Cellular context-specific gene regulation using inhibitory RNAs

Abstract

The present invention relates to methods of context-specific or cellular context-specific gene regulation and related nucleic acid constructs, cells and therapeutic uses and methods. In particular, the present invention relates to the context-specific expression of inhibitory RNA in a cell to regulate the expression of one or more target genes in said cell in a context-specific manner.

Description

[Technical Field]

[0001] The present invention relates to methods of context-specific and cellular context-specific gene regulation and related nucleic acid constructs, cells and therapeutic uses and methods. In particular, the present invention relates to the context-specific expression of inhibitory RNA in a cell to regulate the expression of one or more target genes in said cell in a context-specific manner. [Background technology]

[0002] The following discussion is provided to aid the reader in understanding the present disclosure and does not constitute any admission as to the content or relevance of prior art.

[0003] GEiGS® (or GEiGS) is a gene silencing technology that works by redirecting endogenous non-coding RNAs (ncRNAs) with RNAi-mediated silencing specificity to desired target sequences (e.g., gene transcripts). In humans, the redirected ncRNAs are almost exclusively microRNAs (miRNAs). Because these target their targets through sequence complementarity, altering their sequences can shift their silencing activity toward optimally targeted targets. Precise gene editing is performed on the sequences of endogenous miRNAs, providing them with the ability to engage and silence novel desired targets through perfect sequence complementarity via the RNAi pathway.

[0004] GEiGS® therefore works by hijacking miRNAs already expressed in cells and redirecting them to silence / modulate the expression of desired targets. GEiGS targets are silenced via the RNAi pathway. However, unlike siRNA or shRNA, where silencing is induced by the administration of double-stranded oligos or transgenes, respectively, GEiGS utilizes endogenous ncRNAs as vectors to silence desired targets.

[0005] While modifying the sequence of endogenous ncRNA to redirect its activity is achieved through genome editing technology, GEiGS is technology-independent and can therefore be used with CRISPR, TALENs, zinc finger nucleases, and their derivatives. The GEiGS "engine" (how targeted silencing is achieved) is based on the RNAi mechanism / pathway, and the implementation of the technology (how endogenous ncRNA is hijacked to target desired genes) is based on genome editing technology. For these reasons, GEiGS draws on the power of both RNAi and genome editing to enable stable, tunable, and programmable gene silencing.

[0006] Thus, GEiGS (gene editing-induced gene silencing) uses endogenous miRNA loci to drive the expression of specifically designed interfering RNAs (RNAi) against novel mRNA targets. The present invention is based, at least in part, on the recognition that GEiGS silencing by redirecting miRNAs or other inhibitory RNAs with context-specific activity (e.g., developmentally regulated, cell state-specific, or induced in cells in response to stimuli) can be selectively active in certain contexts (e.g., cell or tissue types, or cells under certain conditions) but not expressed in other contexts (e.g., cell / tissue types or under different conditions). Cells can be programmed to silence pathway genes via gene editing, for example, when differentiating from stem cells or after their stimulation by extracellular signals. In this aspect, GEiGS is particularly powerful because other RNAi approaches cannot silence genes in such a context-specific manner.

[0007] While the use of a GEiGS approach is preferred in some embodiments of the present invention, other approaches for partially or completely silencing gene expression using inhibitory RNAs are known in the art, for example, by inserting a suitable expression construct into the genome of a cell to drive expression of the inhibitory RNA, by providing a suitable expression construct to drive expression of the inhibitory RNA episomally (e.g., in a suitable plasmid or other vector), or by direct delivery of the inhibitory RNA to the cell. Such approaches can also be applied in the present invention, so long as the inhibitory RNA exhibits appropriate context-specific activity. For example, a suitable inhibitory RNA construct can be inserted into the genome, for example, into a locus encoding one or more endogenous miRNAs. Such an approach, which can be adapted to the present invention, is described in Senis et al. ("TALEN / CRISPR-mediated engineering of a promoterless antiviral RNAi hairpin into an endogenous miRNA locus," Nucleic Acids Research, 2017, Vol. 45, No. 1, e3 doi: 10.1093 / nar / gkw805). Thus, in some embodiments of the present invention, a sequence encoding an inhibitory RNA (preferably a promoterless sequence encoding an inhibitory RNA) may preferably be inserted into an endogenous miRNA locus without disrupting or inactivating any endogenous miRNA.

[0008] There remains a need for improved approaches to silencing gene expression in a more selective or specific manner, e.g., partially or completely, in a context-specific manner. The present invention relates to a novel approach for the use of inhibitory RNA for context-specific silencing of target gene expression.

[0009] Non-limiting examples of potential utility of the present invention include context-specific silencing of MHC-I associated with the stage of functional specification in therapeutic cells to prevent host immune-mediated rejection in appropriate circumstances, targeting mechanisms in macrophages to prevent forced conversion of macrophages into immunosuppressive cells in response to the tumor microenvironment, and silencing immune checkpoints in T cells to minimize their exhaustion and cell death.

[0010] These and other advantages and uses of the present invention will become apparent from the disclosure that follows. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO2019 / 058253 [Patent Document 2] WO2020 / 183414 [Patent Document 3] WO2020 / 183419 [Patent Document 4] WO2006 / 040763 [Patent Document 5] U.S. Patent No. 5,486,359 [Patent Document 6] WO2013126963 [Patent Document 7] WO2014107763 [Patent Document 8] WO91 / 10470 [Patent Document 9] WO91 / 10425 [Patent Document 10] U.S. Patent No. 5,837,234 [Patent Document 11] U.S. Patent No. 5,011,472 [Patent Document 12] U.S. Patent No. 4,892,538 [Patent Document 13] WO2012 / 145384 [Non-licensed literature]

