donor cells and host brain tissue at the time of grafting, and both facilitate anatomical integration between the graft and host brain tissue. This is of importance if it is required that the donor cells become an integral part of the host brain and survive for the life of the host.

[0222] Glial progenitor cells can also be delivered intracallosally as described in U. S. Patent Application Publication No. 20030223972 to Goldman, which is hereby incorporated by reference in its entirety. The glial progenitor cells can also be delivered directly to the forebrain subcortex, specifically into the anterior and posterior anlagen of the corpus callosum. Glial progenitor cells can also be delivered to the cerebellar peduncle white matter to gain access to the maj or cerebellar and brainstem tracts. Glial progenitor cells can also be delivered to the spinal cord.64182281269.1UR 6-24056 / FR: 161118.06801

[0223] Alternatively, the cells may be placed in a ventricle, e.g, a cerebral ventricle. Grafting cells in the ventricle may be accomplished by injection of the donor cells or by growing the cells in a substrate such as 30% collagen to form a plug of solid tissue which may then be implanted into the ventricle to prevent dislocation of the graft cells. For subdural grafting, the cells may be injected around the surface of the brain after making a slit in the dura.

[0224] Suitable techniques for glial cell delivery are described supra. In one embodiment, said preparation of glial progenitor cells is administered to one or more sites of the brain, brain stem, spinal cord, or combinations thereof.

[0225] Delivery of the cells to the subject can include either a single step or a multiple step injection directly into the nervous system. Although adult and fetal oligodendrocyte precursor cells disperse widely within a transplant recipient's brain, for widespread disorders, multiple injections sites can be performed to optimize treatment. Injection is optionally directed into areas of the central nervous system such as white matter tracts like the corpus callosum (e.g., into the anterior and posterior anlagen), dorsal columns, cerebellar peduncles, cerebral peduncles. Such injections can be made unilaterally or bilaterally using precise localization methods such as stereotaxic surgery, optionally with accompanying imaging methods (e.g., high resolution MRI imaging). One of skill in the art recognizes that brain regions vary across species; however, one of skill in the art also recognizes comparable brain regions across mammalian species.

[0226] In one embodiment, the method of treating a subject in need of a preparation of cells described herein involves treating a subject having a condition mediated by a loss or dysfunction of oligodendrocytes or by a loss or dysfunction of myelin, which is produced by oligodendrocytes. This method involves administering to the subject a preparation of cells comprising the recombinant nucleic acid molecule as described herein, where the preparation of cells is a preparation of glial progenitor cells or oligodendrocyte-biased progenitor cells. In accordance with this method, the cells are administered in an amount sufficient and under conditions effective to treat the condition mediated by the loss or dysfunction of oligodendrocytes or by the loss or dysfunction of myelin.

[0227] Oligodendrocytes produce myelin, an insulating sheath required for the salutatory conduction of electrical impulses along axons (Goldman et al., “How to Make an Oligodendrocyte,” Development 142(23):3983-3985 (2015), which is hereby incorporated by reference in its entirety). As described herein, oligodendrocyte loss results in demyelination,65182281269.1UR 6-24056 / FR: 161118.06801 which leads to impaired neurological function in a broad array of diseases ranging from pediatric leukodystrophies and cerebral palsy, to multiple sclerosis and white matter stroke.

[0228] Conditions mediated by a loss of myelin or by dysfunction or loss of oligodendrocytes that can be treated in accordance with the methods and cell preparations comprising the recombinant genetic construct as described herein include hypomyelination disorders and demyelinating disorders. In one embodiment, the condition is an autoimmune demyelination condition, such as e.g, multiple sclerosis, Schilder’s Disease, neuromyelitis optica, transverse myelitis, and optic neuritis. In another embodiment, the myelin-related disorder is a vascular leukoencephalopathy, such as e.g., subcortical stroke, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-related white matter disease, and spinal cord injury. In another embodiment, the myelin-related condition is a radiation induced demyelination condition. In another embodiment, the myelin-related disorder is a pediatric leukodystrophy, such as e.g., Pelizaeus-Merzbacher Disease, Tay-Sach Disease, Sandhoff s gangliosidoses, Krabbe's disease, metachromatic leukodystrophy, mucopolysaccharidoses (i.e., Sly’s disease). Niemann-Pick A disease, adrenoleukodystrophy, Canavan’s disease. Vanishing White Matter Disease, and Alexander Disease. In yet another embodiment, the myelin-related condition is periventricular leukomalacia or cerebral palsy.

[0229] Methods of generating glial progenitor cells or oligodendrocyte-biased progenitor cells suitable for treatment of a subject having a condition mediated by a loss or dysfunction of oligodendrocytes or myelin are known in the art. see e.g, U. S. Patent No.9,790,553 to Goldman et al., U. S. Patent No. 10,190,095 to Goldman et al., and U. S. Patent Application Publication No. 2015 / 0352154 to Goldman et al., each of which are hereby incorporated by reference in their entirety. These cells are modified in accordance with the present disclosure to comprise the recombinant nucleic acid molecule at any point prior to transplantation. For example, in one embodiment, the recombinant nucleic acid molecule is introduced into the glial progenitor or oligodendrocyte-biased progenitor cells just prior to transplant. In another embodiment, the recombinant nucleic acid molecule is introduced into a precursor cell of the glial progenitor or oligodendrocyte-biased progenitor cells, e.g., neural progenitor cells or pluripotent stem cells.

[0230] In another embodiment, the method of treating a subject in need of a preparation of cells described herein involves treating a condition mediated by a loss or dysfunction of astrocytes. This method involves administering to the subject a preparation of cells comprising the recombinant nucleic acid molecule as described herein, where the preparation of cells is a 66182281269.1UR 6-24056 / FR: 161118.06801 preparation of glial progenitor cells or astrocyte-biased progenitor cells. The cells are administered in an amount sufficient and under conditions effective to treat the condition mediated by the loss or dysfunction of astrocytes.

[0231] Astrocytes are the largest and most prevalent type of glial cell in the central nervous system. Astrocytes contribute to formation of the blood-brain barrier, participate in the maintenance of extracellular ionic and chemical homeostasis, are involved in the response to injury, and affect neuronal development and plasticity.

[0232] Thus, in some embodiments, the condition mediated by a loss or dysfunction of astrocytes is a neurodegenerative disorder. Neurodegenerative disorders associated with a loss of astrocytes that can be treated in accordance with the methods and cell preparations of the present disclosure include, without limitation, Parkinson's Disease (PD). Alzheimer's disease (AD) and other dementias, degenerative nerve diseases, encephalitis, epilepsy, genetic brain disorders, head and brain malformations, hydrocephalus, multiple sclerosis, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease), Huntington's disease (HD), prion diseases, frontotemporal dementia, dementia with Lew bodies, progressive supranuclear palsy, corticobasal degeneration, multiple system atrophy, hereditary spastic paraparesis, spinocerebellar atrophies, amyloidoses, motor neuron diseases (MND), spinocerebellar ataxia (SCA), and stroke and spinal muscular atrophy (SMA).

[0233] Methods of generating glial progenitor cells or astrocyte-biased progenitor cells suitable for treatment of a subject having a condition mediated by a loss or dysfunction of astrocytes are known in the art, see e.g, U. S. Patent Application Publication No. 2015 / 0352154 to Goldman et al., which is hereby incorporated by reference in its entirety. These cells are modified in accordance with the present disclosure to comprise the recombinant nucleic acid molecule at any point prior to transplantation into the subject in need thereof. For example, in one embodiment, the recombinant nucleic acid molecule is introduced into the glial progenitor or astrocyte-biased progenitor cells just prior to transplant. In another embodiment, the recombinant nucleic acid molecule is introduced into a precursor cell of the glial progenitor or astrocyte-biased progenitor cells, e.g., neural progenitor or pluripotent stem cells.Therapies for Neuron-related Disorders

[0234] In some embodiments, the method of treating a subject in need of a preparation of cells described herein involves treating a condition mediated by a loss or dysfunction of neurons. This method involves administering to the subject a preparation of cells comprising the recombinant nucleic acid molecule / genetic construct, cassette, or expression vector as 67182281269.1UR 6-24056 / FR: 161118.06801 described herein, where the preparation of cells is a preparation of neuronal progenitor cells. The cells are administered in an amount sufficient and under conditions effective to treat the condition mediated by the loss or dysfunction of neurons.

[0235] In accordance with this embodiment, the condition to be treated may be a condition mediated by the loss or dysfunction of a particular type of neuron. For example, in one embodiment the condition to be treated is a condition mediated by the loss or dysfunction of cholinergic neurons. Exemplary conditions mediated by the loss or dysfunction of cholinergic neurons include Alzheimer’s disease, corticobasal degeneration, dementia with Lewy bodies, frontotemporal dementia, multiple system atrophy, Parkinson’s disease, Parkinson’s disease dementia, and progressive supranuclear palsy (Roy et al., “Cholinergic Imaging in Dementia Spectrum Disorders,” Eur. J. Nucl. Med. Mol. Imaging. 43:1376-1386 (2016), which is hereby incorporated by reference in its entirety).

[0236] In another embodiment, the conditions to be treated is a condition mediated by the loss or dysfunction of dopaminergic neurons. Exemplary conditions mediated by the loss or dysfunction of dopaminergic neurons include Parkinson’s disease, Parkinsonian-like disorders (e.g., juvenile parkinsonism, Ramsey-Hunt paralysis syndrome), and mental disorders (e.g., schizophrenia, depression, drug addiction).

[0237] In another embodiment, the condition to be treated is a condition mediated by the loss or dysfunction of medium spiny neurons and / or cortical interneurons. Exemplary conditions mediated by the loss or dysfunction of medium spiny neurons and / or cortical interneurons include Huntington’s disease, epilepsy, anxiety,, and depression (Powell et al., “Genetic Disruption of Cortical Interneuron Development Causes Region- and GABA Cell Type-Specific Deficits, Epilepsy, and Behavioral Dysfunction,” J. Neurosci. 23(2):622-631 (2003). which is hereby incorporated by reference in its entirety).

[0238] Methods of generating neuronal progenitor cells suitable for treatment of a subject having a condition mediated by a loss or dysfunction of neurons are known in the art, see e.g., Goldman, et al., " Transplanted Neural Progenitors Bridge Gaps to Benefit Cord-Injured Monkeys." Nat. Med. 24(4):388-390 (2018); Roy et al., “Functional Engraftment of Human ES Cell-Derived Dopaminergic Neurons Enriched by Coculture with Telomerase-Immortalized Midbrain Astrocytes,” Nat. Med. 12(11): 1259-1268 (2006); Nunes et al., “Identification and Isolation of Multipotential Neural Progenitor Cells from the Subcortical White Matter of the Adult Human Brain,” Nat. Med. 9(4):439-447 (2003), U. S. Patent No.6,812,027 to Goldman et al.; U.S. Patent No. 7,150,989 to Goldman et al.; U.S. Patent No.68182281269.1UR 6-24056 / FR: 161118.06801 7,468,277 to Goldman et al.; U. S. Patent No. 7,785,882 to Goldman; U. S. Patent No.8,263,406 to Goldman et al.; U. S. Patent No. 8,642,332 to Goldman et al.; and U. S. Patent No.8,945,921 to Goldman et al., each of which is hereby incorporated by reference in its entirety. These cells are modified in accordance with the present disclosure to comprise the recombinant nucleic acid molecule at any point prior to transplantation into the subject in need thereof. For example, in one embodiment, the recombinant nucleic acid molecule is introduced into the neuronal progenitor cells just prior to transplant. In another embodiment, the recombinant nucleic acid molecule is introduced into a precursor cell of the neuronal progenitor cells, e.g., neural progenitor or pluripotent stem cells.

[0239] In carrying out the methods of the present disclosure involving cell replacement in central nervous system, the preparation of cells described herein can be administered systemically into the circulation, or administered directly to one or more sites of the brain, the brain stem, the spinal cord, or a combination thereof.

[0240] When the preparation of cells is injected systemically into the circulation, the preparation of cells may be placed in a syringe, cannula, or other injection apparatus for precise placement at a preselected site. The term “injectable” means the preparation of cells can be dispensed from syringes under normal conditions under normal pressure.

[0241] Methods for direct administration of (i.e., transplanting) various nerve tissues / cells into a host brain are well known in the art. In some embodiments, the preparation is administered intraventricularly, intracallosally, or intraparenchymally.

[0242] Intraparenchymal administration, i.e., within the host brain (as compared to outside the brain or extraparenchymal transplantation) is achieved by injection or deposition of cells within the brain parenchyma at the time of administration. Intraparenchymal transplantation can be performed using two approaches: (i) injection of the preparation of cells into the host brain parenchyma or (ii) preparing a cavity by surgical means to expose the host brain parenchyma and then depositing the preparation of cells into the cavity. Both methods provide parenchymal deposition between the preparation of cells and the host brain tissue at the time of administration, and both facilitate anatomical integration between the graft (z. e., the preparation of cells) and the host brain tissue.

[0243] Alternatively, the cell graft may be placed in a ventricle, e.g. a cerebral ventricle or subdurally, i.e. on the surface of the host brain where it is separated from the host brain parenchyma by the intervening pia mater or arachnoid and pia mater. Grafting to the ventricle69182281269.1UR 6-24056 / FR: 161118.06801 may be accomplished by injection of the donor cells or by growing the cells in a substrate such as 3% collagen to form a plug of solid tissue which may then be implanted into the ventricle to prevent dislocation of the graft. For subdural grafting, the cells may be injected around the surface of the brain after making a slit in the dura.

[0244] For transplantation into cavities, which may be preferred for spinal cord grafting, tissue is removed from regions close to the external surface of the CNS to form a transplantation cavity, by removing bone overlying the brain and stopping bleeding with a material such a gelfoam. Suction may be used to create the cavity. The preparation of cells is then placed in the cavity. More than one preparation of cells may be placed in the same cavity. In some embodiments, the site of implantation is dictated by the CNS disorder being treated.

[0245] Injections into selected regions of the host brain may be made by drilling a hole and piercing the dura to permit the needle of a microsyringe to be inserted. The microsyringe is preferably mounted in a stereotaxic frame and three dimensional stereotaxic coordinates are selected for placing the needle into the desired location of the brain or spinal cord. The cells may also be introduced into the putamen. nucleus basalis. hippocampus cortex, striatum, substantia nigra or caudate regions of the brain, as well as the spinal cord.Therapies for Pancreatic Cell-related Disorders

[0246] In some embodiments, this disclosure provides methods for treating disorders related to a loss of or dysfunctional pancreatic cells, in particular pancreatic neuroendocrine cells or pancreatic endocrine cells, by transplanting pancreatic islet progenitor cells comprising the recombinant nucleic acid molecule / genetic construct, cassette, or expression vector described above.

[0247] In one embodiment this disclosure provides for the treatment of type 1 diabetes, type 2 diabetes, pre-diabetes, and the treatment of other metabolic diseases or disorders associated with a deficiency in beta cell number (e.g, a reduction in the number of pancreatic cells) or an insufficient level of beta cell biological activity (e.g, a deficiency in glucose-stimulated insulin secretion, a deficiency in insulin production). For example, this disclosure provides compositions for the treatment of diabetic patients who lack sufficient levels of insulin due to a decrease in the number or activity of insulin-producing pancreatic cells. Many diseases associated with a deficiency in cell number are characterized by beta cell loss or an increase in beta cell death. Methods of this disclosure ameliorate such type 1 diabetes, type 2 diabetes, and related diseases, disorders, by generating cells (e.g., insulinexpressing cells) that can supplement the deficiency.70182281269.1UR 6-24056 / FR: 161118.06801

[0248] The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a cellular composition described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method).