[0012] [Non-licensed Document 1] Senisら ("TALEN / CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus" Nucleic Acids Research, 2017, Volume 45, No. 1 e3 doi: 10.1093 / nar / gkw805) [Non-licensed Document 2] Amaralら, 2017, EMBO J, EMBO J. 2017 Feb 1;36(3):346~360 pages [Non-licensed Document 3] Rothら, 2018, Nature Nature. 2018 Jul;559(7714):405~409 pages [Non-licensed Document 4] Altschulら1990 (J Mol Biol 215: 403~10 pages) [Non-licensed Document 5] Tatusova and Madden 1999 (FEMS Microbiol Lett 174: pages 247~250) [Non-licensed Document 6] Smith and Waterman (1981) Adv. Appl. Math. 2:482 pages [Non-licensed Document 7] Needleman and Wunsch (1970) J. Mol. Biol. 48:443 pages [Non-licensed Document 8] Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444 pages [Non-licensed Document 9] Higgins and Sharp (1988) Gene 73:237~44 pages [Non-licensed Document 10] Higgins and Sharp (1989) CABIOS 5:151~3 pages [Non-licensed Document 11] Corpet (1988) Nucleic Acids Res. 16:10881~90 pages [Non-licensed Document 12] Huangら(1992) Comp. Appl. Biosci. 8:155~65 pages [Non-licensed Document 13] Pearsonら(1994) Methods Mol. Biol. 24:307~31 pages [Non-licensed Document 14] Tatianaら(1999) FEMS Microbiol. Lett. 174:247~page 50 [Non-licensed Document 15] ParkらReprogramming of human somatic cells to pluripotency with defined factors. Nature (2008) 451:141~146 pages [Non-licensed Document 16] Doetschmanら, 1988, Dev Biol. 127: 224~7 pages [Non-licensed Document 17] Iannacconeら, 1994, Dev Biol. 163: pages 288~92 [Non-licensed Document 18] Giles, 1993, Mol Reprod Dev. 36: 130-8 pages [Non-licensed Document 19] Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424~33 pages [Non-licensed Document 20] Notarianni, 1991, J Reprod Fertil Suppl. 43: 255-60 [Non-licensed Document 21] Wheeler 1994, Reprod Fertil Dev. 6: 563~8 pages [Non-licensed Document 22] Mitalipova, 2001, Cloning. 3: pages 59~67 [Non-licensed Document 23] Thomson, 1995, Proc Natl Acad Sci US A. 92: 7844~8 pages [Non-licensed Document 24] Thomson, 1996, Biol Reprod. 55: 254~9 pages [Non-licensed Document 25] Dominici, M, (2001) J. Biol. Regul. Homeost. Agents. 15: 28~37 [Non-licensed Document 26] Alison, MR J Pathol. (2003) 200(5): 547~50 [Non-licensed Document 27] Eventov-Friedman Sら[PLoS Med. (2006) 3: e215] [Non-licensed Document 28] Robert Lanze, Elsevier Academic Press, 2004, Chapter 54, pp. 609-614 [Non-licensed Document 29] Jones EAら, 2002, Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells, Arthritis Rheum. 46(12): 3349~60 pages [Non-licensed Document 30] Stratら, Nucleic Acids Research, 2006, Volume 34, No. 13, pages 3803~3810 [Non-licensed Document 31] Bhargava AらBrain Res. Protoc. 2004;13:115~125 pages [Non-licensed Document 32] Diallo M., Oligonucleotides. 2003;13:381~392 pages [Non-licensed Document 33] Paddison PJ, Proc. Natl Acad. Sci. USA. 2002, 99:1443~1448 pages [Non-licensed Document 34] Tran N., FEBS Lett. 2004, 573:127~134 pages [Non-licensed Document 35] Bartel 2004, Cell pp. 116-281 [Non-licensed Document 36] Lewisら2005 Cell 120~15 [Non-licensed Document 37] Krekら(2005, Nat Genet pages 37~495) [Non-licensed Document 38] Rao DD, Senzer N, Cleary MA, Nemunaitis J. Cancer Gene Ther. 2009 Nov 16(11):807~9. Doi: 10.1038 / cgt.2009.53. Epub 2009 Aug 28 PMID: 19713999 [Non-licensed Document 39] Panwar Bioinformatics. May 15, 2017; 33(10): 1554-1560 [Non-licensed Document 40] Kavakiotis ら, "DIANA-miTED: a microRNA tissue expression database", Nucleic Acids Research, Volume 50, Issue D1, January 7, 2022, Pages D1055-D1061 http: / / www.microrna.gr / mited) [Non-licensed Document 41] Schmittgen and Livak. Nat Protoc (2008) 3: 1101-1108 [Non-licensed Document 42] Wake, BMC Genomics (2016) 17(1):1 page [Non-licensed Document 43] Xing Y et al. Tumor Immune Microenvironment and Its Related miRNAs in Tumor Progression. Front Immunol. 2021 May 18;12:624725. doi: 10.3389 / fimmu.2021.624725. PMID: 34084160; PMCID: PMC8167795 [Non-Patent Document 44] Chatterjee, et al., MicroRNAs: As Critical Regulators of Tumor-Associated Macrophages. Int J Mol Sci. 2020 Sep 27;21(19):7117. doi: 10.3390 / ijms21197117. PMID: 32992449; PMCID: PMC7582892 [Non-Patent Document 45] Katzenelenbogen et al. (2020). Coupled scRNA-Seq and Intracellular Protein Activity Reveal an Immunosuppressive Role of TREM2 in Cancer. Cell, 182(4), pp. 872~885.e19. https: / / doi.org / 10.1016 / J.CELL.2020.06.032] [Non-Patent Document 46] Virtakoivuら[Virtakoivu, R., Rannikko, J.H., Viitala, M., Vaura, F., Takeda, A., Lonnberg, T., Koivunen, J., Jaakkola, P., Pasanen, A., Shetty, S., de Jonge, M.J.A., Robbrecht, D., Ma, Y.T., Skytta, T., Minchom, A., Jalkanen, S., Karvonen, MK, Mandelin, J., Bono, P., & Hollmen, M. (2021). Systemic Blockade of Clever-1 Elicits Lymphocyte Activation Alongside Checkpoint Molecule Downregulation in Patients with Solid Tumors: Results from a Phase I / II Clinical Trial. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 27(15), pp. 4205~4220. https: / / doi.org / 10.1158 / 1078-0432.CCR-20-4862]

non-patent document47

Non-Patent Document 48

Non-Patent Document 49

Non-Patent Document 50

Non-Patent Document 51

Non-licensed Document 70

Non-licensed Document 71

Non-licensed Document 72

Non-licensed Document 73

Non-licensed Document 77

Non-licensed Document 78

Non-licensed Document 79

Non-licensed literature 80

Outdoor Track 84

Outdoor Track 85

[0013] SUMMARY OF THE INVENTION In a first aspect, the present invention provides a method for modulating expression of a target gene in a cell in a context-specific manner, comprising: - providing the cell with a nucleic acid construct adapted to express an inhibitory RNA suitable for inhibiting expression of the target gene; Includes - the inhibitory RNA is active in a cell in a context-specific manner, such that inhibition of target gene expression occurs specifically in the first context in which the inhibitory RNA is active in said cell.

[0014] Optionally, inhibition does not occur or occurs to a reduced extent in at least a second situation in which the inhibitory RNA is inactive or less active.

[0015] The present invention thus relates to the context-specific activity of inhibitory RNAs that provide context-specific inhibition of target genes. Thus, inhibition of target gene expression occurs only, or occurs to a greater extent, in a first context, and inhibition is absent or reduced in at least a second context. The present invention thus enables the provision of cells (preferably cells for use in therapy) that have been modified with conditionally active inhibitory RNAs to inhibit target gene expression in specific contexts.

[0016] In some preferred embodiments, the nucleic acid construct is a modified form of an endogenous sequence encoding an inhibitory RNA, preferably an miRNA, that has been modified to target a transcript from a target gene.

[0017] In some preferred embodiments, the cell is adapted to express at least one additional inhibitory RNA (e.g., a second, third, fourth, etc. inhibitory RNA) that is active in the cell in a context-specific manner, such that inhibition of expression of a second or additional target gene occurs specifically in a first context in which the at least one additional inhibitory RNA is active in the cell. Alternatively, the at least one additional inhibitory RNA (e.g., the second, third, fourth, etc.) may be active in a second or additional context that is different from the first context. Thus, the method optionally comprises providing to the cell a nucleic acid construct suitable for inhibiting expression of a target gene, or adapted to express two or more inhibitory RNAs suitable for inhibiting expression of two or more target genes.

[0018] In some embodiments, the cells are adapted to express two, three, four, five, or more inhibitory RNAs that are active in the cells in a context-specific manner. Optionally, the inhibitory RNAs target more than one target gene. For example, two, three, four, five, or more inhibitory RNAs can target two, three, four, five, or more target genes. In some embodiments, two or more inhibitory RNAs can target a single target gene.

[0019] In some embodiments, cells are provided with two or more nucleic acid constructs adapted to express inhibitory RNAs suitable for inhibiting expression of one or more target genes in a context-specific manner, hi some embodiments, a single nucleic acid construct is adapted to express two or more inhibitory RNAs suitable for inhibiting expression of one or more target genes in a context-specific manner.

[0020] In some embodiments, a cell is provided with a first nucleic acid construct adapted to express a first inhibitory RNA suitable for inhibiting expression of a first target gene in a context-specific manner, and at least a second nucleic acid construct adapted to express a second inhibitory RNA suitable for inhibiting expression of a second target gene in a context-specific manner. In some embodiments, a cell is provided with a first nucleic acid construct adapted to express a first inhibitory RNA suitable for inhibiting expression of a first target gene in a context-specific manner, and at least a second nucleic acid construct adapted to express a second inhibitory RNA suitable for inhibiting expression of a second target gene.

[0021] In some embodiments, cells are provided with a single nucleic acid construct adapted to express two or more inhibitory RNAs, one of the two or more inhibitory RNAs suitable for inhibiting expression of a first target gene in a context-specific manner and another of the two or more inhibitory RNAs suitable for inhibiting expression of a second target gene in a context-specific manner. In some embodiments, cells are provided with a single nucleic acid construct adapted to express two or more inhibitory RNAs, one of the two or more inhibitory RNAs suitable for inhibiting expression of a first target gene in a context-specific manner and another of the two or more inhibitory RNAs suitable for inhibiting expression of a second target gene.

[0022] In some preferred embodiments, the methods involve genetically modifying (editing) an endogenous (usually genomic) sequence encoding an endogenous inhibitory RNA to alter its targeting specificity.

[0023] In a further aspect of the invention there is provided a cell or cell population obtained by the method of the first aspect of the invention.

[0024] In a further aspect of the present invention there is provided a genetically modified cell comprising a nucleic acid construct adapted to express an inhibitory RNA suitable for inhibiting expression of a target gene, Inhibitory RNAs are active in cells in a context-specific manner, such that inhibition of target gene expression occurs specifically in the first context in which the inhibitory RNA is active in said cells.

[0025] The optional and preferred features of the first aspect as described above naturally relate to the second and other aspects described herein.

[0026] Optionally, inhibition is absent or reduced in at least a second situation in which the inhibitory RNA is inactive or less active.

[0027] In some preferred embodiments, the nucleic acid construct is a modified endogenous sequence encoding an inhibitory RNA, preferably an miRNA, that has been modified to target a transcript from a target gene.

[0028] In some preferred embodiments, the cell is adapted to express at least one additional inhibitory RNA (e.g., a second, third, fourth, etc. inhibitory RNA) that is active in the cell in a context-specific manner, such that inhibition of expression of the gene, or a second or additional target gene, occurs specifically in a first context in which the at least one additional inhibitory RNA is active in the cell. Alternatively, the at least one additional inhibitory RNA (e.g., the second, third, fourth, etc.) may be active in a second or additional context that is different from the first context. Thus, the method optionally comprises providing to the cell a nucleic acid construct suitable for inhibiting expression of the target gene, or adapted to express two or more inhibitory RNAs suitable for inhibiting expression of two or more target genes.

[0029] In some embodiments, the cells are adapted to express two, three, four, five, or more inhibitory RNAs that are active in the cell in a context-specific manner. Optionally, the inhibitory RNAs target more than one target gene. For example, two, three, four, five, or more inhibitory RNAs can target two, three, four, five, or more target genes. In some embodiments, two or more inhibitory RNAs can target a single target gene. Optionally, the inhibitory RNAs can be active in different contexts (e.g., at least a first inhibitory RNA is active in a first context and at least a second inhibitory RNA is active in a second context).

[0030] In some embodiments, the cell is provided with two or more nucleic acid constructs adapted to express inhibitory RNAs suitable for inhibiting expression of the target gene in a context-specific manner, hi some embodiments, a single nucleic acid construct is adapted to express two or more inhibitory RNAs suitable for inhibiting expression of the target gene in a context-specific manner.

[0031] In some embodiments, a cell is provided with a first nucleic acid construct adapted to express a first inhibitory RNA suitable for inhibiting expression of a first target gene in a context-specific manner, and at least a second nucleic acid construct adapted to express a second inhibitory RNA suitable for inhibiting expression of a second target gene in a context-specific manner. In some embodiments, a cell is provided with a first nucleic acid construct adapted to express a first inhibitory RNA suitable for inhibiting expression of a first target gene in a context-specific manner, and at least a second nucleic acid construct adapted to express a second inhibitory RNA suitable for inhibiting expression of a second target gene.

[0032] In some embodiments, cells are provided with a single nucleic acid construct adapted to express two or more inhibitory RNAs, one of the two or more inhibitory RNAs suitable for inhibiting expression of a first target gene in a context-specific manner and another of the two or more inhibitory RNAs suitable for inhibiting expression of a second target gene in a context-specific manner. In some embodiments, cells are provided with a single nucleic acid construct adapted to express two or more inhibitory RNAs, one of the two or more inhibitory RNAs suitable for inhibiting expression of a first target gene in a context-specific manner and another of the two or more inhibitory RNAs suitable for inhibiting expression of a second target gene.

[0033] In a further aspect of the invention, nucleic acid constructs are provided for the context-specific expression of inhibitory RNA suitable for inhibiting expression of a target gene.

[0034] In yet another aspect of the present invention, there is provided a nucleic acid construct for context-specific inhibition of expression of a target gene in a cell, the nucleic acid construct encoding an inhibitory RNA suitable for inhibiting expression of the target gene, wherein the inhibitory RNA is active in the cell in a context-specific manner, such that inhibition of expression of the target gene occurs in a first context in which the inhibitory RNA is active in the cell.

[0035] Optionally, inhibition is absent or reduced in at least a second situation in which the inhibitory RNA is inactive or less active.

[0036] In yet another aspect of the present invention, there is provided the use of a context-active inhibitory RNA or a nucleic acid construct encoding a context-active inhibitory RNA in a method for context-specific inhibition of expression of a target gene in a cell, wherein the inhibitory RNA is active in a cell in a context-specific manner, such that inhibition of expression of the target gene occurs in a first context in which the inhibitory RNA is active in said cell.

[0037] Optionally, inhibition is absent or reduced in at least a second situation in which the inhibitory RNA is inactive or less active.

[0038] According to a further aspect of the invention there is provided a composition comprising the cells of the invention.

[0039] According to a further aspect of the invention there is provided a cell or composition of the invention for use in therapy.

[0040] According to a further aspect of the invention there is provided a method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutic amount of a cell of the invention or a composition of the invention.

[0041] According to a further aspect of the invention there is provided use of a cell of the invention or a composition of the invention in the manufacture of a medicament for treating a disease in a subject.

[0042] In a further aspect, a reporter nucleic acid is provided that comprises or consists of a constitutive promoter operably linked to a gene encoding a fluorescent protein and a target gene (or a portion of the target gene), wherein the fluorescent protein and the target gene are transcribed as a polycistronic mRNA.

[0043] In some preferred embodiments, the target gene is inserted into the 3' untranslated region downstream of the fluorescent protein. In some embodiments, the target gene sequence is inserted into the 3' untranslated region of the fluorescent protein as a contiguous sequence. In some preferred embodiments, the fluorescent protein and the target gene are transcribed as a single RNA construct.

[0044] In some embodiments, the portion of the target gene is one or more exons.

[0045] In some preferred embodiments, the target gene is a target gene of one or more inhibitory RNAs. In some preferred embodiments, the target gene is a target gene of one or more inhibitory RNAs according to the present invention. The target gene can be any target gene disclosed herein. In some preferred embodiments, the target gene is PDCD-1, B2M, PPARG, IRF4, KDM6B, FOXP3, or STAT6.

[0046] The constitutive promoter may be selected from the simian virus early promoter (SV40), human ubiquitin C promoter (UBC), human elongation factor 1 alpha promoter (EF1A), mouse phosphoglycerate kinase 1 promoter (PGK) and human phosphoglycerate kinase 1 (PGK1).

[0047] In some preferred embodiments, the constitutive promoter is EF1a.

[0048] The fluorescent protein may be selected from green fluorescent protein (GFP), blue fluorescent protein (BFP), and red fluorescent protein (e.g., dsRed, mCherry, RFP). In some preferred embodiments, the fluorescent protein is GFP, optionally CopGFP (a rapidly maturing bright variant).

[0049] In some embodiments, the reporter nucleic acid further comprises a WPRE (Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element). In some preferred embodiments, the reporter nucleic acid is flanked by long terminal repeat (LTR) sequences.