[0249] The therapeutic methods described herein (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of a cellular composition described herein, to subjects, particularly humans, suffering from, having susceptibility to, or at risk of having type 1 diabetes, type 2 diabetes, pre-diabetes, or another metabolic disease or disorder associated with a deficiency in beta cell number (e.g., a reduction in the number of pancreatic cells) or an insufficient level of beta cell biological activity (e.g.. a deficiency in glucose-stimulated insulin secretion, a deficiency in insulin production). Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, family history, and the like).

[0250] In one embodiment, the disclosure provides a method of monitoring treatment progress in connection with type 1 diabetes, type 2 diabetes, pre-diabetes, and the treatment of other metabolic diseases or disorders associated with a deficiency in beta cell number (e.g, a reduction in the number of pancreatic cells) or an insufficient level of beta cell biological activity (e.g., a deficiency in glucose-stimulated insulin secretion, a deficiency in insulin production). The method includes the step of determining a level of diagnostic marker (e.g., any target delineated herein in a subject suffering from or susceptible to a disorder or symptoms thereof associated with a defect in beta cell number or activity, in which the subject has been administered a therapeutic amount of a cellular composition described herein sufficient to treat the disease or symptoms thereof. The level of marker determined in the method can be compared to known levels of marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of marker can then be compared to the level of marker in the subject after the treatment commences, to determine the efficacy of the treatment.71182281269.1UR 6-24056 / FR: 161118.06801

[0251] Cells useful in the methods include virtually any cell type that expresses markers that are typically expressed in a beta cell or beta cell progenitor. In particular embodiments, cells useful in the invention can be induced to acquire glucose-stimulated insulin secretion. In particular embodiments, cells useful include, but are not limited to, adult or embryonic stem cells or other multi- or pluripotent stem cells that express or that can be induced to express one or more pancreatic islet beta cell markers, endocrine markers or beta cell transcription factors. Exemplary beta cell transcription factors include Pdx1, Mafa, Math, Nkx6.1, NeuroD1, Foxa2, Hnf4a, Nkx2.2, Pax6 and HNF4a. In particular embodiments, the beta cell transcription factor is a factor expressed at higher levels in an adult islet cell relative to a neonatal islet cell. Beta cell transcription factors expressed at increased levels in an adult islet include Pdx1, MafA and Nkx6-1 compared with neonatal islets. In other embodiments, a cell described herein or a progeny thereof expresses a beta cell marker including, but not limited to, Insulin 1, Insulin 2, a-cell marker glucagon and 5-cell marker, somatostatin. In other embodiments, the cell or progeny expresses an endocrine marker including, but not limited to, Insulinl, Insulin2, Glucagon and Somatostatin.

[0252] In particular embodiments, human induced pluripotent stem cells are derived from somatic cells (iPSC) or from embryonic stem cells. Cells of the disclosure may be maintained, for example, on matrigel (BD) coated dishes in virtually any culture media that supports growth or maintenance of the cells (e.g., complete TeSR Media). For pancreatic differentiation, pluripotent stem cells are infected with a human insulin reporter lentivirus (pGreenZero lent! reporter human insulin. System biosciences) or using any other standard transfection method.

[0253] Methods for producing the pancreatic islet progenitor cells are known in the art and described herein, for example, where an embryonic stem cell or induced pluripotent stem cell is recombinantly modified to express any one or more of Oct4, Nanog, Soxl7, FoxA2, Pdxl, Nkx6.1, and / or Ngn3. Beta cells generated by such methods express one or more of the following markers: insulin, Pdxl, Mafa, Pax6, Glut2, NeuroDl, glucokinase, glucagon, somatostatin, chromogranin A, and Vamp2. See also, Pagliuca et al.. Dev. 140:2472-2483, 2013, which is incorporated herein by reference in its entirety.

[0254] '‘Pancreatic endocrine cells” or ‘'Pancreatic neuroendocrine cells” as used interchangeably herein, refer to cells capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, ghrelin, and pancreatic polypeptide. In addition to these hormones, markers characteristic of pancreatic endocrine cells include one or more of 72182281269.1UR 6-24056 / FR: 161118.06801 NGN3, NeuroDl, ISL1, PDX1, NKX6.1, PAX4, ARX, NKX2.2, and PAX6. Pancreatic endocrine cells expressing markers characteristic of (3 cells can be characterized by their expression of insulin and at least one of the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroDl, ISL1, HNF30, MAFA and PAX6.

[0255] “Pancreatic endocrine progenitor cells” or “pancreatic islet progenitor cells” are used interchangeably herein to refer to pancreatic endoderm cells capable of becoming a pancreatic hormone expressing cell. Such progenitor cells express at least one of the following markers: NGN3; NKX2.2; NeuroDl; ISL1; PAX4; PAX6; or ARX. Pancreatic endocrine progenitor cells may be characterized by their expression of NKX2.2 and NeuroDl. Examples of such progenitor cells include the pancreatic progenitor 1 cells, and pancreatic progenitor 2 cells described in US Patent NO. 10927350, the contents of which is incorporated by reference. “Pancreatic progenitor 1 cells” refers to endoderm cells that give rise to the esophagus, lungs, stomach, liver, pancreas, gall bladder, and a portion of the duodenum. Pancreatic progenitor 1 cells express at least one of the following markers: PDX1, FOXA2, CDX2, SOX2, and HNF4a. Pancreatic progenitor 1 cells may be characterized by an increase in expression of PDX1, compared to gut tube cells. For example, greater than fifty percent of the cells in Stage 3 cultures typically express PDX1. “Pancreatic progenitor 2 cells,” as used herein, refers to cells that express at least one of the following markers: PDX1, NKX6.1, HNF6, NGN3, SOX9, PAX4, PAX6, ISL1, gastrin, FOXA2, PTFla, PROXI and HNF4a. Pancreatic progenitor 2 cells may be characterized as positive for the expression of PDX1, NKX6.1, and SOX9.

[0256] A “precursor of pancreatic endocrine progenitor cells” refers to any cell that is capable of differentiating into a pancreatic endocrine progenitor cell, including for example, a pluripotent stem cell, a definitive endoderm cell, a gut tube cell, or a pancreatic progenitor cell, when cultured under conditions suitable for differentiating the precursor cell into the pancreatic pro endocrine cell.

[0257] In one embodiment, pancreatic islet progenitor cells are administered to a subject by route known to those skilled in the art including intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, subcutaneous, kidney capsule, intratumoral, epidural, nasal, oral, vaginal, rectal, topical, local, otic, inhalational, buccal (e.g., sublingual), and transdermal administration or any route. In another embodiment, other modes of administration also are contemplated. In some embodiments, the administration is by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral 73182281269.1UR 6-24056 / FR: 161118.06801 injection, intrachoroidal injection, intracameral injection, subconjunctival injection, subTenon’s injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.

[0258] In another embodiment, the pancreatic islet progenitor cells can be administered at any suitable location in the subject. For example, the islet progenitor cells are administered to the kidney, forearm, mouth, anus, nose, upper arm, hip, thigh, buttocks, liver, spleen, muscle, subcutaneous tissue, or white adipose tissue of the subject. In some embodiments, the engineered cells are administered to the liver, muscle, or white adipose tissue of the subject. In some embodiments, the white adipose tissue is omentum.

[0259] In another embodiment, the islet progenitor cells may be implanted into the subject. The islet progenitor cells may be implanted as dispersed cells or formed into clusters.

[0260] In another embodiment, the condition to be treated is a condition associated with islet cell related disease. In one embodiment, the beta cell related disorder is a metabolic disorder. A metabolic disorder may occur when abnormal chemical reactions in the body of a subject disrupts metabolic processes (e.g., processes related to the metabolism, or breakdown, of energy into sugars and acids or the storage of said energy). In some embodiments, the metabolic disorder affects the breakdown of amino acids, carbohydrates, or lipids in a subject’s body. In some embodiments, the metabolic disorder affects the subject’s mitochondria (e.g., mitochondrial diseases). In one embodiment, the metabolic disorder develops when the subject’s organs, such as the liver or pancreas, become disease and / or do not function normally. Exemplary metabolic disorders herein may comprise, but are not limited to, any disease or disorder characterized by increased blood pressure, high blood sugar, excess body fat around the waist, and abnormal cholesterol or triglyceride levels. In some embodiments, the metabolic disorder is familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe disease, maple syrup urine disease, metachromatic leukodystrophy, mitochondrial encephalopathy, lactic acidosis, stroke-hke episodes (MELAS), Niemann-Pick disease, phenylketonuria (P KU), porphyria, Tay-Sachs disease, Wilson's disease, Type I diabetes, Type II diabetes, obesity, hypertension, dyslipidemia, or carbohydrate intolerance. In one embodiment, the diabetes is Type I or Type II diabetes.

[0261] In accordance with an embodiment of the present disclosure, a method for treating disorders related to a loss or dysfunctional pancreatic cells entails administering to a subject in need thereof an isolated population of islet progenitor cells described above or a microcapsule comprising the cells encapsulated therein. In some embodiments, the subject is in need of additional [3 cells. In some embodiments, the subject has. or has an increased risk of 74182281269.1UR 6-24056 / FR: 161118.06801 developing diabetes. A pancreatic islet progenitor cell or population of the cells generated by a method of the present disclosure can be administered to a subject for treatment of type 1 or type 2 diabetes. In some embodiments, the subject has, or has an increased risk of developing, a metabolic disorder. In some embodiments, administering to the subject comprises implanting pancreatic islet progenitor cells described herein, a microcapsule comprising the pancreatic islet progenitor cells, or a macroencapsulation device comprising the pancreatic islet progenitor cells into the subject. The subject may be a human subject or an animal subject. In some embodiments, the cells may be implanted as dispersed cells or formed into clusters that may be infused into the hepatic portal vein. In some embodiments, cells may be provided in biocompatible degradable polymeric supports, porous non-degradable devices or encapsulated to protect from host immune response. Cells may be implanted into an appropriate site in a recipient. The implantation sites include, for example, the liver, natural pancreas, renal subcapsular space, omentum, peritoneum, sub serosal space, intestine, stomach, or a subcutaneous pocket.

[0262] Artificial Islet or Pancreas

[0263] In accordance with an embodiment of the present disclosure, an artificial islet or pancreas is provided. The artificial islet or pancreas can be constructed using the pancreatic islet progenitor cells generated according to the methods described herein.

[0264] An artificial pancreas is a device that encapsulates and nurtures islets of Langerhans to replace the islets and [3 cells destroyed by type 1 diabetes. An artificial pancreas may contain a million islets or more, and may be implanted in the peritoneal cavity or under the skin where it can respond to changing blood glucose levels by releasing hormones, such as insulin. An artificial pancreas may be made using living (e.g., glucose-sensing and insulin secreting islets) and nonliving components (e.g., to shield the islets from the diabetic's body and its destructive immune mechanism while permitting the islets to thrive).

[0265] The present disclosure contemplates using (3 cells in any artificial pancreas. In some embodiments, the artificial pancreas comprises microencapsulated or coated islets comprising pancreatic islet progenitor cells generated according to the methods herein. In some embodiments, the artificial pancreas comprises a macroencapsulation device into which islet cells comprising pancreatic islet progenitor cells generated according to the methods herein are grouped together and encapsulated. In some embodiments, the macroencapsulation device comprises a PVA hydrogel sheet for an artificial pancreas of the present invention (Qi et al., 2004). In some embodiments, the artificial islet comprises pancreatic islet progenitor 75182281269.1UR 6-24056 / FR: 161118.06801 cells generated according to the methods herein, along with other islet cells (a, 5, etc.) in the form of an islet sheet. The islet sheet comprises a layer of artificial human islets comprising the pancreatic islet progenitor cells macroencapsulated within a membrane (e.g., of ultra-pure alginate). The sheet membrane is reinforced with mesh and may be coated on the surface to prevent or minimize contact between the cells encapsulated inside and the transplantation recipient's host immune response. Oxygen, glucose, and other nutrients readily diffuse into the sheet through the membrane nurturing the islets, and hormones, such as insulin readily diffuse out. Additional examples of membranes designed for macroencapsulation / implantation of an artificial islet or pancreas can be found in the literature (Isayeva et al. 2003). Another example of a macroencapsulated implant suitable for an artificial islet or pancreas can be found in the literature (Aurelien, etal. 2014).

[0266] In all of the methods described herein, to enhance further differentiation, survival or activity’ of the implanted cells in vivo, additional factors (such as growth factors, antioxidants, anti-inflammatory agents, and / or immunosuppressants) can be administered before, simultaneously with, or after the administration of the cells. Some of these factors can be secreted by endogenous cells and exposed to the administered cells in situ. Implanted cells can be induced to differentiate by any combination of endogenous and exogenously administered growth factors known in the art.

[0267] In some embodiments, the method of treatment further comprises incorporating the cells into a three-dimensional support prior to implantation. The cells can be maintained in vitro on this support prior to implantation into the patient. Alternatively, the support containing the cells can be directly implanted in the patient without additional in vitro culturing. The support can optionally be incorporated with at least one pharmaceutical agent that facilitates the survival and function of the transplanted cells.

[0268] The number of cells used in implantation depends on a number of various factors including the patient's condition and response to the therapy, and can be determined by one skilled in the art. The number of cells in a given volume can be determined by w ell-known and routine procedures and instrumentation. The percentage of the cells in a given volume of a mixture of cells can be determined by much the same procedures. Cells can be readily counted manually or by using an automatic cell counter. Specific cells can be determined in a given volume using specific staining and visual examination and by automated methods using specific binding reagent, typically antibodies, fluorescent tags, and a fluorescence activated cell sorter.76182281269.1UR 6-24056 / FR: 161118.06801

[0269] The preparation of cells can be administered in dosages and by techniques well known to those skilled in the medical and veterinary arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the formulation that will be administered. The dose appropriate to be used in accordance with various embodiments described herein will depend on numerous factors. It may vary considerably for different circumstances. The parameters that will determine optimal doses to be administered for primary and adjunctive therapy generally will include some or all of the following: the disease being treated and its stage; the species of the subject, their health, gender, age, weight; the subject’s immunocompetence; other therapies being administered; and expected potential complications from the subject’s history or genotype. The parameters may also include whether the cells are syngeneic, autologous, allogeneic, or xenogeneic; their potency (specific activity); the site and / or distribution that must be targeted for the cells / medium to be effective; and such characteristics of the site such as accessibility to cells / medium and / or engraftment of cells. Additional parameters include co-administration with other factors (such as growth factors and cytokines). The optimal dose in a given situation also will take into consideration the way in which the cells / medium are formulated, the way they are administered, and the degree to which the cells / medium will be localized at the target sites following administration. Finally, the determination of optimal dosing necessarily will provide an effective dose that is neither below the threshold of maximal beneficial effect nor above the threshold where the deleterious effects associated with the dose outweighs the advantages of the increased dose.