[0050] In some embodiments, the reporter nucleic acid further comprises a gene for a cell surface protein operably linked to a constitutive promoter. In some preferred embodiments, the cell surface protein is a tNGFR (low affinity NGF receptor) protein. The constitutive promoter operably linked to the gene for the cell surface protein may be selected from the group consisting of simian virus early promoter (SV40), human ubiquitin C promoter (UBC), human elongation factor 1 alpha promoter (EF1A), mouse phosphoglycerate kinase 1 promoter (PGK), and human phosphoglycerate kinase 1 (PGK1). In some embodiments, the constitutive promoter operably linked to the gene for the cell surface protein is PGK. Optionally, cell surface expression of tNGFR is used to identify successfully transfected cells using a labeled antibody.

[0051] In some embodiments, the reporter nucleic acid is for use in detecting the inhibition or silencing of a target gene by one or more inhibitory RNAs. The reporter nucleic acid is particularly useful in detecting the inhibition or silencing of a target gene, where the target gene does not encode a cell surface protein in living cells.

[0052] In a further aspect, there is provided the use of a reporter nucleic acid according to the invention in detecting the inhibition or silencing of a target gene by one or more inhibitory RNAs.

[0053] In a further aspect, - a reporter nucleic acid according to the invention, and - an expressible nucleic acid comprising a constitutive promoter operably linked to a nucleic acid sequence encoding an inhibitory RNA; An expression system comprising:

[0054] In some embodiments, the expressed nucleic acid comprises a constitutive promoter operably linked to a nucleic acid sequence encoding two or more inhibitory RNAs. In some embodiments, the expression system comprises two or more expressed nucleic acids, each of which comprises a constitutive promoter operably linked to a nucleic acid sequence encoding an inhibitory RNA. Expression systems having nucleic acid sequences encoding two or more inhibitory RNAs or two or more expressed nucleic acids are particularly useful for screening multiple inhibitory RNAs.

[0055] In some embodiments, the inhibitory RNA can be any inhibitory RNA according to any of the embodiments disclosed herein (e.g., in the "Inhibitory RNA" section). In some embodiments, the inhibitory RNA is a sequence encoding an endogenous non-coding RNA, e.g., a sequence encoding an endogenous inhibitory RNA, modified to retarget the endogenous inhibitory RNA to silence or partially silence a target gene. In some embodiments, the inhibitory RNA is a sequence encoding an endogenous non-coding RNA, e.g., a sequence encoding an endogenous inhibitory RNA, modified to retarget the endogenous inhibitory RNA to silence or partially silence a target gene via a GEiGS approach. In some embodiments, the inhibitory RNA is a GEiGS mature sRNA sequence (guide), a GEiGS solution sequence (pre-miRNA hairpin), or a GEiGS extended solution. In some preferred embodiments, the inhibitory RNA is a GEiGS extended solution.

[0056] In some preferred embodiments, the expressed nucleic acid is flanked by long terminal repeat (LTR) sequences.

[0057] In some embodiments, the reporter nucleic acid and the expression nucleic acid are provided on a single plasmid. In some embodiments, the reporter nucleic acid and the expression nucleic acid are provided on two separate plasmids.

[0058] In some embodiments, the reporter nucleic acid and the expression nucleic acid are provided in a single viral vector.In some embodiments, the reporter nucleic acid and the expression nucleic acid are provided in a single retroviral vector.

[0059] In some embodiments, the reporter nucleic acid and the expression nucleic acid are provided in two separate viral vectors. In some embodiments, the reporter nucleic acid and the expression nucleic acid are provided in two separate retroviral vectors.

[0060] In some embodiments where the expression system comprises two or more expression nucleic acids, the two or more expression nucleic acids may be provided in two or more separate plasmids or two or more separate viral vectors. In some embodiments where the expression system comprises two or more expression nucleic acids, the two or more expression nucleic acids may be provided in two or more separate lentiviral vectors.

[0061] In some embodiments, the inhibitory RNA is incorporated into or comprises an miRNA scaffold. The miRNA scaffold can be any miRNA scaffold disclosed herein. In some preferred embodiments, the inhibitory RNA is incorporated into an miRNA scaffold, the reporter nucleic acid is provided in a lentiviral vector, and the expression construct is provided in a lentiviral vector. Embodiments in which the inhibitory RNA is incorporated into or comprises an miRNA scaffold are advantageous because the inhibitory RNA incorporated into or comprising an miRNA scaffold is shorter than the usual length used for shRNA in lentiviral constructs. This is expected to lead to more efficient processing of the inhibitory RNA incorporated into the miRNA scaffold compared to usual shRNA constructs.

[0062] In some embodiments, the expressed nucleic acid further comprises a gene encoding an additional fluorescent protein operably linked to a constitutive promoter. The additional fluorescent protein may be selected from green fluorescent protein (GFP), blue fluorescent protein (BFP), and red fluorescent protein (e.g., dsRed, mCherry, RFP). In some embodiments, the additional fluorescent protein is dsRed. In some embodiments, the nucleic acid sequence encoding the inhibitory RNA and the gene encoding the additional fluorescent protein are transcribed as a single RNA construct. In some embodiments, the nucleic acid sequence encoding the inhibitory RNA and the gene encoding the fluorescent protein are transcribed as a single RNA construct separated by a T2A sequence.

[0063] In some embodiments, the constitutive promoter in the expressed nucleic acid is selected from simian virus early promoter (SV40), human ubiquitin C promoter (UBC), human elongation factor 1 alpha promoter (EF1A), mouse phosphoglycerate kinase 1 promoter (PGK), and human phosphoglycerate kinase 1 (PGK1). In some embodiments, the constitutive promoter in the expressed nucleic acid is EF1a.

[0064] In some preferred embodiments, the expression system has nucleic acid sequences encoding two or more inhibitory RNAs or two or more expressible nucleic acids, and the reporter nucleic acid and the expressible nucleic acid are provided in one or more viral vectors, preferably lentiviral vectors.

[0065] In some preferred embodiments, the expression system comprises two or more expressible nucleic acids, wherein the reporter nucleic acid is provided in a lentiviral vector and the two or more expressible nucleic acids are provided in separate lentiviral vectors. This embodiment is particularly useful for screening inhibitory RNAs (in pools) in an efficient, cost-effective manner, as detailed in Examples 21-22 and Example 10.

[0066] In a further aspect, there is provided a method for detecting the inhibitory or silencing activity of one or more inhibitory RNAs against a target gene, comprising: (i) transfecting a cell(s) with an expression system according to the invention; (ii) binding of inhibitory RNA to target genes within polycistronic mRNAs; (iii) degradation of polycistronic mRNAs, and (iv) a reduction in the fluorescent signal from the fluorescent protein in the cell(s). A method is provided that includes:

[0067] In some embodiments, binding of the inhibitory RNA to a target gene within the polycistronic mRNA results in degradation of the polycistronic mRNA, hi some embodiments, degradation of the polycistronic mRNA results in a reduction in fluorescent signal from the fluorescent protein in the cell(s).

[0068] In some embodiments, the method of detecting inhibitory or silencing activity further comprises incubating the cell(s) under conditions suitable for expression of the reporter nucleic acid and the expressed nucleic acid, hi some embodiments, the method of detecting inhibitory or silencing activity further comprises detecting a reduction in fluorescent signal.

[0069] In some embodiments, the reduction in the fluorescent signal from the fluorescent protein in the cell(s) is detected by flow cytometry, a fluorescent microscope, or any fluorescent imaging system. In some embodiments, the reduction in the fluorescent signal from the fluorescent protein in the cell(s) is detected by flow cytometry.

[0070] The cell(s) can be any cell disclosed herein. In some preferred embodiments, the cell is a macrophage. In some preferred embodiments, the cell is an iPSC. In some preferred embodiments, the cell is a T cell. In some preferred embodiments, the cell is a primary T cell. In some preferred embodiments, the cell is a T cell line, preferably a Jurkat.