[0270] For fairly pure preparations of cells, optimal doses in various embodiments will range from about 104to about 109cells per administration. In some embodiments, the optimal dose per administration will be between about 105to about 107cells. In many embodiments the optimal dose per administration will be about 5xl05to about 5xl06cells.

[0271] It is to be appreciated that a single dose may be delivered all at once, fractionally, or continuously over a period of time. The entire dose also may be delivered to a single location or spread fractionally over several locations.

[0272] Human subjects are treated generally longer than experimental animals; but treatment generally have a length proportional to the length of the disease process and the effectiveness of the treatment. Those skilled in the art will take this into account in using the results of other procedures carried out in humans and / or in animals, such as rats, mice, nonhuman primates, and the like, to determine appropriate doses for humans. Such determinations, based on these considerations and taking into account guidance provided by the present 77182281269.1UR 6-24056 / FR: 161118.06801 disclosure and the prior art will enable the skilled artisan to do so without undue experimentation.

[0273] Suitable regimens for initial administration and further doses or for sequential administrations may all be the same or may be variable. Appropriate regimens can be ascertained by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art.

[0274] In some embodiments, the preparation of cells is administered to a subject in one dose. In others, the preparation of cells is administered to a subject in a series of two or more doses in succession. In some other embodiments where the preparation of cells is administered in a single dose, in two doses, and / or more than two doses, the doses may be the same or different, and they are administered with equal or with unequal intervals between them.

[0275] The preparation of cells may be administered in many frequencies over a wide range of times. In some embodiments, they are administered over a period of less than one day. In other embodiments, they are administered over two, three, four, five, or six days. In some embodiments, they are administered one or more times per week, over a period of weeks. In other embodiments, they are administered over a period of weeks for one to several months. In various embodiments, they may be administered over a period of months. In others they may be administered over a period of one or more years. Generally, lengths of treatment will be proportional to the length of the disease process, the effectiveness of the therapies being applied, and the condition and response of the subject being treated.

[0276] The choice of formulation for administering the composition for a given application will depend on a variety of factors. Prominent among these will be the species of subject, the nature of the disorder, dysfunction, or disease being treated and its state and distribution in the subject, the nature of other therapies and agents that are being administered, the optimum route for administration, survivability via the route, the dosing regimen, and other factors that will be apparent to those skilled in the art. In particular, for instance, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form.

[0277] For example, cell survival can be an important determinant of the efficacy of cell-based therapies. This is true for both primary and adjunctive therapies. Another concern arises when target sites are inhospitable to cell seeding and cell growth. This may impede access to the site and / or engraftment there of therapeutic cells. Thus, measures may be taken78182281269.1UR 6-24056 / FR: 161118.06801 to increase cell survival and / or to overcome problems posed by barriers to seeding and / or growth.

[0278] Final formulations may include an aqueous suspension of cells / medium and, optionally, protein and / or small molecules, and will typically involve adjusting the ionic strength of the suspension to isotonicity (i.e., about 0.1 to 0.2) and to physiological pH (i.e., about pH 6.8 to 7.5). The final formulation will also typically contain a fluid lubricant, such as maltose, which must be tolerated by the body. Exemplary lubricant components include glycerol, glycogen, maltose, and the like. Organic polymer base materials, such as polyethylene glycol and hyaluronic acid as well as non-fibrillar collagen, such as succinylated collagen, can also act as lubricants. Such lubricants are generally used to improve the injectability, intrudability, and dispersion of the injected material at the site of injection and to decrease the amount of spiking by modifying the viscosity of the compositions. This final formulation is by definition the cells described herein in a pharmaceutically acceptable carrier.

[0279] Multiple preparations of cells may be administered concomitantly to different locations such as combined administration intrathecally and intravenously to maximize the chance of targeting into affected areas.Methods of Administration

[0280] The above-described molecule, or polynucleotide, or vector (e.g, vector genome, rAAV vector) may be administered to a subject (e.g.. a patient) or a target cell in order to treat the subject. Administration of a vector to ahuman subject, or an animal in need thereof, can be by any means known in the art for administering a vector. Examples of a target cell include cells of the CNS, preferably oligodendrocytes or the progenitor cells thereof.

[0281] A vector can be administered in addition to, and as an adjunct to. the standard of care treatment. That is, the vector can be co-administered with another agent, compound, drug, treatment or therapeutic regimen, either simultaneously, contemporaneously, or at a determined dosing interval as would be determined by one skilled in the art using routine methods. Uses disclosed herein include administration of an rAAV vector of the disclosure at the same time, in addition to and / or on a dosing schedule concurrent with, the standard of care for the disease as known in the art.

[0282] In some embodiments, a combination composition includes one or more immunosuppressive agents. In some embodiments, a combination composition includes an rAAV vector comprising a transgene (e.g, a polynucleotide encoding an RNA molecule disclosed herein) and one or more immunosuppressive agents. In some embodiments, a method 79182281269.1UR 6-24056 / FR: 161118.06801 includes administering or delivering an rAAV vector comprising the transgene to a subject and administering an immunosuppressive agent to the subject either prophylactically prior to administration of the vector, or after administration of the vector (z.e., either before or after symptoms of a response against the vector and / or the protein provided thereby are evident).

[0283] In one embodiment, a vector of the disclosure (e.g., an rAAV vector) is administered systemically. Exemplary methods of systemic administration include, but are not limited to, intravenous (e.g., portal vein), intraarterial (e.g., femoral artery, hepatic artery), intravascular, subcutaneous, intradermal, intraperitoneal, transmucosal, intrapulmonary, intralymphatic and intramuscular administration, and the like, as well as direct tissue or organ injection. One skilled in the art would appreciate that systemic administration can deliver a nucleic acid to all tissues. In some embodiments, direct tissue or organ administration includes administration to areas directly affected by oligodendrocyte deficiency (e.g., brain and / or central nervous system). In some embodiments, vectors of the disclosure, and pharmaceutical compositions thereof, are administered to the brain parenchyma (i.e., by intraparenchymal administration), to the spinal canal or the subarachnoid space so that it reaches the cerebrospinal fluid (CSF) i.e., by intrathecal administration), to a ventricle of the brain (i.e., by intracerebroventricular administration) and / or to the cistema magna of the brain (i.e., by intracistemal magna administration).

[0284] Accordingly, in some embodiments, a vector of the present disclosure is administered by direct injection into the brain (e.g, into the parenchyma, ventricle, cistema magna, etc.) and / or into the CSF (e.g., into the spinal canal or subarachnoid space) to treat a disorder of myelin. A target cell of a vector of the present disclosure includes a cell located in the cortex, subcortical white matter of the corpus callosum, striatum and / or cerebellum. In some embodiments, a target cell of a vector of the present disclosure is an oligodendrocyte or a progenitor cell thereof. Additional routes of administration may also comprise local application of a vector under direct visualization, e.g., superficial cortical application, or other stereotaxic application.

[0285] In some embodiments, a vector of the disclosure is administered by at least two routes. For example, a vector is administered systemically and also directly into the brain. If administered via at least two routes, the administration of a vector can be, but need not be, simultaneous or contemporaneous. Instead, administration via different routes can be performed separately with an interval of time between each administration.80182281269.1UR 6-24056 / FR: 161118.06801

[0286] The above-described recombinant nucleic acid molecule, or polynucleotide, or vector genome, or a vector (e.g. an rAAV vector) comprising the polynucleotide may be used for transduction of a cell ex vivo or for administration directly to a subject (e.g., directly to the CNS of a patient with a disease). In some embodiments, a transduced cell (e.g., a host cell) is administered to a subject to treat or prevent a disease, disorder or condition (e.g., cell therapy for the disease). For example, an rAAV vector comprising a therapeutic nucleic acid (e.g, encoding a protein) can be preferably administered to an oligodendrocyte or a progenitor cell thereof in a biologically effective amount.

[0287] The dosage amount of a vector depends upon, e.g., the mode of administration, disease or condition to be treated, the stage and / or aggressiveness of the disease, individual subject's condition (age, sex, weight, etc.), particular viral vector, stability of protein to be expressed, host immune response to the vector, and / or gene to be delivered. Generally, doses range from at least 1 x 108, or more, e.g., 1 x 109, 1 x 1010, 1 x 1011, 1 x 1012, 1 x 1013, 1 x 1014, 1 x 1015or more vector genomes (vg) per kilogram (kg) of body weight of the subject to achieve a therapeutic effect.

[0288] In some embodiments, a recombinant nucleic acid molecule, or polynucleotide, or vector genome, or a vector described herein may be administered as a component of a DNA molecule (e.g, a recombinant nucleic acid) having a regulatory element (e.g, a promoter) appropriate for expression in a target cell (e.g., oligodendrocytes). The polynucleotide may be administered as a component of a plasmid or a viral vector, such as an rAAV vector. An rAAV vector may be administered in vivo by direct delivery of the vector (e.g, directly to the CNS) to a patient in need of treatment. An rAAV vector may be administered to a patient ex vivo by administration of the vector in vitro to a cell from a donor patient in need of treatment, followed by introduction of the transduced cell back into the donor (e.g, cell therapy).

[0289] Compositions comprising cells or their progenitors / descendants as described herein can be provided systemically or directly to a subject for the treatment or prevention of a disorder described herein. In one embodiment, the cells and / or their progenitors are directly injected into an organ of interest (e.g.. pancreas). Alternatively, compositions comprising the cells and / or their progenitors are provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g, the pancreatic vasculature). Expansion and differentiation agents can be provided prior to, during or after administration of the cells and / or their progenitors to increase production of cells having the desired phenotype (e.g., insulinproducing potential) in vitro or in vivo. The cells and / or their progenitors can be administered 81182281269.1UR 6-24056 / FR: 161118.06801 in any physiologically acceptable vehicle, normally intravascularly, although they may also be introduced into another convenient site where the cells may find an appropriate site for regeneration and differentiation.

[0290] In one approach, at least 100,000, 250,000, or 500,000 cells are injected. In other embodiments, 750,000, or 1,000,000 cells are injected. In other embodiments, at least about 1×105cells will be administered, 1×106, 1×107. or even as many as 1×108to 1×1010, or more are administered. Preferable ranges of purity in populations comprising selected cells are about 50 to about 55%, about 55 to about 60%, and about 65 to about 70%. More preferably the purity is at least about 70%, 75%, or 80% pure, more preferably at least about 85%, 90%, or 95% pure. In some embodiments, the population is at least about 95% to about 100% selected cells. Dosages can be readily adjusted by those skilled in the art (e.g, a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, or the like.

[0291] The cell compositions described herein include pharmaceutical compositions comprising genetically modified cells or their progenitors and a pharmaceutically acceptable carrier. Administration can be autologous or heterologous. For example, beta-like cells, or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Selected cells of the disclosure or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering atherapeutic composition, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0292] In another pharmaceutical use, stem / progenitor cells of the present disclosure can be genetically modified prior to their administration to a subject. For this purpose, the cells can be transformed with a nucleic acid encoding the protein that is to be produced in the cells. The nucleic acid can be introduced into cells of the invention using any of the various methods that are well known to the skilled person, for example, using a viral vector and / or a lipid containing transfection composition such as IBAfect (IBA GmbH. Goettingen, Germany), Fugene (Roche), GenePorter (Gene Therapy Systems), Lipofectamine (Invitrogen), Superfect (Qiagen), Metafecten (Biontex) or those ones described in the PCT application WO 01 / 015755). In a related embodiment, the cells described herein, after being transformed with a nucleic acid encoding a polypeptide of choice, can be used to recombinantly produce this polypeptide.82182281269.1UR 6-24056 / FR: 161118.06801 Kit

[0293] The present disclosure provides a kit with packaging material and one or more components therein. A kit ty pically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo or ex vivo, of the components therein. A kit can contain a collection of such components, e.g., the above-described recombinant nucleic acid molecule, polynucleotide, expression cassette, expression vector (e.g., viral vector genome, expression vector, rAAV vector), and host cell or progenies thereof, and optionally a second active agent such as a compound, therapeutic agent, drug or composition.

[0294] A kit refers to a physical structure that contains one or more components of the kit. Packaging material can maintain the components in a sterile manner and can be made of material commonly used for such purposes (e.g, paper, glass, plastic, foil, ampules, vials, tubes, etc.).

[0295] In one embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of a cell described herein in unit dosage form. In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic cellular composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0296] A label or insert can include identifying information of one or more components therein, dose amounts, clinical pharmacology' of the active ingredients(s) including mechanism of action, pharmacokinetics and pharmacodynamics. A label or insert can include information identifying manufacture, lot numbers, manufacture location and date, expiration dates. A label or insert can include information on a disease (e.g, an inherited or acquired disorder of myelin such as HD) for which a kit component may be used. A label or insert can include instructions for a clinician or subject for using one or more of the kit components in a method, use or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency of duration and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimens described herein.

[0297] A label or insert can include information on potential adverse side effects, complications or reactions, such as a warning to a subject or clinician regarding situations where it would not be appropriate to use a particular composition.83182281269.1UR 6-24056 / FR: 161118.06801 Definitions

[0298] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a,” "an" and ‘ he” are intended to include the plural forms as well, unless the context clearly indicates otherwise. The following terms have the meanings given:

[0299] As used herein, the term "about." or "approximately" refers to a measurable value such as an amount of the biological activity, homology or length of a polynucleotide or polypeptide sequence, dose, time, temperature, and the like, and is meant to encompass variations of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% 1 %, 0.5% or even 0.1 %, in either direction (greater than or less than) of the specified amount unless otherwise stated, otherwise evident from the context, or except where such number would exceed 100% of a possible value.

[0300] As used herein, the term “homologous,” or “homology,” refers to two or more reference entities (z. e., a nucleic acid or polypeptide sequence) that share at least partial identity over a given region or portion. For example, when an amino acid position in two peptides is occupied by identical amino acids, the peptides are homologous at that position. Notably, a homologous peptide will retain activity or function associated with the unmodified or reference peptide and the modified peptide will generally have an amino acid sequence “substantially homologous” with the amino acid sequence of the unmodified sequence. When referring to a polypeptide, nucleic acid or fragment thereof, “substantial homology” or “substantial similarity,” means that when optimally aligned with appropriate insertions or deletions with another polypeptide, nucleic acid (or its complementary strand) or fragment thereof, there is sequence identity in at least about 70% to 99% of the sequence. The extent of homology (identity) between two sequences can be ascertained using computer program or mathematical algorithm known in the art. Such algorithms that calculate percent sequence homology (or identity) generally account for sequence gaps and mismatches over the comparison region or area.

[0301] A nucleic acid or polynucleotide refers to a DNA molecule (e.g, a cDNA or genomic DNA), an RNA molecule (e.g, an mRNA), or a DNA or RNA analog. A DNA or84182281269.1UR 6-24056 / FR: 161118.06801 RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably double-stranded DNA.

[0302] An isolated or recombinant nucleic acid refers to a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukary ote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA. a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. The nucleic acid described above can be used to express the protein of this disclosure. For this purpose, one can operatively link the nucleic acid to suitable regulatory sequences to generate an expression vector.

[0303] A "recombinant nucleic acid” is a combination of nucleic acid sequences that are joined together using recombinant technology and procedures used to join together nucleic acid sequences.

[0304] As used herein, the term “nucleotide sequence'’ and “nucleic acid sequence’' are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA / RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural. or derivatized nucleotide bases. In the context of the recombinant genetic construct of the present disclosure, the nucleotide sequence may be a nucleotide sequence that “encodes” a protein if, in its native state or when manipulated by methods well known to those skilled in the art, the nucleotide sequence can be transcribed and / or translated to produce the mRNA for the protein and / or a fragment thereof. Nucleotide sequences of the recombinant genetic construct may also “encode” an agent that has an effector function if, in it its native state or when manipulated by methods well known in the art, can be transcribed to produce the agent with the desired effector function of a polypeptide (e g., immune checkpoint proteins) or an RNA (e.g, shRNA, siRNA, microRNA, guide RNA, etc.).85182281269.1UR 6-24056 / FR: 161118.06801

[0305] The terms “heterologous” DNA molecule and “heterologous” nucleic acid, as used herein, each refer to a molecule that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of shuffling or recombination. When used to describe two nucleic acid segments, the terms mean that the two nucleic acid segments are not from the same gene or, if from the same gene, one or both of them are modified from the original forms. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA molecule. Thus, the terms refer to a nucleic acid segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous RNAs or polypeptides. A "homologous DNA molecule" is a DNA molecule that is naturally associated with a host cell into which it is introduced.

[0306] A "regulatory sequence" includes promoters, enhancers, and other expression control elements (e g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and / or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein or RNA desired, and the like. The expression vector can be introduced into host cells to produce an RNA or a polypeptide of interest. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes RNAs to be initiated at high frequency.

[0307] " Expression cassette" as used herein means a nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, which may include a promoter operably linked to the nucleotide sequence of interest that may be operably linked to termination signals. It also may include sequences required for proper translation of the nucleotide sequence. The coding region usually codes for an RNA or protein of interest. The expression cassette including the nucleotide sequence of interest may be chimeric. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of a regulatable promoter that initiates transcription only when the host cell is exposed to some86182281269.1UR 6-24056 / FR: 161118.06801 particular stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.

[0308] A vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The vector may or may not be capable of autonomous replication or integrated into a host DNA. Examples of the vector include a plasmid, cosmid, or viral vector. The vector includes a nucleic acid in a form suitable for expression of a nucleic acid of interest in a host cell. Preferably the vector includes one or more regulators’ sequences operatively linked to the nucleic acid sequence to be expressed.

[0309] As used herein, the term “therapeutically effective amount” refers to an amount that produces the desired therapeutic effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.

[0310] As used herein, the term "stem cells" refers to cells with the ability to both replace themselves and to differentiate into more specialized cells. Their self-renewal capacity generally endures for the lifespan of the organism. A pluripotent stem cell can give rise to all the various cell types of the body. A multipotent stem cell can give rise to a limited subset of cell types. For example, a hematopoietic stem cell can give rise to the various types of cells found in blood, but not to other types of cells. Multipotent stem cells can also be referred to as somatic stem cells, tissue stem cells, lineage-specific stem cells, and adult stem cells. The nonstem cell progeny of multipotent stem cells are progenitor cells (also referred to as restricted-progenitor cells). Progenitor cells give rise to fully differentiated cells, but a more restricted set of cell types than stem cells. Progenitor cells also have comparatively limited self-renewal capacity; as they divide and differentiate, they are eventually exhausted and replaced by new progenitor cells derived from their upstream multipotent stem cell.

[0311] As used herein, "therapeutic cells" refers to a cell population that ameliorates a condition, disease, and / or injury in a patient. Therapeutic cells may be autologous (i.e., derived 87182281269.1UR 6-24056 / FR: 161118.06801 from the patient), allogeneic (i.e., derived from an individual of the same species that is different from the patient) or xenogeneic (z. e., derived from a different species than the patient). Therapeutic cells may be homogenous (i.e., consisting of a single cell type) or heterogeneous (i.e., consisting of multiple cell types). The term "therapeutic cell" includes both therapeutically active cells as well as progenitor cells capable of differentiating into a therapeutically active cell.EXAMPLESExample 1

[0312] This example describes material and methods used in the other examples below.

[0313] Design of GPR17-GFAP Dual Promoter: To create a promoter that allows for specific expression in both oligodendrocytes and astrocytes, the oligodendrocyte-specific GPR17 promoter was cloned upstream of an MVM intron (Gray et al., Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous systems using self-complementary vectors. Hum Gene Ther. 22(9), 1143-53 (2011) into which the astrocyte-specific minimal GFAP (ABC1D) promoter (Lee et al., GFAP promoter elements required for region-specific and astrocyte-specific expression. Glia 56, 481-493 (2008).) was inserted to allow for alternative splicing with expression from either GPR17 or GFAP. The GFAP (ABC ID) promoter was obtained from constructs described in Benraiss et al., Cell-intrinsic glial pathology is conserved across human and murine models of Huntington’s disease. Cell Reports. 36(1), 109308 (2011). The 2400 bp upstream of the GPR17 gene NM_001161417.2 was cloned out of HEK293 genomic DNA, purified, and used to clone the 2197 bp upstream of the GPR17 gene from 5’-GGATACGGAAGAGATCCAATCGCAG-3’ (SEQ ID NO: 55) to 5 -GCCCTTGCCGCTGTGGGTG-3’ (SEQ ID NO: 56) that was used as the promoter. The dual promoter is cloned upstream of EGFP-MIR124T in lentivirus vector. The sequence 1 below depicts the expression cassette as cloned into the lentivirus.

[0314] Cre Recombinase To allow for the cloning of the cassette containing both complement lox sites and cre recombinase in the same construct with self-excision in bacteria, a 92 bp corresponding to the human GAPDH (NM 001289745.3) intron 6 (5’CTGCAAAGAAAGAGGGAGCGGGGCGCAAGTCAGGGGAGCGTGTCCATAGGGT GCCAGGCTGGCCGCTTCTCCACGGGCCCTGCCTTCCTCAC-3 ) (SEQ ID NO: 57) was inserted between amino acids Q133 and A134. Functional intron splicing was confirmed using 88182281269.1UR 6-24056 / FR: 161118.06801 a constitutive CAG promoter driving the cre and a lox flip switch EGFP expression cassette in HEK293 cells.

[0315] All-in-one Cre and lox construct To establish conditional and simultaneous immuno-cloaking in astrocytes and oligodendrocytes, we modified an AAVS1 insertion vector, pAAVSl-P-CAG-EGFP (Addgene plasmid no. 80492), in which EGFP was replaced with an overexpression cassette. In the 5’ to 3‘ direction, this cassette consists of a loxP and sequence-optimized B2M in tandem with self-cleavage peptide and fluorescent reporter tRFP followed by a lox2272 and bGH-PolyA signal. Directly facing the bGH-PolyA signal in the 3’ to 5’ direction is the dual astrocyte / oligodendrocyte-specific GPR17 / GFAP promoter. After this promoter, a Cre recombinase containing a GAPDH intron to allow for bacterial cloning and two C-terminal HA tags was connected to an SV40PA followed by a loxP. EGFP-FLAG, and an hGH-PolyA sequence. Two additional constructs were made by substituting the EGFP-FLAG for a CD47-323-P2A-EGFP-FLAG or a truncated CD47-293-P2A-EGFP-FLAG. The sequence 2 below depicts the expression cassette as cloned into the AAVS1 integration vector.

[0316] A truncated CD47 XM 005247909.3 isoform, CD47-293 amino acids, missing its C-terminal domain was generated by truncation of optimized full-length CD47-323 amino acids and replaced into the above cassette.

[0317] Viral construct and injection for validating the specificity of the dual promoter for targeting Glial progenitors / astrocytes we generated pTANK-GPR17-EGFP-WPRE and pTANK-GPR17 / GFAP-EGFP-WPRE. Viral particles pseudotyped w ith Vesicular Stomatitis Virus G glycoprotein were produced. One microliter of viral suspension was injected into the striatum of ten-week-old C57BL6 / J mice bilaterally in the following coordinates from the bregma: coordinates: +1.1 mm anterior / posterior, ±1.5 mm medial / lateral, -2.3 mm dorsal / ventral from the dura.