[0071] In embodiments in which the cell is a T cell, the reporter nucleic acid and the expression nucleic acid are preferably provided in a single lentiviral vector or in two or more separate lentiviral vectors. Plasmid-based approaches are limited in their ability to transduce T cells, and using lentiviral delivery is beneficial in detecting the inhibitory or silencing activity of multiple inhibitory RNAs.

[0072] In some preferred embodiments, the expression system has nucleic acid sequences encoding two or more inhibitory RNAs or two or more expressible nucleic acids, and the reporter nucleic acid and the expressible nucleic acid are provided in one or more viral vectors, preferably lentiviral vectors. This embodiment is particularly useful for screening inhibitory RNAs in an efficient, cost-effective manner, as detailed in Examples 21-22 and Example 10.

[0073] In some embodiments, where the cell is an iPSC, the reporter nucleic acid and the expression nucleic acid are preferably provided in a single lentiviral vector, or in two or more separate lentiviral vectors.Plasmid-based screening in iPSC is limited in the number of plasmids that can be transduced, and requires multiple inhibitory RNAs to be screened in arrays.On the other hand, lentiviral delivery allows multiple inhibitory RNAs to be screened at any one time.

[0074] In a further aspect, there is provided a method for selecting an inhibitory RNA(s) that exhibits silencing or inhibition of a target gene from a pool of test inhibitory RNAs, comprising: (i) transfecting a cell(s) with an expression system according to the invention; (iii) selecting the inhibitory RNA(s) transfected into the cell(s) that exhibit a reduced fluorescent signal from the fluorescent protein; A method is provided that includes:

[0075] In some embodiments, the expression system comprises a nucleic acid sequence encoding each of a pool of test inhibitory RNAs or two or more expressible nucleic acids each encoding one of the pool of test inhibitory RNAs.

[0076] In some embodiments, the selection method further comprises detecting and selecting the inhibitory RNA that exhibits the highest silencing or inhibition of the target gene among the selected inhibitory RNAs. In some embodiments, the detecting and selecting step is carried out by detecting distinct cell populations corresponding to each inhibitory RNA by flow cytometry and selecting the inhibitory RNA transfected into the cell population that has the highest silencing or inhibition of the target gene.

[0077] In some embodiments, binding of the inhibitory RNA to a target gene within the polycistronic mRNA results in degradation of the polycistronic mRNA, hi some embodiments, degradation of the polycistronic mRNA results in a reduction in fluorescent signal from the fluorescent protein in the cell(s).

[0078] In some embodiments, the method of selecting an inhibitory RNA further comprises incubating the cell(s) under conditions suitable for expression of the reporter nucleic acid and the expressed nucleic acid, hi some embodiments, the method of selecting an inhibitory RNA further comprises detecting a decrease in the fluorescent signal.

[0079] In some embodiments, the reduction in the fluorescent signal from the fluorescent protein in the cell(s) is detected by flow cytometry, a fluorescent microscope, or any fluorescent imaging system. In some embodiments, the reduction in the fluorescent signal from the fluorescent protein in the cell(s) is detected by flow cytometry.

[0080] The cell(s) can be any cell disclosed herein. In some preferred embodiments, the cell is a macrophage. In some preferred embodiments, the cell is an iPSC. In some preferred embodiments, the cell is a T cell. In some preferred embodiments, the cell is a primary T cell. In some preferred embodiments, the cell is a T cell line, preferably a Jurkat.

[0081] In embodiments in which the cell is a T cell, the reporter nucleic acid and the expression nucleic acid are preferably provided in a single lentiviral vector or in two or more separate lentiviral vectors. Plasmid-based approaches are limited in their ability to transduce T cells, and using lentiviral delivery is beneficial in detecting the inhibitory or silencing activity of multiple inhibitory RNAs.

[0082] In some embodiments, where the cell is iPSC, the reporter nucleic acid and the expression nucleic acid are preferably provided in a single lentiviral vector, or in two or more separate lentiviral vectors.The plasmid-based screening in iPSC is limited in number, and requires multiple inhibitory RNAs to be screened in arrays.Lentiviral delivery allows multiple inhibitory RNAs to be screened at any one time.

[0083] Various aspects, embodiments and examples of the invention are described in further detail below.

[0084] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating the plural as well as the singular unless the context otherwise requires.

[0085] It is to be understood that any property, integer, feature, compound, chemical moiety, or group described in connection with a particular aspect, embodiment, or example of the invention is applicable to any other aspect, embodiment, or example described herein, to the extent not incompatible therewith. [Brief explanation of the drawings]