[0318] Histology Animals were killed using sodium pentobarbital and perfused transcardially with saline followed by 4% paraformaldehyde, and their brains were processed for immunocytochemistry. Sagittal sections (20 pm) spanning corpus callosum were processed for immunostaining with anti-EGFP (Chicken anti-Anti-GFP; Rockland), in combination with anti-01ig2 (Goat anti-olig2; R& D Systems), anti-Ng2 (Rabbit anti-NG2; Millipore), anti-GFAP (Mouse monoclonal anti-human GFAP; Covance Research), ALDH1L1 (Abeam, AB56777), NeuN (Invitrogen, ABN78MI), Ng2 (Millipore, AB5320), Sox9 (Abeam, abl84547), or PDGFRa (Cell Signaling, 3471T).89182281269.1UR 6-24056 / FR: 161118.06801 Sequence 1 / Construct 1 (SEQ ID NO: 58):< GP / ?77>< SD>intron< GFAP>intron< SD>< SA>< EGFP><m / f?7247>< WPRE> GGATACGGAAGAGATCCAATCGCAGACCCAAGATCCCCACCCAGGTTATGGTGGGCAGACCCCAGATGCCAGGGC CACCCATTCAGCATCCCTCCCTGGACCCCAGGACCTGCTACTGCTGGGTGTCTGGACTCCATCCTGCACAGCACT GTGCTCCATCTGCCCTGGGGTGTCTCATCATCAGCTGTGTGCAGGGC AGGGGCCC AAC AAGGCCCAGCAGTC ACTGGCTAAGCTGCCGACTGGCTCTCTGTGCCTCCCCAAGACCCTATGTGCCCAGCAGGGGGCAACAGCTCAGGG TCAGCTGACCGAATGCCTCGGTGAATGAATGACTCTACAAGAGAGGAAGGGAGCCTCGGTGGGCATCATCTCCCC TCGACTACTGGCCAGAGCCCTGGCTCTTACACCCCAGCGACGGGAAGCAGTTGTGGCCTGTGGCTTCAGTCTTCA TCACCACAATCCCTGAAGCCCACCCTTGCCCAGACACC GTGCCCCAGCCCCAACCCCAGGCCACC CC CAGCA GGTCTGGGGCTGAGCTGCCCCACCTGGCGCCTATGGCGGCCAGCCCATGCCCCCTGCGGTGCCTCTGTCCCAGAC TCAGCATGTAGGCCCCATGACCCCACTCCACATTCTGGTGACTCCTCCTGAGCGTCAGGACAACACTCAACCCAC GAGGAATTATTTCTGTCTCAAAGATGCAGGAATCAGCTCAACGCCTCAAAACTCCATCACCACGGTCAATGCCCT TGAAGCCATCGACAGTGATCACCCCAATAACAGAAGGTCTGTGAGCCCAGAAATGCCCTGCTCAGGGTGGTTAGC TTCAAGCCACCACCTTTCCAACCAGCCTGGGCCAGTTCTTCCAGACAGCCGCCTGCGGGCACAACAGGAAAGAGA CCTGCGCCCCGGCTCAGACACCTCACACCCAGCTGGCTCTCAGGCCAGACAAACTGGGAAGCCCATCTCTCTTGA AGGAAGTCCAGATGGGAAACAGCTTCTCAACAGACCAGATCACAGCATCAGATCTAAAGGTGGCCTTCAGAATTC TTTTTCAGGTTGAATTAGGATCAAATCTAAGAATTCTAAATTCAAAATGCAGCAGAAAAACAAAACACACACACA CACGGAGCCTAAGTTCTGGAGTGACATGTGCTTGGGTTCAAATCCTGGCTCTGTTGCTTCCTACTGTTTGTTGAT GGGTGAGTTTCTTCATTTGCCTGAGCCTCAGTTTCCTTGTCTGTAAAATGGGGCAATAATCCCAGCTGCACAGGG TGATGTGAAGAGACAAATTTAAGACACTGCCCCTTA ATGCTAGCCACATACATACAGTTTTCAATGTTTAAACA ACAAAATGTAAAGTCTTTTGAAACCAGGAAGGGTGATTTGGTTTCCCATGTTGCTGGATGTATCATTTTCAGAAA GACAGAGAGAAATGAACTTTGTTCACTCAGTCTCAGAGGCGGCCGCCGGCAGCATTCAAAGGCACCCCAGCCCGG AGCCACCCCAGGGAGGAGCCCCAGGCCAGCGGTCAGATTCATGGGCTTCCGTGCAGAAGGGGAGCTGCACCGGCG AGCACCCGGCCTCTGAGCTGAGCCGCATCCTCACGGACAGGACAGCGCCCCATTATGAGGCTCCTGCAGCTGTTC CTCGCTCCAGATAAAGGCCATGATTTATTCTGTGTGCCCAAATGGGGCCTCATTATACAGGGCAGGACACAAGGA CCCTACAGCAAGTGTCCTCAAAGAGTCGCCTCTCACTCCGTGAGCAAGACTCCTCGGCCTCCCACCCTCCGTTCA CAGGCCCCCTCCGCCGTCTGCGGGCGCAGGCCTGGGAGCGCCGCCTGTTGCCATGACAGCCGGCCCCTCCCTGCC CCCCATCAGTAGGAAATCATCCCCTTCTGAAACGTCCTGTTGTGTCCCTCAGCTCCAGCCCAAGCCCCCCACCCA GCCCCCGCCTGCTCTGAGTCTCTGAGACAGTCACACACTCAGACTATGTGGCCAAGCTGGGGGCGGGGGGCATGG GCTAGGGACACACTAGAATATTCACGCTCCGGTGGCAGCAGCAGCAGCAGCCAGAGGAGCAGCCCGACACAACAA GGGACCCCTCAGGAATGAAGCAGCCTTTCAGGGCCAGAGGGGCTGTGGTCTCCCTTCCTCTCCTTAAATAGCCAG CGTTCCACCCACAGCGGCAAGGGCg-cgccgg-aGTcgctgcgcgctgccttcgccccgtgccccgctccgccgtcg ACATATCCTGGTGTGGAGTAGGGGACGCTGCTCTGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGGG AGGAGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGCCCCCCAGGGCCTCCTCT TCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTCGGGGTGGGCACAGTGCCTGCTTCCCGCCGCAC CCCAGCCCCCCTCAAATGCCTTCCGAGAAGCCCATTGAGCAGGGGGCTTGCATTGCACCCCAGCCTGACAGCCTG GCATCTTGGGATAAAAGCAGCACAGCCCCCTAGGGGCTGCCCTTGCTGTGTGGCGCCACCGGCGGTGGAGAACAA GGCTCTATTCAGCCTGTGCCCAGGAAAGGGGATCAGGGGATGCCCAGGCATGGACAGTGGGTGGCAGGGGGGGAG AGGAGGGCTGTCTGCTTCCCAGAAGTCCAAGGACACAAATGGGTGAGGGGAGAGCTCTCCCCATAGCTGGGCTGC GGCCCAACCCCACCCCCTCAGGCTATGCCAGGGGGTGTTGCCAGGGGCACCCGGGCATCGCCAGTCTAGCCCACT CCTTCATAAAGCCCTCGCATCCCAGGAGCGAGCAGAGCCAGAGCAGGTTGGAGAGGAGACGCATCACCTCCGCTG CTCGCGGTcgacccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcggg acggcccttctcctccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtat taatgtttaattacctggagcacctgcctgaaatcactttttttcAGGGCGCGCCCCACCGGTCGCCACCATGGT GAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAA GTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGG CAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAA GGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAA GGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTA TATCATGGCCGACAAGCAG AGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGT GCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCT GAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGC CGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAc tatgctcgagattaagccgaatt caatggcatt caccgcgtgcct taaacggatc ct ggtcgagggaatt cc gat aTCAACCTCTGGATTACAAAATTTGTGAAAGA TTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCT ATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGG CCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACC90182281269.1UR 6-24056 / FR: 161118.06801 ACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTT GCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTT CCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAAT CCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACG AGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGGGSequence 2 / Construct2 (SEQID NO: 59):< CAG-Promo terXLOXPXB 2M-0ptimiz edXT2AX tRFPXLOX2272 X BGH POLY (A) SIGNALXHGH POLY (A) SIGNAL><egfpXP2 AXCD47 XLQXPXSV40 poly (a) signalxCre- C' TERMINALXGAPDH-INTRONXCre-N' TERMINAL>< LOX2272Xmvm intronXGFAP PROMOTERXmvm intronXGPRll PROMOTER>GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCA TATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGA CCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCC ATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTAT CATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGC CCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTA TTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCA CCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGG GGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAG GTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGG CGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCG CCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACT CCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAAT GACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTG TGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGG CTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCA GTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGG AACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGT CGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTG CGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTG GGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGG CCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAAT CGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGG CGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAAT GGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGG GCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGG CGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACA GCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCATAACTT CGTATAGCATACATTATACGAAGTTAT C G G T A C C a t g a g c a g g a g c g t g g c c c t g g c c g t g c tggccctgctgagcctgagcggcctggaggccatccagag acccccaagatcca gtctac agcaggcaccccgccgagaacggcaaga caacttcctgaact ctacgtgagcggcttcca ccccagcgacatcgaggtggacctgctgaagaacggcgagaggatcgagaaggtggagcaca gcgacctgagcttcagcaaggactggagcttctacctgctgtactacaccgagttcaccccc accgagaaggacgagtacgcctgcagggtgaaccacgtgaccctgagccagccca gatcgt g a a g t g g g a c a g g g a c a t g G G AA G C G GAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGATG 91182281269.1UR 6-24056 / FR: 161118.06801 T GGAG G AGAAT C CT GGAC C TATGAGCGAGCTGATCAAGGAGAACATGCACATGAAGCTGTAC ATGGAGGGCACCGTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTA CGAGGGCACCCAGACCATGAAGATCAAGGTGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCG ACATCCTGGCTACCAGCTTCATGTACGGCAGCAAAGCCTTCATCAACCACACCCAGGGCATC CCCGACTTCTTTAAGCAGTCCTTCCCTGAGGGCTTCACATGGGAGAGAATCACCACATACGA AGACGGGGGCGTGCTGACCGCTACCCAGGACACCAGCTTCCAGAACGGCTGCATCATCTACA ACGTCAAGATCAACGGGGTGAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAAAACACGC GGCTGGGAGGCCAACACCGAGATGCTGTACCCCGCTGACGGCGGCCTGAGAGGCCACAGCCA GATGGCCCTGAAGCTCGTGGGCGGGGGCTACCTGCACTGCTCCTTCAAGACCACATACAGAT CCAAGAAACCCGCTAAGAACCTCAAGATGCCCGGCTTCCACTTCGTGGACCACAGACTGGAA AGAATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAGCACGAGATGGCTGTGGCCAAGTA CTGCGACCTCCCTAGCAAACTGGGGCACAGATAAGGTACCGATACTGACTACGACCCTGACC TCATAACTTCGTATAGGATACTTTATACGAAGTTATCTGTGCCTTCTAGTTGCCAGCCATCT GTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTC CTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCG GTGGGCTCTATGGACGCGTGACAGTGGTCGCAACACAAGCAGGACAGGGAAGGGAGCAGTGG TTCACGCCTGTAATCCCAGCAATTTGGGAGGCCAAGGTGGGTAGATCACCTGAGATTAGGAG TTGGAGACCAGCCTGGCCAATATGGTGAAACCCCGTCTCTACCAAAAAAACAAAAATTAGCT GAGCCTGGTCATGCATGCCTGGAATCCCAACAACTCGGGAGGCTGAGGCAGGAGAATCGCTT GAACCCAGGAGGCGGAGATTGCAGTGAGCCAAGATTGTGCCACTGCACTCCAGCTTGGTTCC CAATAGACCCCGCAGGCCCTACAGGTTGTCTTCCCAACTTGCCCCTTGCTCCATACCACCCC CCTCCACCCCATAATATTATAGAAGGACACCTAGTCAGACAAAATGATGCAACTTAATTTTA TTAGGACAAGGCTGGTGGGCACTGGAGTGGCAACTTCCAGGGCCAGGAGAGGCACTGGGGAG GGGTCACAGGGATGCCACCCGCCTGCAGGT C ACT T GT C GT C AT C GT CT T T GT AGT C c 11 g t a cagctcgtccatgccgagagtgatcccggcggcggtcacgaactcca caggaccatgtgat cgcgcttctcgttggggtctttgctcagggcggactgggtgctcaggtagtggttgtcgggc agcagcacggggccgtcgccgatgggggtgttct ctggtagtggtcggcgagctgcacgct gccgtcctcgatgttgtggcggatcttgaagttcaccttgatgccgttcttctgcttgtcgg ccatgatatagacgttgtggctgttgtagttgtactccagcttgtgccccaggatgttgccg tcctccttgaagtcgatgcccttcagctcgatgcggttcaccagggtgtcgccctcgaactt cacctcggcgcgggtcttgtagttgccgtcgtccttgaagaagatggtgcgctcctggacgt agccttcgggcatggcggacttgaagaagtcgtgctgcttcatgtggtcggggtagcggctg aagcactgcacgccgtaggtcagggtggtcacgagggtgggccagggcacgggcagcttgcc ggtggtgcagatgaacttcagggtcagcttgccgtaggtggcatcgccctcgccctcgccgg acacgctgaacttgtggccgtttacgtcgccgtccagctcgaccaggatgggcaccaccccg g t a a c a c t c c t c g c c c t t c t c a c c a t CGGTCCAGGGTTCTCCTCCACGTCTCCAGCCTG CTTCAGCAGGCTGAAGTTAGTAGCT C C G CT AC C GGTTTCATCATTCATCATTCCTTTTGATT CTTTGAATGCATTAAGGGGTTCCTCTACAGCTTTCCTAGGAGGTTGTATAGTCTTCTGATTG GAAGCCACAAATTTCATATAAACTAATCCAAGTAATTGTGCTAGAGCTAAGATACTCAAACC TGAAATCAGAAGAGGGCCATGCATTGGTATACACGCCGCAATACAGAGACTCAGTCCAACCA CAGCGAGGATATAGGCTATCACCTGAATAACCAATATGGCAATGACGAAGGAGGTTAATCCA ATCGCTGTACTAAACACATAGTAGTGAAGTAATATTAATATCCCTGTAGAAGTCACAATTAA ACCAAGGCCAGTAGCATTCTTTAATGAATATTCACCTGGGACGAAAAGAATGGCTCCAACAA TGACAATGACAGTGATCACAAGTCCAGCAACAAGTAAAGCAATTGTTTTCTCATCCATACCA CCGGATCTATATTTAAGTGTTTTAATACCAAACTGTCCCCAGAACAGGAGTATAGCAAAAAT TGGGAAAATAACAATAAGAATATTTTCATTTGGAGAAAACCATGAAACAACACGATATTTTA GCTCGATGATCGTTTCACCTTCTCTGGTTAATTCTGTTACTTCACAAGTGTAGTTTCCTGTG TGTGAGACAGCATCACTCTTATCCATCTTCAAAGAGGCATCTCCTTTTAGTAATTGTGAGAC TTCAATTTTTGCACTACTAAAGTCAGTGGGGACAGTGGACTTGTTTAGAGCTCCATCAAAGG92182281269.1UR 6-24056 / FR: 161118.06801 TGTAAATATCTCTTCCTTTAAATTTCCACTTTACGTATACTTCAGTAGTGTTTTGTGCCTCC ATATTAGTAACAAAGCATGGAATGACGACAGTGTCATTACAAAACGTGAACTCTACAGATTT TGTTTTATTAAATAGTAGCTGAGCTGATCCGCAGCACGCCGAGCCCAGCAACAGCGCCGCTA CCAGGGGCCACATGGACCGGTATAACTTCGTATAATGTATGCTATACGAAGTTATacttqtt tattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagCAC TCTGGTATGACAAGCCCTGCGGATCCTCAAGCGTAATCGGGAACATCGTAGGGATAAGCGTA AT CT G GAAC AT C GT AAGGAT AATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGTTTCAC TATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAGCTTGCATG ATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCGCGGCTCCGACACGGGCACTGTG TCCAGACCAGGCCAGGTATCTCTGACCAGAGTCATCCTTAGCGCCGTAAATCAATCGATGAG TTGCTTCAAAAATCCCTTCCAGGGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCA ACACCATTTTTTCTGACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGAC GGAAATCCATCGCTCGACCAGTTTAGTTACCCCCAGGCTAAGTGCCTTCTCTACACCTGCGG TGCTAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAG ATATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCCC CAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGAGTGAACGAACCT GGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGCAAAGAAAGAGGGAGCGGGGCGCAAGTC AGGGGAGCGTGTCCATAGGGTGCCAGGCTGGCCGCTTCTCCACGGGCCCTGCCTTCCTCACC TGTTTTGCACGTTCACCGGCATCAACGTTTTCTTTTCGGATGCGCCGCATAACCAGTGAAAC AGCATTGCTGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCA AATGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCCTGAAGATATAGAAGATAATCGCGA ACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCACGA CCGGCAAACGGACTGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTGAACA TGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGACAGGTAATGCAGGCAAATTT TGGTGTACGGTCAGTAAATTGGACACCTTCCTCTTCTTCTTGGGCATG G T G GATAACTTCGT ATAAAGTATCCTATACGAAGTTATGAAT TCGGCGCGCCctgaaaaaaagtgatttcaggcag gtgctccaggtaa ttaaacattaataccccaccaaccaacca tcccttaaacccttacctct tgctcagctaat tacagcccggagga gaagggccgtcccgcccgctcacctgtgggagtaac gcggtcagtcagagccggggcgggcggcgcgaggcgggtcgaCCGCGAGCAGCGGAGGTGAT GCGTCTCCTCTCCAACCTGCTCTGGCTCTGCTCGCTCCTGGGATGCGAGGGCTTTATGAAGG AGTGGGCTAGACTGGCGATGCCCGGGTGCCCCTGGCAACACCCCCTGGCATAGCCTGAGGGG GTGGGGTTGGGCCGCAGCCCAGCTATGGGGAGAGCTCTCCCCTCACCCATTTGTGTCCTTGG ACTTCTGGGAAGCAGACAGCCCTCCTCTCCCCCCCTGCCACCCACTGTCCATGCCTGGGCAT CCCCTGATCCCCTTTCCTGGGCACAGGCTGAATAGAGCCTTGTTCTCCACCGCCGGTGGCGC CACACAGCAAGGGCAGCCCCTAGGGGGCTGTGCTGCTTTTATCCCAAGATGCCAGGCTGTCA GGCTGGGGTGCAATGCAAGCCCCCTGCTCAATGGGCTTCTCGGAAGGCATTTGAGGGGGGCT GGGGTGCGGCGGGAAGCAGGCACTGTGCCCACCCCGAACATTGTGTCTGTGCCAAGGTGAGT CATTCACTGGGCATGAAGAGGAGGCCCTGGGGGGCGGCCAGCCCAATGCTGCCAGGTCTGCT TGCAGACAAGGCCTGGCTGTCTGCCTCCTCCCCAGCTCACAGAGCCAGCTCAGGCCCCCGAG CCTCTGTCAGAGCAGCGTCCCCTACTCCACACCAGGATATGTcga cggcgga gcggggca eg gggegaaggea gegegea gega c tccGGCGCGCCCTTGCCGCTGTGGGTGGAACGCTGGCTA TTTAAGGAGAGGAAGGGAGACCACAGCCCCTCTGGCCCTGAAAGGCTGCTTCATTCCTGAGG GGTCCCTTGTTGTGTCGGGCTGCTCCTCTGGCTGCTGCTGCTGCTGCCACCGGAGCGTGAAT ATTCTAGTGTGTCCCTAGCCCATGCCCCCCGCCCCCAGCTTGGCCACATAGTCTGAGTGTGT GACTGTCTCAGAGACTCAGAGCAGGCGGGGGCTGGGTGGGGGGCTTGGGCTGGAGCTGAGGG ACACAACAGGACGTTTCAGAAGGGGATGATTTCCTACTGATGGGGGGCAGGGAGGGGCCGGC TGTCATGGCAACAGGCGGCGCTCCCAGGCCTGCGCCCGCAGACGGCGGAGGGGGCCTGTGAA CGGAGGGTGGGAGGCCGAGGAGTCTTGCTCACGGAGTGAGAGGCGACTCTTTGAGGACACTT GCTGTAGGGTCCTTGTGTCCTGCCCTGTATAATGAGGCCCCATTTGGGCACACAGAATAAAT CATGGCCTTTATCTGGAGCGAGGAACAGCTGCAGGAGCCTCATAATGGGGCGCTGTCCTGTC93182281269.1UR 6-24056 / FR: 161118.06801 CGTGAGGATGCGGCTCAGCTCAGAGGCCGGGTGCTCGCCGGTGCAGCTCCCCTTCTGCACGG AAGCCCATGAATCTGACCGCTGGCCTGGGGCTCCTCCCTGGGGTGGCTCCGGGCTGGGGTGC CTTTGAATGCTGCCGGCGGCCGCCTCTGAGACTGAGTGAACAAAGTTCATTTCTCTCTGTCT TTCTGAAAATGATACATCCAGCAACATGGGAAACCAAATCACCCTTCCTGGTTTCAAAAGAC TTTACATTTTGTTGTTTAAACATTGAAAACTGTATGTATGTGGCTAGCATTTAAGGGGCAGT GTCTTAAATTTGTCTCTTCACATCACCCTGTGCAGCTGGGATTATTGCCCCATTTTACAGAC AAGGAAACTGAGGCTCAGGCAAATGAAGAAACTCACCCATCAACAAACAGTAGGAAGCAACA GAGCCAGGATTTGAACCCAAGCACATGTCACTCCAGAACTTAGGCTCCGTGTGTGTGTGTGT TTTGTTTTTCTGCTGCATTTTGAATTTAGAATTCTTAGATTTGATCCTAATTCAACCTGAAA AAGAATTCTGAAGGCCACCTTTAGATCTGATGCTGTGATCTGGTCTGTTGAGAAGCTGTTTC CCATCTGGACTTCCTTCAAGAGAGATGGGCTTCCCAGTTTGTCTGGCCTGAGAGCCAGCTGG GTGTGAGGTGTCTGAGCCGGGGCGCAGGTCTCTTTCCTGTTGTGCCCGCAGGCGGCTGTCTG GAAGAACTGGCCCAGGCTGGTTGGAAAGGTGGTGGCTTGAAGCTAACCACCCTGAGCAGGGC ATTTCTGGGCTCACAGACCTTCTGTTATTGGGGTGATCACTGTCGATGGCTTCAAGGGCATT GACCGTGGTGATGGAGTTTTGAGGCGTTGAGCTGATTCCTGCATCTTTGAGACAGAAATAAT TCCTCGTGGGTTGAGTGTTGTCCTGACGCTCAGGAGGAGTCACCAGAATGTGGAGTGGGGTC ATGGGGCCTACATGCTGAGTCTGGGACAGAGGCACCGCAGGGGGCATGGGCTGGCCGCCATA GGCGCCAGGTGGGGCAGCTCAGCCCCAGACCTGCTGAGGAGGTGGCCTGGGGTTGGGGCTGG GGCACAGGTGTCTGGGCAAGGGTGGGCTTCAGGGATTGTGGTGATGAAGACTGAAGCCACAG GCCACAACTGCTTCCCGTCGCTGGGGTGTAAGAGCCAGGGCTCTGGCCAGTAGTCGAGGGGA GATGATGCCCACCGAGGCTCCCTTCCTCTCTTGTAGAGTCATTCATTCACCGAGGCATTCGG TCAGCTGACCCTGAGCTGTTGCCCCCTGCTGGGCACATAGGGTCTTGGGGAGGCACAGAGAG CCAGTCGGCAGCTTAGCCAGTGACTGCTGGGCCTTTGTTTGGGCCCCTTGCCCTGCACACAG CTGATGATGAGACACCCCAGGGCAGATGGAGCACAGTGCTGTGCAGGATGGAGTCCAGACAC CCAGCAGTAGCAGGTCCTGGGGTCCAGGGAGGGATGCTGAATGGGTGGCCCTGGCATCTGGG GTCTGCCCACCATAACCTGGGTGGGGATCTTGGGTCTGCGATTGGATCTCTTCCGTATCCExample 2

[0319] This example shows validation of the self-excision and recombination of full knockin approach described herein. A cassette similar to that shown in Fig. 3 was made in the manner described above. This cassette is shown in Fig 3, except the first promoter and the second promoter were replaced with a TRE promoter. In addition, an intro sequence was inserted into the Cre-coding sequence so that the Cre recombinase could express in a eukaryotic cell but not in a prokaryotic cell. Lentivirus particles containing this cassette were generated and used to transduce 293FT cells in the manner described above.

[0320] As shown in Fig. 9, 293FT cells knockin clones transduced with rtTA-3G-expressing lentivirus were generated. In the absence of doxycycline, the Cre recombinase was not expressed, and the cassette did not undergo Cre-mediated site-specific recombination. As a result, the sequence encoding the red fluorescent protein (RFP) was transcribed under the constitutive CAG promoter leading to expression of the red fluorescent protein. In contrast, upon induction of the TRE promoter with doxycycline (Dox+), the Cre recombinase was expressed and mediated the site-specific recombination. As a result of the recombination, the 94182281269.1UR 6-24056 / FR: 161118.06801 sequences encoding synB2M, RFP, and Cre were removed while the sequences encoding the green fluorescent protein (EGFP) and CD47 were flipped and operably lined to the constitutive CAG promoter. The green fluorescent protein was then expressed by the cells.

[0321] RT-PCT assays were carried out using primer pairs designed to anneal to a site in the CAG promoter and a site in CD47 coding sequence, respectively. The sites and primers sequences were designed in such a way that they produced an amplicon of a predetermined size only after the sequences encoding the EGFP) and CD47 were flipped and operably lined to the constitutive CAG promoter. As shown in Fig. 10, the amplicons were obtained in three clones after induction of the TRE promoter with doxycycline.

[0322] Fluorescent microscopy also confirmed the post-recombination expressions of EGFP and CD47. See Fig. 12.

[0323] These results demonstrates self-excision and recombination of the Knockin cassette at the genomic level.Example 3

[0324] A similar Knockin cassette encoding PD-L1 instead of CD47 was made and used to transduce 293FT cells in the manner described above. In the absence of induction of the TRE promoter with doxycycline (Dox+), only the expressions of the B2M protein and the tRFP were detected in the cells. In contrast, upon induction of the TRE promoter with doxycycline (Dox+) and subsequent expression of the Cre recombinase and Cre-mentioned site specific recombination, only the PD-L1 protein expression and EGEP expression were detected. These results are shown in Fig. 11, further validating gene expression following recombination.