[0086] [Figure 1] FIG. 1 shows a schematic diagram of MHC-I on the surface of cells and the immune surveillance system. [Figure 2] FIG. 2 shows a schematic illustration of the interaction of cells containing non-self MHC-I and those completely lacking MHC-I with cells of the adaptive and innate immune systems. [Figure 3] FIG. 3 illustrates a strategy for generating hypoimmune cells based on downregulating MHC-I expression to an optimal level that can avoid both T cell- and NK cell-mediated responses. [Figure 4] FIG. 4 shows a schematic illustration of the use of the GEiGS workflow to develop, test and implement modified cells according to the present invention. [Figure 5] Figure 5 shows the plasmid map of plasmid VB210602-1567ytv, which was used for ectopic (episomal) expression to assess the ability of miRNA sequences determined by GEiGS ("GEiGS Solutions") to silence B2M expression. [Figure 6]Figure 6 shows a graph of small RNA real-time PCR (RT-PCR) in iPSCs transfected with a plasmid expressing either a negative control expression plasmid or B2M-targeted Solution 43. GEiGS Solution 43 is detected only in the corresponding samples, demonstrating the specificity of the assay. Solution 43 is expressed at levels comparable to a panel of endogenous miRNAs. [Figure 7] FIG. 7 shows the flow cytometry method and gating to assess the ability of a given GEiGS silencing RNA to reduce B2M expression. [Figure 8] FIG. 8 shows a graph illustrating the ability or range of inhibitory / silencing RNA sequences developed through the GEiGS process ("GEiGS solutions") to reduce B2M expression in cells as determined by flow cytometry. Each GEiGS solution is a combination of a miRNA scaffold and a specific sequence change complementary to B2M mRNA that causes degradation of the B2M mRNA. Solutions are developed through a computational workflow to reduce B2M expression. Series 1 illustrates the results for all cells expressing DsRed (all successfully transfected cells), while Series 2 shows the results for the top 20%, i.e., cells expressing high levels of DsRed and, therefore, high levels of GEiGS solution (silencing RNA, sRNA). [Figure 9] Figure 9 shows the average silencing activity of GEiGS in clonal cell lines in which the GEiGS solution was knocked into one or both alleles corresponding to the genomic location of the original encoded miRNA using gene editing techniques. Data are expressed as residual B2M expression measured by flow cytometry, averaged across multiple clones of the same genotype. [Figure 10]Figure 10 shows the average silencing activity of GEiGS in clonal cell lines in which the GEiGS solution was knocked into one or both alleles corresponding to the genomic location of the original encoded miRNA using gene editing techniques. Data are expressed as residual B2M expression measured by flow cytometry, averaged across multiple clones of the same genotype. [Figure 11] Figure 11 shows the miRNA expression of endogenous miRNAs in Solutions 12 and 30 in iPSCs and pancreatic progenitor cells. Expression of Solution 30 miRNA (hsa-mir-21) is specific to pancreatic progenitor cells, while expression of Solution 12 miRNA (hsa-mir-302c) is specific to iPSCs. [Figure 12a] Figure 12a) shows results demonstrating reduced surface B2M expression. Approximately 24% of cells in a CD3-positive, mixed T cell population exhibited silencing of B2M, with an 80% reduction in mean fluorescence intensity compared to control, unedited cells. [Figure 12b] Figure 12b) shows quantification of B2M silencing in helper (CD4+) and cytotoxic (CD8+) T cells, demonstrating efficient silencing of B2M (approximately 90%) in both compartments. [Figure 13] FIG. 13 shows a schematic illustration of the use of the GEiGS approach to promote T cell activity in the tumor microenvironment. [Figure 14] Figure 14 shows a schematic illustration of a strategy for using GEiGS in macrophage therapy in glioblastoma. GEiGS is used to prevent macrophages from adopting a tumor-promoting state (i.e., tumor-associated macrophages, TAMs) and promote an active inflammatory phenotype in the tumor microenvironment (TME). Context-specific GEiGS is used to target the IL-4-inducible genes STAT6 and IRF4 to promote inflammatory gene expression. [Figure 15]Figure 15 shows a schematic illustration of how GEiGS-modification can be used to specifically silence additional pathways, for example, only once T cells reach a tumor. Context-specific GEiGS-mediated inhibition of mitochondrial fission in the TME by redirecting induced miRNAs to conditionally silence DRP1 favors memory T cell differentiation and persistence, allowing mitochondrial dynamics to continue and contribute to mitochondrial homeostasis (e.g., mitophagy). [Figure 16] Figure 16 shows a scatter plot of the transcriptome of primary T cells, illustrating the fold change (from untreated to stimulated) in counts per million. This demonstrates that stimulation-induced miRNAs can be detected in primary T cells. Candidate upregulated miRNAs are highlighted by large circles (data from Amaral et al., 2017, EMBO J, EMBO J. 2017 Feb 1;36(3):346-360). [Figure 17] Figure 17 is a graph illustrating primary T cell editing efficiency with CRISPR knock-in (KI) (GFP KI at the RAB11A locus) demonstrating that T cells can be edited with high efficiency by CRISPR KI (data from Roth et al., 2018, Nature Nature. 2018 Jul;559(7714):405-409). [Figure 18a] Figure 18a shows differential expression analysis of miRNAs and mRNAs in primary macrophages. Plots show expression level (x-axis, log2 of normalized counts) versus fold change (y-axis, log2 transformed). Comparisons are between pro-inflammatory (M1) and anti-inflammatory (M2a). Dots represent individual genes, and highlighted values ​​indicate statistically significant differences between conditions. Values ​​above 0 are high anti-inflammatory and low pro-inflammatory; values ​​below 0 are low anti-inflammatory and high pro-inflammatory. [Figure 18b]Figure 18b shows efficient differentiation and polarization of THP-1 cells into pro- and anti-inflammatory macrophage-like cells. The plot shows flow cytometry detection of known macrophage polarization markers (CD80 for pro-inflammatory, CD209 for anti-inflammatory). [Figure 18c] FIG. 18c shows a correlation analysis of miRNA expression in polarized primary macrophages and THP-1 cells, demonstrating a high degree of correlation between the two experimental models. [Figure 19a] Figure 19a shows efficient generation of monocytes from iPSCs. Using iPSC lines (GEiGS Solution 30 (S30 / S30 genotype) and isogenic controls), enriched monocytes were generated using published protocols, and over 90% of the derived cells were double positive for the known monocyte markers CD14 and CD45. [Figure 19b] Figure 19b shows context-specific silencing of B2M in iPSC-derived monocytes. iPSC lines (GEiGS Solution 30 and isogenic control) were used to generate monocytes, which were then stained for residual cell surface B2M expression using flow cytometry (as described in Figures 9, 10, and 12). When expanded as undifferentiated iPSCs, over 93% of cells expressed B2M for both the isogenic control and GEiGS Solution 30 clonal lines. However, after differentiation into monocytes, the GEiGS Solution 30 line showed a significant reduction in the number of cells with the same amount of cell surface-expressed B2M compared to the isogenic control line (77% compared to 16% of cells with residual B2M). [Figure 20a] FIG. 20a illustrates an illustration of the process of identifying context-specific GEiGS miRNAs and performing an ectopic screen to assess target RNA silencing in target cells using a dual reporter plasmid system. [Figure 20b]FIG. 20b shows the plasmid maps of the plasmids used to perform an ectopic expression screen to identify GEiGS solutions targeting the genes PPARγ, IRF4, KDM6B, and STAT6 in monocyte-derived macrophages (see Example 9). [Figure 20c] FIG. 20c shows the plasmid maps of the plasmids used to perform an ectopic expression screen to identify GEiGS solutions targeting the genes PPARγ, IRF4, KDM6B, and STAT6 in monocyte-derived macrophages (see Example 9). [Figure 20d] Figure 20d shows a dual reporter construct containing the 3' untranslated sequence of a target gene in macrophages for analysis by GFP regression, the specificity of the GEiGS solution. [Figure 21a] Figure 21a shows the results of context-specific silencing of B2M in iPSC-derived monocytes, in relation to the results shown in Figure 19. Figure 21a shows the expression of hsa-mir-21 in iPSCs compared to macrophages; miR-21 is highly expressed in macrophages but not in iPSCs (NGS quantification was used). [Figure 21b] Figure 21b illustrates the results of context-specific silencing of B2M in iPSC-derived monocytes, in relation to the results shown in Figure 19. Figure 21b shows the relative B2M expression in GEiGS-modified iPSCs, monocytes, and macrophages compared to control cells in which endogenous miR-21 was knocked out (KO / KO) (miR-21 redirected to target B2M, i.e., Solution 30). GEiGS and control lines were differentiated in vitro first into monocytes and then into macrophages. B2M was assayed by flow cytometry and showed effective silencing (84-88% silencing) in myeloid cells but not at the iPSC stage. [Figure 21c]FIG. 21c shows that B2M silencing in myeloid cells results in a reduction of cell surface HLA-A / B / C (and therefore MHC-I) in monocytes and macrophages. [Figure 21d] FIG. 21d shows that B2M silencing in myeloid cells results in a reduction of cell surface HLA-A / B / C (and therefore MHC-I) in monocytes and macrophages. [Figure 21e] FIG. 21e shows that B2M silencing in myeloid cells results in a reduction of cell surface HLA-A / B / C (and therefore MHC-I) in monocytes and macrophages. [Figure 21f] FIG. 21f shows that B2M silencing in myeloid cells results in a reduction of cell surface HLA-A / B / C (and therefore MHC-I) in monocytes and macrophages. [Figure 22a] Figure 22a shows the results of a study demonstrating that GEiGS is highly specific and has no off-target effects. In particular, the GEiGS approach described herein for silencing B2M is highly specific and has no significant off-target effects on expression other than B2M. The MA plot of differential gene expression analysis shows the log2 of mean counts per million on the x-axis and the log2 of fold change on the y-axis. Dots represent individual genes tested. Significantly downregulated genes are highlighted in blue, and significantly upregulated genes are highlighted in red. Genes without significant changes in expression levels are shown in black. Figure 22a shows transcriptome data from cells modified by CRISPR B2M knockout compared to the control WT / WT parental cell line. The significantly reduced expression level of B2M compared to the control is highlighted. No other significant changes in expression levels were evident for other genes. [Figure 22b]Figure 22b shows the results of a study demonstrating that GEiGS is highly specific and has no off-target effects. In particular, the GEiGS approach described herein for silencing B2M is highly specific and has no significant off-target effects on expression other than B2M. The MA plot of differential gene expression analysis shows the log2 of mean counts per million on the x-axis and the log2 of fold change on the y-axis. Dots represent individual genes tested. Significantly downregulated genes are highlighted in blue, and significantly upregulated genes are highlighted in red. Genes without significant changes in expression levels are shown in black. Figure 22b shows transcriptome data from cells with hsa-mir-20a knockout compared to the control WT / WT parental cell line. Note that hsa-mir-20a is the miRNA edited when "Solution 29" is introduced by GEiGS. No significant changes in expression levels are evident as a result of hsa-mir-20a knockout. [Figure 22c] Figure 22c shows the results of a study demonstrating that GEiGS is highly specific and has no off-target effects. In particular, the GEiGS approach described herein for silencing B2M is highly specific and has no significant off-target effects on expression other than B2M. The MA plot of differential gene expression analysis shows the log2 of mean counts per million on the x-axis and the log2 of fold change on the y-axis. Dots represent individual genes tested. Significantly downregulated genes are highlighted in blue, and significantly upregulated genes are highlighted in red. Genes without significant changes in expression levels are shown in black. Figure 22c shows transcriptome data for cells with miRNA solution 29 introduced by GEiGS compared to the control WT / WT parental cell line. As can be seen, similar to CRISPR KO, only B2M shows significant changes in expression. No other significant changes in expression levels are evident for other genes. [Figure 23]Figure 23 shows the effect of homology arm length on the efficiency of homology-dependent repair (HDR). Genomic sequences flanked by 40 base pairs in length (homology arms) show a dose-dependent superior effect on HDR efficiency (% HDR) compared to flanking genomic sequences 350 bp in length or 150-350 bp in length, which are only slightly better than the donor-less template alone. [Figure 24] Figure 24 shows that partial knockdown of B2M results in reduced specific lysis by activated NK cells in tissue culture (corresponding to enhanced cell survival after exposure to the innate (NK-mediated) immune system). NK and target cells were cultured under different experimental conditions, including exposure to the NK cell-activating cytokines IL-2 and / or IL-12. Control iPSCs showed reduced levels of specific lysis compared to the positive control K562 cell line, which is known to be a potent stimulator of activated NK cell-mediated killing. Because the complete absence of B2M results in the complete absence of MHC-I (a "loss of self" phenotype) and detection and lysis by activated NK cells, undifferentiated B2M- / - iPSCs showed increased levels of specific lysis. The undifferentiated, GEiGS-mediated iPSC line (iPSCS29 / 29) consistently showed reduced specific lysis by NK cells compared to the B2M- / - cell line across all mixed culture conditions tested. [Figure 25] Figure 25 shows the ability of Solution 29 to buffer the induction of B2M expression (as measured by MHC-I detection) in edited cells following exposure to an inflammatory stimulus (IFN-γ). [Figure 26] Figure 26 shows the ability of Solution 30 heterozygous iPSC lines to differentiate into CXCR4-expressing definitive endoderm cells. The heterozygous knock-in line (iPSC miR-21S30 / +1) showed a higher percentage of CXCR4-positive cells (approximately 97%) compared to both control (unmodified, 50%) and B2M- / - iPSCs (95%). [Figure 27a]Figure 27a shows the reduction of B2M expression in the percentage of CXCR4-expressing cells generated by in vitro differentiation of the Solution 30 heterozygous iPSC line. The miR-21S30 / +1 iPSC line showed approximately an 80% reduction in B2M expression compared to the control (unmodified) iPSC line. As expected, B2M- / - iPSCs do not express B2M protein. [Figure 27b] Figure 27b shows the expression of pancreatic and duodenal homeobox-1 (PDX-1) and B2M in pancreatic progenitor cells. The results show that a significant proportion of specific progenitor cells is positive for PDX-1, a marker gene required for pancreatic development and beta cell maturation. The majority of PDX-1-positive cells also stained for B2M. iPSC miR-21S30 / +1 clonal lines showed a high proportion of PDX-1-expressing cells that had significantly reduced B2M expression compared with control (unmodified) iPSCs. [Figure 28] FIG. 28 shows the flow cytometry gating strategy used for the determination of GEiGS solution "hits" against macrophage target genes using a dual reporter system. [Figure 29] FIG. 29 shows the identification of macrophage target genes (STAT6, IRF4, and KDM6B) GEiGS solutions from an ectopic screen using dual reporter system constructs. [Figure 30] FIG. 30 illustrates an ectopic screening strategy using a dual reporter system to identify "hits" from engineered GEiGS solutions against T cell targets of interest. [Figure 31a] FIG. 31a shows the constructs used to validate the use of lentiviral transduction and pooled screening strategies. [Figure 31b] FIG. 31b illustrates the use of lentiviral transduction and pooled screening strategies to identify "hits" from GEiGS solutions engineered using primary human T cells. [Figure 32a] FIG. 32a shows validation of the lentiviral-based pooled screening strategy in primary human T cells using validated solutions for both eGFP and B2M. [Figure 32b] FIG. 32b shows DsRed and B2M knockdown corresponding to individual tested solutions (selected from Example 2) and as combined solutions delivered via lentivirus in the Jurkat cell line. [Figure 33a] Figure 33a shows that multiple solutions can silence their target genes when ectopically expressed from a single construct with a constitutive promoter in iPSCs. Figure 33a shows a plasmid map for the expression of one or more solutions. The plasmid construct is represented using a miR-30-based solution targeting eGFP downstream of a dsRed reporter in a multiple cloning site (MCS) driven by a constitutive EF1a promoter. The eGFP-targeting solution is replaced with one or more solutions designed against B2M and eGFP or B2M alone (see Example 23). [Figure 33b] Figure 33b shows that multiple solutions can silence their target genes when ectopically expressed in iPSCs from a single construct with a constitutive promoter. Figure 33b shows that solution 101, consisting of miRNA-30 repurposed to target GFP alone, demonstrated efficient silencing of GFP without affecting the level of B2M expression in undifferentiated iPSCs, and solution 179, consisting of two separate engineered miRNA scaffolds (miRNA-20a and miRNA-30), was effective in silencing both B2M and eGFP with apparently comparable efficiency. [Figure 34]Figure 34 shows that Solution 177, consisting of modified miRNA-518b (Solution 43) and miRNA-20a (Solution 29), separate solutions with diverse sequences targeting the B2M transcript (trigger), appeared more effective at silencing B2M compared to Solution 43 alone, demonstrating minimal additive effects and that the two separate solutions do not interfere with each other's ability to silence B2M. On the other hand, Solution 178, which contains two copies of Solution 29 and the same trigger sequence, has no additive effect on silencing B2M compared to Solution 29 alone. DETAILED DESCRIPTION OF THE INVENTION