[0325] The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present disclosure as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present disclosure as set forth in the claims. Such variations are not regarded as a departure from the scope of the disclosure, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entireties.95182281269.1UR 6-24056 / FR: 161118.06801 Sequence 3 / Construct 3 (Allele 1: GPR17_GFAP-Cre-WPRE w / Insulator) (SEQ ID NO: 60):< HALX5AXT2AXPuro><bGHpAXcHS4 InsulatorXSRF UCOEXGPR17 Pr omo t e r XHybr id_Intr on>< GFAP_Promo terXHybrid IntronXLox 71 > < Cre>< GAPDH IntronXCreXUA Tag sXWPREXbGH pAXSV40 pA> < Lox66XHA-R> CAGCAGGAGAATCATGCCAATGGGCCAATATACATTCTGACCCACAGTTTCATAATAAAATA AAATGGTTGTGGTTGTAAGCCACTATGTTTCAGAGTGGTTTGTTACACAGCAATAAATAACT AATATAGTAGGCATACCATCAAGTCCAAAGTAGGTAGAGAAGAATGTAAATAGCAGAGCAAA ACAGCATGACTGGTGGCTGGGAGGCTTAAAACTGGGACAGGATCAGAGTCATGAAAGAAGTC AAAGAAATGGTTCAGAAGTAAGGCTGAGACTGACTTACAAAAGCTGAAAGTCCCTTTAAGTT GGTGTTTGGTGCATTGGCAGGGGCAGGTATGGTGACTTAAAAGAGCCATGCTCAACAAGATC AAGCACAACACAATCACGGGTCACCCCAGCAGACCTTAGCGAGTCTAGCCATTTCTTTGGTG GTGGTCACAGTCATGCTTCAGCCCAGTTTCCACTTGGACAAATGGTACATATTTTCAATGAG ATGAAAATTAAGATACAATCCATGTGCTCAGAGAGTGATCACAGCTCTGACTAAACACTGTG CCCCAAAGTGTTGAGGAATTGGGAAAACCTAGCTGAGTTAGTGGTCTGTTTTCTGTTACAAT AAAGCTCATAATGAAAATTAGCCTTCTTTGTTCTTCCCCAAGTCTTTCTTTCTAGACGAAAC TACTTTCAACTGTTTTAACTTCCTTACTGTTAACTTCCATATTTCTAGATAGTATGGTCAGA CTGTTATTTCTTGACTTTTAAATGTAAGATATTATCTACTGACTTCCTTCTATGTAAGATGA GGATTGAGCTCTCTTACCCTTCactagaagcttCTGACCTCTTCTCTTCCTCCCACAGGgcc tcgagagatctggcagcgg aGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAG AATCCCGGCCCTa g g c t c g a gATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGA CGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCC ACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACG CGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTG GACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCG AGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGG CCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGG TCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCT TCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACC GCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTG ACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCT GGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGA GTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAA GACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGgctagcgATTCTCACTGACTCC GTCCTGGAGTTGGATGAGAGATAATGGCCTTACGTTGTGCCAGGGGAGGGTCGGGCTGGATT TAGCAAGATTTACCTTCTCCAAAGAGCGGTGCTGCAGTGGCACAGCTGCCCACGGAGGTGGG GGGGTCACCGTCCCTGGAGGTGATGAAGAACTGTGGGGATGTGGCACTGAGGGACATGGCCA GTGGGCACGGTGGGTGGGTTGGGGTTGGTCTTGGGGATCTTGGAGGGCTTTTCCAGCCTTCA TGATTTGACGATTGTATGAACATCTACATGGCAATTCTCCAGCTGCCTGTCCCAGTCCTACT GACCCAGCTGTATCTCTCCAGGCAAGCTCTTCCACCCCTTCTGCTTGCATCCAGACACCATC AAACATGCAGGCTCAGACACAGGGACCAGCAGTGTCTGTGGCCTTTTTGTGCTCCTCTCCAT GCTGGGTTTTAACTTGCTCTTTGTCCTTCTATCCTATCTTCTTATCCTTAAGGCTGTTCTGA ACGCTGTGACTTGGAGAGTGTCCCAGAGCCCTCAACACCTGCATGTCCCACGTCCATGCTGT CCTGCACTTCCTTATCCCCAAGATCTGCCTCTCCGTGATGCACTGAATTGGCAAACATGTGT96182281269.1UR 6-24056 / FR: 161118.06801 CACCCCAGACCAACAATGTCACAGCAAACTCCCCCTTGATAGGACAAGGGGGAATGGCTTTA CACTGAGACAGGGGAGGTTTGGGTTGGATATGAGGAGGCAGTTTTTCCCCCAGAGGGTGGTG ACGCACTGAACAGGTTGCCCAAGGAGGCTGTGGATGCCCCATCCCTGCAGGCATTCAAGGCC AGGCTGGATGTGGCTCTGGGCAGCCTGGGCTGCTGGTTGATGACCCTGCACATAGCAGGGGG TTGGATCTGGATGAGCACTGTGCTCCTTTGCAACCCAGGCCGTTCTATGATTCTGTCATTCT AAATCTCTCTTTCAGCCTAAAGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAG AGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGTCC CCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGC TCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGC TGTCCCCGTGAGCTCCACGTGCCTAGGGCACACGACCACAATTCCACTGAAAGCATTTTAAT ACGGAACTTGTCACTCCCAGGGAGCCTCCGCTCAGCCGGCAGTTGGTTCATTTCAATCCCCA CGACAACCCTTCAAAGTGCAGGGCAGACAGCAGGTGGCTCTGCCCAGGCGCCTGGATCACAG CCCGGCCTGCAGCCCTCACCTGGGCGCGGGGAGACCCTGAGGACGCTCCTCCAGGCGGCGCT GGCCGGGGCCTGCGGACACGGACGGGCGGGCTGAGCTCCGGGACCCCTCCCCGCGCCCCGCA CCCCGCACCCCGCACCCCGCACCCCGCACCCGGCGCTCACCCGTCCCAGCCCCGCCGCCCGC AGCCCCAGCTGCAACGCAGCCACCGCCGCCATCGCACCCGGCCCCGCGGGCGCTTCCGGGAC GCAGGAGGCATCTGCATCCGGGGCGCCGCTGAGTCCCGCCCAGAGCCCCGCCCCCGGCTCCA GGTTCTGCGAGCGGCTTCCGCCGGGCTGCTCCGCGGGCGCGTCGGCCATGAGCGAGTTGCCG GGCGACGTGCGGGCGTTTCTGCGGGAGCACCCGAGCCTGCGGCTCCAGACGGACGCCCGCAA GGTTCGCAGCGCGGGAGGGGAACGGAGTGGCGGAGAAGGGCGCAGTTGGGATGAGGGGCTGA GGGGAGGGCAGGGGAGAGGAGAGGGCAGGGGAGAGGGGAGAGGGGAGAGCAGGAGAGAGGGG AAGGCAGGGGAGAGGGCGCGGCGGGATCAGGGGAGGAGAGGGAAGGGGGCGCGGCAGGAGGG GGCACCAGGGAGCGGAGCCCTGGCCCTCCTGACGTCCTGCCCGCCCACGCGTCCGCAGGTGA GGTGCATCCTGACAGGTCACGAGCTGCCCTGCCGCCTGCCGGAGCTCCAGGTCTACACCCGC GGCAAAAAGTACCAGCGGCTGGTCCGCGCCTCCCCGGCCTTCGACTATGCAGAGTTCGAGCC GCACATCGTGCCCAGCACCAAGAACCCGTAGGTGGTG GAT AC G G AAG AG AT C C AAT C G C AG A CCCAAGATCCCCACCCAGGTTATGGTGGGCAGACCCCAGATGCCAGGGCCACCCATTCAGCA TCCCTCCCTGGACCCCAGGACCTGCTACTGCTGGGTGTCTGGACTCCATCCTGCACAGCACT GTGCTCCATCTGCCCTGGGGTGTCTCATCATCAGCTGTGTGCAGGGCAAGGGGCCCAAACAA AGGCCCAGCAGTCACTGGCTAAGCTGCCGACTGGCTCTCTGTGCCTCCCCAAGACCCTATGT GCCCAGCAGGGGGCAACAGCTCAGGGTCAGCTGACCGAATGCCTCGGTGAATGAATGACTCT ACAAGAGAGGAAGGGAGCCTCGGTGGGCATCATCTCCCCTCGACTACTGGCCAGAGCCCTGG CTCTTACACCCCAGCGACGGGAAGCAGTTGTGGCCTGTGGCTTCAGTCTTCATCACCACAAT CCCTGAAGCCCACCCTTGCCCAGACACCTGTGCCCCAGCCCCAACCCCAGGCCACCTCCTCA GCAGGTCTGGGGCTGAGCTGCCCCACCTGGCGCCTATGGCGGCCAGCCCATGCCCCCTGCGG TGCCTCTGTCCCAGACTCAGCATGTAGGCCCCATGACCCCACTCCACATTCTGGTGACTCCT CCTGAGCGTCAGGACAACACTCAACCCACGAGGAATTATTTCTGTCTCAAAGATGCAGGAAT CAGCTCAACGCCTCAAAACTCCATCACCACGGTCAATGCCCTTGAAGCCATCGACAGTGATC ACCCCAATAACAGAAGGTCTGTGAGCCCAGAAATGCCCTGCTCAGGGTGGTTAGCTTCAAGC CACCACCTTTCCAACCAGCCTGGGCCAGTTCTTCCAGACAGCCGCCTGCGGGCACAACAGGA AAGAGACCTGCGCCCCGGCTCAGACACCTCACACCCAGCTGGCTCTCAGGCCAGACAAACTG GGAAGCCCATCTCTCTTGAAGGAAGTCCAGATGGGAAACAGCTTCTCAACAGACCAGATCAC AGCATCAGATCTAAAGGTGGCCTTCAGAATTCTTTTTCAGGTTGAATTAGGATCAAATCTAA GAATTCTAAATTCAAAATGCAGCAGAAAAACAAAACACACACACACACGGAGCCTAAGTTCT GGAGTGACATGTGCTTGGGTTCAAATCCTGGCTCTGTTGCTTCCTACTGTTTGTTGATGGGT GAGTTTCTTCATTTGCCTGAGCCTCAGTTTCCTTGTCTGTAAAATGGGGCAATAATCCCAGC TGCACAGGGTGATGTGAAGAGACAAATTTAAGACACTGCCCCTTAAATGCTAGCCACATACA97182281269.1UR 6-24056 / FR: 161118.06801 TACAGTTTTCAATGTTTAAACAACAAAATGTAAAGTCTTTTGAAACCAGGAAGGGTGATTTG GTTTCCCATGTTGCTGGATGTATCATTTTCAGAAAGACAGAGAGAAATGAACTTTGTTCACT CAGTCTCAGAGGCGGCCGCCGGCAGCATTCAAAGGCACCCCAGCCCGGAGCCACCCCAGGGA GGAGCCCCAGGCCAGCGGTCAGATTCATGGGCTTCCGTGCAGAAGGGGAGCTGCACCGGCGA GCACCCGGCCTCTGAGCTGAGCCGCATCCTCACGGACAGGACAGCGCCCCATTATGAGGCTC CTGCAGCTGTTCCTCGCTCCAGATAAAGGCCATGATTTATTCTGTGTGCCCAAATGGGGCCT CATTATACAGGGCAGGACACAAGGACCCTACAGCAAGTGTCCTCAAAGAGTCGCCTCTCACT CCGTGAGCAAGACTCCTCGGCCTCCCACCCTCCGTTCACAGGCCCCCTCCGCCGTCTGCGGG CGCAGGCCTGGGAGCGCCGCCTGTTGCCATGACAGCCGGCCCCTCCCTGCCCCCCATCAGTA GGAAATCATCCCCTTCTGAAACGTCCTGTTGTGTCCCTCAGCTCCAGCCCAAGCCCCCCACC CAGCCCCCGCCTGCTCTGAGTCTCTGAGACAGTCACACACTCAGACTATGTGGCCAAGCTGG GGGCGGGGGGCATGGGCTAGGGACACACTAGAATATTCACGCTCCGGTGGCAGCAGCAGCAG CAGCCAGAGGAGCAGCCCGACACAACAAGGGACCCCTCAGGAATGAAGCAGCCTTTCAGGGC CAGAGGGGCTGTGGTCTCCCTTCCTCTCCTTAAATAGCCAGCGTTCCACCCACAGCGGCAAG GGCgcgccGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGTCGACATATCC TGGTGTGGAGTAGGGGACGCTGCTCTGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGC TGGGGAGGAGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCG CCCCCCAGGGCCTCCTCTTCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTCG GGGTGGGCACAGTGCCTGCTTCCCGCCGCACCCCAGCCCCCCTCAAATGCCTTCCGAGAAGC CCATTGAGCAGGGGGCTTGCATTGCACCCCAGCCTGACAGCCTGGCATCTTGGGATAAAAGC AGCACAGCCCCCTAGGGGCTGCCCTTGCTGTGTGGCGCCACCGGCGGTGGAGAACAAGGCTC TATTCAGCCTGTGCCCAGGAAAGGGGATCAGGGGATGCCCAGGCATGGACAGTGGGTGGCAG GGGGGGAGAGGAGGGCTGTCTGCTTCCCAGAAGTCCAAGGACACAAATGGGTGAGGGGAGAG CTCTCCCCATAGCTGGGCTGCGGCCCAACCCCACCCCCTCAGGCTATGCCAGGGGGTGTTGC CAGGGGCACCCGGGCATCGCCAGTCTAGCCCACTCCTTCATAAAGCCCTCGCATCCCAGGAG CGAGCAGAGCCAGAGCAGGTTGGAGAGGAGACGCATCACCTCCGCTGCTCGCGGTCGACCCG CCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGAC GGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGT TGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGACTA G TACCGTTCGTATAGCATACATTATACGAAGTTATcGGCGCGCCkCCATGCCCAAGAAGAA GAGGAAGGTGTCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCTGTCGATGCAA CGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAG CATACCTGGAAAATGCTTCAGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAATAA CCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGC GCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGG TCCGGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGCATCCGAAA AGAAAACGTTGATGCCGGTGAACGTGCAAAACAGgt ga ggaaggca gggcccgtgga gaagc ggccagcctggcacccta tggacacgctcccctgact tgcgccccgctccctct t tct t tgc agGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGAT CGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTAT AGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAA TCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTG GGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAA CTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTAT CAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGAT GACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGA TATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAA98182281269.1UR 6-24056 / FR: 161118.06801 ATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTG GAAGATGGCGATTATCCTTACGATGTTCCAGATTACGCTTATCCCTACGATGTTCCCGATTA CGCTTGAg g c g c g GATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTC TTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCT ATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTA TGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAA CCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCC CTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCG GCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGC TCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTC AATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCG CCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGGccGACGCACGGTCTACCCTG GTCCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTG CCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGC ATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGG GGGAGGATTGGGAAGACAA TAGCAGGCA TGCTGGGGA TGCGG TGGGCTCTA PGGGGT AC CAC TTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAA AGCACTCTGGTATGACAAGCCCTGCaccggtatttttttTACCGTTCGTATAATGTATGCTA TACGAAGTTATggcatcggagcctcactacgcgtgaattcccctcgagttattaaTTCCTCA TCCTTCCAACATAAATATATTTTGGGATTATATCAACATTCAATGTTACTTAAAGTGACCTT GTAAATATTTTCACAACTGAGCCATGTTTGATTTGTATACTTATGTTTACTTTACTGTTTTT CCTGAAGTTAATAATTGCCTTGAATTTATTTATTTCTTTAAAAATGTTTCATTACTCAGGAC TGTAGTTTACATTACGATTCTTTGTGTTATACAGTTGATGGGTTTCTTTTCTTTCTTAATTT CTTTAAAAAATAGAGATGGGGTCTTACTATATTACCCAGGCTGGTCTTGAAGTCCTGGGCTC AAGTGATCTTCCTGTCTCAGCCTACCAAGTAGCTGAGACTATAGGTGCAAAAAAGCCACTAT ACCTGGCTAGTTTACAGGTTTTAACAAATGCATTATGCCACGTATCCATTATTACAGGATCA CACAAGATATTTTCATTACCCTGAAAGCATCCCTGTGTTCCACCAATTCATCCTGCCTCCAT GAGCCGCTGGCAACCACTGATCTCTATAGTTTTGCCTTTTCTAAAATGTCATATAATTGGAA TCATACAGTCTGTAGCATTTTCAGACTAGCTTTTAAAATTTGGCAATATGCATTTAAGGTTC CTCCTTAAATGTGAAGGGCATAGCCAATGTGGCTTGATAGCTCATTTCTTTTTATTGGTGAA TATTTCATTGTCTGGATGTTCCACAGTTTGTTTATCCATTCACCTATTCAATTTGCTTTTTT TCTGTGTATCTATCACTAATTCAATACTGGACTCTCCAACAGAGCCSequence 4 / Construct 4 (Allele 2: CAG: B2M / CD47 w / insulator) (SEQ ID NO: 61): < HA-LXSAXT2AXPuroXbGHpAXcHS4 InsulatorXSRF UCOEXAox 71> < B2MXT2A>< tRFPXWPREX S V40 pAXbGH pAXWPREXCT47XLox66> < HA-R> CAGCAGGAGAATCATGCCAATGGGCCAATATACATTCTGACCCACAGTTTCATAATAAAATA AAATGGTTGTGGTTGTAAGCCACTATGTTTCAGAGTGGTTTGTTACACAGCAATAAATAACT AATATAGTAGGCATACCATCAAGTCCAAAGTAGGTAGAGAAGAATGTAAATAGCAGAGCAAA ACAGCATGACTGGTGGCTGGGAGGCTTAAAACTGGGACAGGATCAGAGTCATGAAAGAAGTC AAAGAAATGGTTCAGAAGTAAGGCTGAGACTGACTTACAAAAGCTGAAAGTCCCTTTAAGTT GGTGTTTGGTGCATTGGCAGGGGCAGGTATGGTGACTTAAAAGAGCCATGCTCAACAAGATC AAGCACAACACAATCACGGGTCACCCCAGCAGACCTTAGCGAGTCTAGCCATTTCTTTGGTG GTGGTCACAGTCATGCTTCAGCCCAGTTTCCACTTGGACAAATGGTACATATTTTCAATGAG ATGAAAATTAAGATACAATCCATGTGCTCAGAGAGTGATCACAGCTCTGACTAAACACTGTG99182281269.1UR 6-24056 / FR: 161118.06801 CCCCAAAGTGTTGAGGAATTGGGAAAACCTAGCTGAGTTAGTGGTCTGTTTTCTGTTACAAT AAAGCTCATAATGAAAATTAGCCTTCTTTGTTCTTCCCCAAGTCTTTCTTTCTAGACGAAAC TACTTTCAACTGTTTTAACTTCCTTACTGTTAACTTCCATATTTCTAGATAGTATGGTCAGA CTGTTATTTCTTGACTTTTAAATGTAAGATATTATCTACTGACTTCCTTCTATGTAAGATGA GGATTGAGCTCTCTTACCCTTCactagaagcttCTGACCTCTTCTCTTCCTCCCACAGGgcc tcgagagatctggcagcgg aGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAG AATCCCGGCCCTaggctcgagATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGA CGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCC ACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACG CGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTG GACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCG AGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGG CCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGG TCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCT TCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACC GCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGAtctagagggcccgtttaaacccgctgatcagcctcgaCTGTGCCTTCTAGTTGCCAGCCAT CTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTT TCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGG TGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATG CGGTGGGCTCTATGGg ctagcggtg g ATTCTCACTGACTCCGTCCTGGAGTTGGATGAGAGA TAATGGCCTTACGTTGTGCCAGGGGAGGGTCGGGCTGGATTTAGCAAGATTTACCTTCTCCA AAGAGCGGTGCTGCAGTGGCACAGCTGCCCACGGAGGTGGGGGGGTCACCGTCCCTGGAGGT GATGAAGAACTGTGGGGATGTGGCACTGAGGGACATGGCCAGTGGGCACGGTGGGTGGGTTG GGGTTGGTCTTGGGGATCTTGGAGGGCTTTTCCAGCCTTCATGATTTGACGATTGTATGAAC ATCTACATGGCAATTCTCCAGCTGCCTGTCCCAGTCCTACTGACCCAGCTGTATCTCTCCAG GCAAGCTCTTCCACCCCTTCTGCTTGCATCCAGACACCATCAAACATGCAGGCTCAGACACA GGGACCAGCAGTGTCTGTGGCCTTTTTGTGCTCCTCTCCATGCTGGGTTTTAACTTGCTCTT TGTCCTTCTATCCTATCTTCTTATCCTTAAGGCTGTTCTGAACGCTGTGACTTGGAGAGTGT CCCAGAGCCCTCAACACCTGCATGTCCCACGTCCATGCTGTCCTGCACTTCCTTATCCCCAA GATCTGCCTCTCCGTGATGCACTGAATTGGCAAACATGTGTCACCCCAGACCAACAATGTCA CAGCAAACTCCCCCTTGATAGGACAAGGGGGAATGGCTTTACACTGAGACAGGGGAGGTTTG GGTTGGATATGAGGAGGCAGTTTTTCCCCCAGAGGGTGGTGACGCACTGAACAGGTTGCCCA AGGAGGCTGTGGATGCCCCATCCCTGCAGGCATTCAAGGCCAGGCTGGATGTGGCTCTGGGC AGCCTGGGCTGCTGGTTGATGACCCTGCACATAGCAGGGGGTTGGATCTGGATGAGCACTGT GCTCCTTTGCAACCCAGGCCGTTCTATGATTCTGTCATTCTAAATCTCTCTTTCAGCCTAAA GCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAG GAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGG ATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGG GGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCCGTGAGCT C C AC G T G CCTAGGGCACACGACCACAATTCCACTGAAAGCATTTTAATACGGAACTTGTCACTCCCAGG GAGCCTCCGCTCAGCCGGCAGTTGGTTCATTTCAATCCCCACGACAACCCTTCAAAGTGCAG GGCAGACAGCAGGTGGCTCTGCCCAGGCGCCTGGATCACAGCCCGGCCTGCAGCCCTCACCT GGGCGCGGGGAGACCCTGAGGACGCTCCTCCAGGCGGCGCTGGCCGGGGCCTGCGGACACGG ACGGGCGGGCTGAGCTCCGGGACCCCTCCCCGCGCCCCGCACCCCGCACCCCGCACCCCGCA CCCCGCACCCGGCGCTCACCCGTCCCAGCCCCGCCGCCCGCAGCCCCAGCTGCAACGCAGCC ACCGCCGCCATCGCACCCGGCCCCGCGGGCGCTTCCGGGACGCAGGAGGCATCTGCATCCGG100182281269.1UR 6-24056 / FR: 161118.06801 GGCGCCGCTGAGTCCCGCCCAGAGCCCCGCCCCCGGCTCCAGGTTCTGCGAGCGGCTTCCGC CGGGCTGCTCCGCGGGCGCGTCGGCCATGAGCGAGTTGCCGGGCGACGTGCGGGCGTTTCTG CGGGAGCACCCGAGCCTGCGGCTCCAGACGGACGCCCGCAAGGTTCGCAGCGCGGGAGGGGA ACGGAGTGGCGGAGAAGGGCGCAGTTGGGATGAGGGGCTGAGGGGAGGGCAGGGGAGAGGAG AGGGCAGGGGAGAGGGGAGAGGGGAGAGCAGGAGAGAGGGGAAGGCAGGGGAGAGGGCGCGG CGGGATCAGGGGAGGAGAGGGAAGGGGGCGCGGCAGGAGGGGGCACCAGGGAGCGGAGCCCT GGCCCTCCTGACGTCCTGCCCGCCCACGCGTCCGCAGGTGAGGTGCATCCTGACAGGTCACG AGCTGCCCTGCCGCCTGCCGGAGCTCCAGGTCTACACCCGCGGCAAAAAGTACCAGCGGCTG GTCCGCGCCTCCCCGGCCTTCGACTATGCAGActcctgggcaacgtgctggttattgtgctg tctcatcattttggcaaaGAATTCACTAGTaaaaaaa taccgttcgta tagca tacattata cgaagtta t<cggcgcgccACCATGAGCAGGAGCGTGGCCCTGGCCGTGCTGGCCCTGCTGA GCCTGAGCGGCCTGGAGGCCATCCAGAGGACCCCCAAGATCCAGGTCTACAGCAGGCACCCC GCCGAGAACGGCAAGAGCAACTTCCTGAACTGCTACGTGAGCGGCTTCCACCCCAGCGACAT CGAGGTGGACCTGCTGAAGAACGGCGAGAGGATCGAGAAGGTGGAGCACAGCGACCTGAGCT TCAGCAAGGACTGGAGCTTCTACCTGCTGTACTACACCGAGTTCACCCCCACCGAGAAGGAC GAGTACGCCTGCAGGGTGAACCACGTGACCCTGAGCCAGCCCAAGATCGTGAAGTGGGACAG GGACATGG G AAG C G GAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGATGTGGAGGAGAATC CTGGACCTATGAGCGAGCTGATCAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACC GTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCA GACCATGAAGATCAAGGTGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCTGGCTA CCAGCTTCATGTACGGCAGCAAAGCCTTCATCAACCACACCCAGGGCATCCCCGACTTCTTT AAGCAGTCCTTCCCTGAGGGCTTCACATGGGAGAGAATCACCACATACGAAGACGGGGGCGT GCTGACCGCTACCCAGGACACCAGCTTCCAGAACGGCTGCATCATCTACAACGTCAAGATCA ACGGGGTGAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAAAACACGCGGCTGGGAGGCC AACACCGAGATGCTGTACCCCGCTGACGGCGGCCTGAGAGGCCACAGCCAGATGGCCCTGAA GCTCGTGGGCGGGGGCTACCTGCACTGCTCCTTCAAGACCACATACAGATCCAAGAAACCCG CTAAGAACCTCAAGATGCCCGGCTTCCACTTCGTGGACCACAGACTGGAAAGAATCAAGGAG GCCGACAAAGAGACCTACGTCGAGCAGCACGAGATGGCTGTGGCCAAGTACTGCGACCTCCC TAGCAAACTGGGGCACAGATAAggtaccGATACGATAATCAACCTCTGGATTACAAAATTTG TGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTT TAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAA TCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTG CACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTT CCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCC CGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCT GACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCT GCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTG CGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTC CCCGCATCGGTGACTACGACCCTGACCTggcgcgccGCAGGGCTTGTCATACCAGAGTGCTT TATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAG TTCTAGACCATAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCCC CTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCTACTCAGACAATGCGATG CAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGC ACGGGGGAGGGGCAAACAACAGATGGCTGGCAACTAGAAGGCACAGGACTCAGTGGTAGACA GTGCGTCaccgCCGATGCGGGGAGGCGGCCCAAAGGGAGATCCGACTCGTCTGAGGGCGAAG GCGAAGACGCGGAAGAGGCCGCAGAGCCGGCAGCAGGCCGCGGGAAGGAAGGTCCGCTGGAT TGAGGGCCGAAGGGACGTAGCAGAAGGACGTCCCGCGCAGAATCCAGGTGGCAACACAGGCG101182281269.1UR 6-24056 / FR: 161118.06801 AGCAGCCATGGAAAGGACGTCAGCTTCCCCGACAACACCACGGAATTGTCAGTGCCCAACAG CCGAGCCCCTGTCCAGCAGCGGGCAAGGCAGGCGGCGATGAGTTCCGCCGTGGCAATAGGGA GGGGGAAAGCGAAAGTCCCGGAAAGGAGCTGACAGGTGGTGGCAATGCCCCAACCAGTGGGG GTTGCGTCAGCAAACACAGTGCACACCACGCCACGTTGCCTGACAACGGGCCACAACTCCTC ATAAAGAGACAGCAACCAGGATTTATACAAGGAGGAGAAAATGAAAGCCATACGGGAAGCAA TAGCATGATACAAAGGCATTAAAGCAGCGTATCCACATAGCGTAAAAGGAGCAACATAGTTA AGAATACCAGTCAATCTTTCACAAATTTTGTAATCCAGAGGTTGATTATCGTTCATTCGTCG TTCATCATACCCTTGCTCTCCTTGAAGGCATTAAGAGGCTCTTCGACCGCCTTGCGCGGGGG TTGGATCGTTTTCTGGTTACTTGCAACAAACTTCATATAGACCAGTCCGAGCAGTTGGGCCA GCGCCAAGATACTAAGACCACTGATCAACAGCGGTCCGTGCATAGGAATGCAGGCTGCGATG CAGAGTGAAAGACCGACCACTGCCAAAATGTATGCGATGACCTGAATGACCAGAATTGCGAT AACAAAAGATGTGAGTCCAATTGCGGTGCTGAACACGTAGTAGTGCAGGAGGATGAGAATGC CCGTTGAAGTCACTATCAATCCCAGCCCTGTTGCATTCTTGAGACTATACTCACCCGGAACA AACAAGATCGCACCTACGATTACAATTACTGTTATGACCAAGCCCGCCACGAGGAGGGCGAT TGTTTTCTCGTCCATGCCTCCGGATCGATACTTGAGCGTTTTGATTCCAAATTGGCCCCAAA AGAGGAGAATTGCGAAAATCGGGAAGATCACGATCAGAATGTTTTCATTTGGAGAAAACCAA GAAACCACTCTATATTTCAGTTCTATAATAGTTTCGCCTTCCCGAGTCAGTTCTGTCACCTC ACAGGTGTAGTTACCCGTATGGCTGACGGCGTCAGACTTATCCATTTTCAAAGACGCATCGC CCTTGAGGAGTTGGCTGACTTCTATCTTCGCTGAAGAAAAGTCTGTCGGAACGGTTGATTTA TTAAGCGCGCCGTCGAATGTGTAAATGTCGCGGCCTTTGAATTTCCACTTCACGTAGACTTC GGTTGTATTCTGCGCCTCCATGTTGGTTACGAAACAGGGGATTACTACTGTATCGTTACAAA AGGTGAAtTCCACGGACTTCGTTTTGTTAAAGAGCAATTGGGCACTCCCGCAGCAAGCGGAA CCCAACAAGAGGGCCGCCACAAGGGGCCACATg gaccggtatttttt tTACCGTTCGTATAA TGTATGCTATACGAAGTTATggcatcqgagcctcact cqcqttagaattcgaqttatt aT TCCTCATCCTTCCAACATAAATATATTTTGGGATTATATCAACATTCAATGTTACTTAAAGT GACCTTGTAAATATTTTCACAACTGAGCCATGTTTGATTTGTATACTTATGTTTACTTTACT GTTTTTCCTGAAGTTAATAATTGCCTTGAATTTATTTATTTCTTTAAAAATGTTTCATTACT CAGGACTGTAGTTTACATTACGATTCTTTGTGTTATACAGTTGATGGGTTTCTTTTCTTTCT TAATTTCTTTAAAAAATAGAGATGGGGTCTTACTATATTACCCAGGCTGGTCTTGAAGTCCT GGGCTCAAGTGATCTTCCTGTCTCAGCCTACCAAGTAGCTGAGACTATAGGTGCAAAAAAGC CACTATACCTGGCTAGTTTACAGGTTTTAACAAATGCATTATGCCACGTATCCATTATTACA GGATCACACAAGATATTTTCATTACCCTGAAAGCATCCCTGTGTTCCACCAATTCATCCTGC CTCCATGAGCCGCTGGCAACCACTGATCTCTATAGTTTTGCCTTTTCTAAAATGTCATATAA TTGGAATCATACAGTCTGTAGCATTTTCAGACTAGCTTTTAAAATTTGGCAATATGCATTTA AGGTTCCTCCTTAAATGTGAAGGGCATAGCCAATGTGGCTTGATAGCTCATTTCTTTTTATT GGTGAATATTTCATTGTCTGGATGTTCCACAGTTTGTTTATCCATTCACCTATTCAATTTGC TTTTTTTCTGTGTATCTATCACTAATTCAATACTGGACTCTCCAACAGAGCC102182281269.1