[0087] Detailed Description of Embodiments and Examples of the Invention While the making and using of various embodiments of the invention are discussed in detail below, it should be understood that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0088] To facilitate understanding of the present invention, several terms are defined below. Terms defined herein have meanings commonly understood by one of ordinary skill in the art in the areas relevant to the present invention. Terms such as "a," "an," and "the" are not intended to refer to only a single entity, but include a general class for which a particular example may be used for illustration. While technical terms are used herein to describe particular embodiments of the present invention, their use does not delimit the scope of the invention, except as outlined in the claims.

[0089] definition "Context-specific activity," "context-specific inhibition," "context-specific manner," and similar phrases refer to the inhibition of a target gene or target RNA by an inhibitory RNA that occurs preferentially, primarily, or only in a given cellular context. In some embodiments, "context-specific" can be "cellular context-specific" inhibition of a target gene or target RNA by an inhibitory RNA that occurs preferentially, primarily, or only in individual cells in a given cellular context. In some embodiments, "context-specific" does not include tissue-specificity.

[0090] "Context" in this context refers to any suitable cell type, cell condition, or cell state, as appropriate. A "first context" is a context in which it is desirable to selectively regulate (e.g., silence) the expression of a target gene. In particular, a first context is typically a cellular context in which there is increased activity of a given endogenous inhibitory RNA (e.g., miRNA) compared to other contexts. Thus, the activity of endogenous RNA can be utilized to provide context-specific gene silencing. A "second context" can be any context in which it is desirable for a target gene to be unsilenced or silenced to a lesser extent. For example, the first or second context of a cell of the present invention can be selected from the following non-exhaustive list: a cell type, a stage of cell differentiation, a disease state, a hypoxic state, a stimulated state, an inflammatory state, a tumor-related state, etc. In some embodiments, the second context is any context other than the first context. In some embodiments, the second context is a different stage of cell differentiation. In some embodiments, the second situation is when the cell is not in its intended location (e.g., in the TME). Further situations are discussed in more detail herein. The target mRNA (i.e., the mRNA that is the target of the inhibitory RNA of the present invention) is usually at least partially silenced in the first situation, and is not silenced or is silenced to a lesser extent in situations other than the first situation. The target mRNA can be, for example, an mRNA that is upregulated in the first situation and is therefore expressed at a high level in the first situation.