Claims

UR 6-24056 / FR: 161118.06801CLAIMS WHAT IS CLAIMED IS:

1. A recombinant nucleic acid molecule comprising:a first expression cassette comprising a first promoter, a second promoter, a first coding-sequence, and second coding-sequence, wherein the first coding-sequence and second coding-sequence are operably linked to the first promoter and the second promoter;a second expression cassette joined to the first expression cassette in an opposite expression direction, comprising a third promoter and a third coding-sequence operably linked to the third promoter;a first pair of sites of recombinase-mediated recombination, anda second pair of sites of recombinase-mediated recombination,wherein a segment of the recombinant nucleic acid molecule between the two members of the first pair or between the two members of the second pair is reversable upon exposure of the two members to a recombinase, andwhereina first member and a second member of the first pair are in the first expression cassette and the second expression cassette, respectively,a first member and a second member of the second pair are in the first expression cassette and the second expression cassette, respectively,the second member of the first pair is between the two members of the second pair, and the first member of the second pair is between the two members of the first pair, wherein the first promoter and the second promoter regulate, respectively, expressions of genes or cassettes of genes sequentially expressed at two different stages of differentiation within a common lineage.

2. The recombinant nucleic acid molecule of claim 1, wherein (i) the recombinant nucleic acid molecule is for conditional, stage- and phenotype-specific expression of one or more transgenes in a differentiated cell derived from a pluripotent stem cell and (ii) the first coding-sequence, second coding-sequence, or the third coding-sequence encodes the transgenes.

3. The recombinant nucleic acid molecule of claim 1, wherein (i) the recombinant nucleic acid molecule is for conditionally rendering a cell hypo-immune and / or non-immunogenic in103182281269.1UR 6-24056 / FR: 161118.06801 a developmentally-restricted and phenotype-specific fashion and (ii) the second coding-sequence encodes for an immune checkpoint protein.

4. The recombinant nucleic acid molecule of claim 1, wherein the first coding-sequence, the second coding-sequence, or the third coding-sequence encodes for one or more proteins or RNAs.

5. The recombinant nucleic acid molecule of any one of the proceeding claims, wherein the first promoter or the second promoter or both are tissue-specific, differentiation stagespecific, cell-specific, or ligand inducible.

6. The recombinant nucleic acid molecule of claim 5, wherein the first promoter regulates gene expression specifically in a progenitor cell.

7. The recombinant nucleic acid molecule of claim 6, wherein the second promoter regulates gene expression specifically in a differentiated cell derived from the progenitor cell.

8. The recombinant nucleic acid molecule of claim 7, wherein the progenitor cell is a glial progenitor cell, a neural progenitor cell, an oligodendrocyte progenitor cell, or an astrocyte progenitor cell.

9. The recombinant nucleic acid molecule of claim 8, wherein the differentiated cell is an astrocyte, an oligodendrocyte, or a neuron.

10. The recombinant nucleic acid molecule of claim 7, wherein the progenitor cell is a pancreatic Islet progenitor cell, a pancreatic Islet beta cell, or a pancreatic Islet alpha cell.

11. The recombinant nucleic acid molecule of claim 7, wherein the differentiated cell is a pancreatic Islet insulin-producing beta cell or glucagon-secreting alpha cell.

12. The recombinant nucleic acid molecule of any one of the preceding claims, wherein the second promoter is embedded within an intron and flanked by a splice donor site and a splice acceptor site.104182281269.1UR 6-24056 / FR: 161118.06801 13. The recombinant nucleic acid molecule of any one of the preceding claims, wherein the second promoter is embedded within an intron and followed by a second splice donor site that is 5’ end to said splice acceptor site.

14. The recombinant nucleic acid molecule of any one of the preceding claims, wherein the first promoter is selected from the group consisting of promoters of Neurogenin2, NeuroD family. GPR17, NG2, NGN3, PDX1, and NKX6.1.

15. The recombinant nucleic acid molecule of any one of the preceding claims, wherein the second promoter is selected from the group consisting of promoters of ELAV4; SYN 1, MAP2, SOXIO, MYRF, MAG, GFAP, AQP4, INS (beta), GCG (alpha), and SST (delta).

16. The recombinant nucleic acid molecule of any one of the preceding claims, wherein the third promoter is a constitutive promoter.

17. The recombinant nucleic acid molecule of any one of the preceding claims, wherein the third promoter is selected from the group consisting of promoters of CAG, CBH, spleen focusforming virus (SFFV), CBG, CMV, EFl alpha, UBC, and PGK.

18. The recombinant nucleic acid molecule of any one of the preceding claims, wherein the first coding-sequence encodes a recombinase.

19. The recombinant nucleic acid molecule of claim 18, wherein the first coding-sequence encoding the recombinase comprises an intron.

20. The recombinant nucleic acid molecule of claim 18 or 19, wherein the recombinase is a Cre recombinase or a FLP recombinase.

21. The recombinant nucleic acid molecule of any one of the preceding claims, wherein the second coding-sequence encodes for an immune checkpoint protein.105182281269.1UR 6-24056 / FR: 161118.06801 22. The recombinant nucleic acid molecule of claim 19, wherein the immune checkpoint protein is selected from the group consisting of CD47, PDL1, HLA-E. CD200, CD36, and CD64.

23. The recombinant nucleic acid molecule of claim 21 or 22, wherein the second coding-sequence further encodes for a first reporter protein.

24. The recombinant nucleic acid molecule of any one of the preceding claims, wherein the third coding-sequence encodes for p2-microglobulin protein.

25. The recombinant nucleic acid molecule of claim 24, wherein the third coding-sequence further encodes for a second reporter protein.

26. The recombinant nucleic acid molecule of any one of the preceding claims, wherein the first expression cassette comprises a first poly adenylation signal (PAI) 3’ end to the first coding-sequence; orwherein the first expression cassette comprises a second poly adenylation signal (PA2) 3’ end to the second coding-sequence; orwherein the second expression cassette comprises a third poly adenylation signal (PA3) 3’ end to the third coding-sequence.

27. The recombinant nucleic acid molecule of claim 26, wherein(A) the first member of the first pair is located between the second promoter and the first coding-sequence,(B) the second member of the first pair is located between the PA3 and the third coding sequence;(C) the first member of the second pair is located between the PAI and the second coding-sequence, and / or(D) the second member of the second pair is located between the third coding sequence and the third promoter.28 A polynucleotide derived from exposing the recombinant nucleic acid molecule of any one of the preceding claims to a recombinase.106182281269.1UR 6-24056 / FR: 161118.0680129. A polynucleotide derived from exposing the recombinant nucleic acid molecule of claim 27 to a recombinase, said polynucleotide comprising from the 5’ end thereof to the 3’ end thereof:the third promoter;the third coding-sequence;the complement of the second promoter, andthe complement of the first promoter.

30. The polynucleotide of claim 29, further comprising the PA2 and the complement of PA3.

31. A vector comprising the recombinant nucleic acid molecule or polynucleotide of any one of the preceding claims.

32. The vector of claim 31, wherein the vector is a viral vector or a plasmid vector.

33. The vector of claim 31 or 32, wherein the vector further comprises one or more elements selected from the group consisting of a DNA nuclear targeting sequence and a barcoding sequence.34 A composition comprising (i) the recombinant nucleic acid molecule of any one of claims 1-27, the polynucleotide of any one of claims 28-30, or the vector of any one of claims 31-33, and (ii) a pharmaceutically acceptable carrier.

35. The composition of claim 34, wherein the carrier comprises a liposome, or a nanoparticle or an exosome.

36. A cell or a progeny thereof, said cell comprising the recombinant nucleic acid molecule of any one of claims 1-27, the polynucleotide of any one of claims 28-30, or the vector of any one of claims 31-33.

37. The cell or progeny of claim 36, wherein the cell is a pluripotent stem cell.107182281269.1UR 6-24056 / FR: 161118.0680138. The cell or progeny of claim 37, wherein the cell is an embryonic stem cell.

39. The cell or progeny of claim 37, wherein the cell is an induced pluripotent stem cell.

40. The cell or progeny of any one of claims 36-39, wherein the cell is a human cell.

41. A method of conditional, stage- and phenotype-specific expression of a transgene in a differentiated cell derived from a pluripotent stem cell, comprising,obtaining a pluripotent stem cell;introducing into the pluripotent stem cell the recombinant nucleic acid molecule of any one of claims 1-27 or the vector of any one of claims 31-33;culturing the pluripotent stem cell containing the recombinant nucleic acid molecule or vector under conditions suitable for (i) differentiation of the pluripotent stem cell and (ii) expression of the proteins encoded by the recombinant nucleic acid molecule under the control of the first promoter or the second promoter; andobtaining one or more differentiated cells derived from the pluripotent stem cell.

42. A method of conditionally rendering or obtaining one or more hypo-immune and / or non-immunogenic cells, comprisingobtaining a starting cell;introducing into the starting cell the recombinant nucleic acid molecule of any one of claims 3-27 or a vector comprising the recombinant nucleic acid molecule, wherein the second coding-sequence encodes for an immune checkpoint protein;culturing the starting cell containing the recombinant nucleic acid molecule or vector under conditions permitting expression of the proteins encoded by the recombinant nucleic acid molecule under the control of the first promoter or the second promoter; andobtaining one or more hypo-immune and / or non-immunogenic cells.

43. The method of claim 42, wherein the starting cell lacks a functional β2-microglobulin protein and / or a functional CIITA protein.108182281269.1

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