[0091] The terms "RNA interference" and "RNAi" are synonymous and refer to the process by which a polynucleotide (e.g., miRNA or siRNA) containing at least one polyribonucleotide unit exerts an effect on a biological process. The process includes, but is not limited to, gene silencing by degrading mRNA, attenuating translation, interacting with tRNA, rRNA, hnRNA, cDNA, and genomic DNA, and methylating DNA with auxiliary proteins. "Inhibitory RNA" is an RNA molecule capable of RNA interference either directly or after cellular processing. Thus, this term includes, inter alia, RNA precursors that are processed to form miRNA, siRNA, or other RNAs that can degrade target mRNA. The term "sRNA" is also used herein to refer to inhibitory RNA (i.e., silencing RNA, abbreviated as sRNA).

[0092] The term "silencing" or "gene silencing" refers to the process by which the expression of a specific gene product is reduced or attenuated, in this case, usually by RNA interference. Target genes are typically silenced as a result of an inhibitory RNA that targets (e.g., binds via complementarity to) a target RNA encoded by the gene. Thus, silencing a gene in this context is typically achieved by targeting a target RNA transcribed from the gene with an inhibitory RNA as described herein. Silenced genes typically encode protein gene products, but may also encode RNA gene products. The level of gene silencing (sometimes referred to as "knockdown" or the degree of reduced expression) can be measured by various means, including, but not limited to, Northern blot analysis, B-DNA technology, transcription-sensitive reporter constructs, expression profiling (e.g., DNA chips), qRT-PCR, and related techniques to measure transcript levels. Alternatively, the level of silencing can be measured by assessing the level of protein encoded by a specific gene. This can be achieved by performing several studies involving antibody-based detection, such as Western analysis and flow cytometry, measuring the expression level of a reporter protein with fluorescent properties (e.g., GFP) or enzymatic activity (e.g., alkaline phosphatase), or several other procedures. Typically, the level of silencing or reduction is compared to the level of expression of a given protein or mRNA in control cells, as appropriate. Depending on the circumstances, silencing of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% may be preferred, for example. Higher levels of silencing are often preferred.

[0093] The terms "microRNA," "miRNA," or "miR" are synonymous and all refer to non-coding RNAs (and, as the context indicates, DNA sequences encoding such RNAs) approximately 19-24 nucleotides in length that can, for example, enter the RNAi pathway and regulate gene expression. A "primary miRNA" or "pri-miRNA" refers to the non-coding transcript before Drosha processing, including the stem-loop structure(s) and adjacent 5' and 3' sequences. A "precursor miRNA" or "pre-miRNA" refers to the non-coding transcript after Drosha processing of the pri-miRNA. The term "mature miRNA" can refer to the double-stranded product resulting from Dicer processing of the pre-miRNA or the single-stranded product introduced into RISC after Dicer processing. In some cases, only one strand of the miRNA enters the RNAi pathway. In other cases, both strands of the miRNA can enter the RNAi pathway. miRNAs are found in a wide range of organisms (eg, insects, mammals, plants, nematodes) and are known to play roles in development, homeostasis, and disease pathogenesis.

[0094] The terms "silencing RNA form" or "silencing RNA" or "silencing RNA molecule," "sRNA" or "trigger sequence / RNA" refer to mature small RNA molecules that are capable of hybridizing to a target RNA (or a fragment thereof) and engage the RNAi pathway.

[0095] The term "target RNA" refers to a specific RNA that is targeted by the RNAi pathway, resulting in a decrease in the functional activity of the RNA. In some cases, the RNA target is an mRNA (usually B2M) whose functional activity is its ability to be translated. In such cases, the RNAi pathway decreases the functional activity of the mRNA by attenuating translation or by cleavage. In the present disclosure, the target RNA is targeted by a non-naturally occurring miRNA. The term "target" can also refer to DNA.

[0096] The term "endogenous miRNA" refers to a miRNA produced in an organism by transcription of a sequence naturally present in the genome of the organism. Endogenous miRNAs may be located, for example, in an intron, an open reading frame (ORF), a 5' or 3' untranslated region (UTR), or an intergenic region. Organisms that produce endogenous miRNAs may include, but are not limited to, humans (and other primates), mice, rats, flies, worms, fish, or other organisms with intact RNAi pathways. In some embodiments of the present invention, endogenous miRNA loci are altered to retarget endogenous miRNAs to novel targets (e.g., see WO2019 / 058253, WO2020 / 183414, and WO2020 / 183419 for some suitable approaches using an approach called GEiGS). Endogenous miRNAs can be modified in situ, i.e., the endogenous miRNA in the genome can be modified directly, or a copy of the endogenous miRNA can be removed from its normal genomic context and modified (e.g., for ectopic expression in the cell, e.g., either at a different genomic locus or episomally in a suitable vector). A modified copy of an endogenous miRNA can also be inserted into the locus of the original endogenous miRNA so that it is co-transcribed with the original endogenous miRNA, which can be achieved without disrupting or inactivating the original endogenous miRNA (e.g., by placing the modified copy of the endogenous miRNA upstream or downstream of the original endogenous miRNA).

[0097] The term "nucleic acid construct adapted to express an inhibitory RNA," as used herein, refers to any polynucleotide sequence suitable for expressing an inhibitory RNA described herein. The nucleic acid construct may be genomic or episomal. In some embodiments, the nucleic acid construct may be, for example, a modified endogenous genomic sequence that has been genetically modified, e.g., gene-edited, to be adapted to express an inhibitory DNA. In some embodiments, the nucleic acid construct may be a synthetic construct, e.g., an expression cassette, in a suitable vector. In other embodiments, the nucleic acid construct may be a synthetic construct that has been inserted into a genome. In some preferred embodiments, the construct is a modified endogenous genomic locus in which an endogenous sequence encoding a non-coding RNA has been modified (in situ) to provide a sequence encoding an inhibitory RNA that has been retargeted so that it can silence a target gene.

[0098] Terms such as "identity" and "identical" refer to similar sequences between two polymer molecules, for example, between two nucleic acid molecules, for example, between two DNA molecules. Sequence alignment and sequence identity determination can be performed, for example, using the Basic Local Alignment Search Tool (BLAST), first described by Altschul et al., 1990 (J Mol Biol 215: 403-10), or the "BLAST2 sequence" algorithm, for example, described by Tatusova and Madden, 1999 (FEMS Microbiol Lett 174: 247-250).

[0099] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described, for example, in Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31, and Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50. A detailed discussion of sequence alignment methods and homology calculations can be found, for example, in Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[0100] The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™; Altschul et al. (1990)) is available from several sources, including the National Center for Biotechnology Information (Bethesda, Maryland) and on the Internet, for use with several sequence analysis programs. Instructions on how to determine sequence identity using this program are available on the Internet under the "Help" section of BLAST™. For comparison of nucleic acid sequences, the "BLAST2 Sequence" function of the BLAST™ (Blastn; Align Sequence Nucleotide BLAST) program can be used using default parameters. Nucleic acid sequences with greater similarity to a reference sequence will exhibit increasing percentage identities when assessed by this method. Percent sequence identity is usually calculated over the entire length of the sequence.

[0101] For example, a global optimal alignment is conveniently found by the Needleman-Wunsch algorithm using the following scoring parameters: match score: +2, mismatch score: -3; gap penalty: gap open 5, gap extension 2. The percentage identity of the resulting optimal global alignment is conveniently calculated by multiplying the ratio of the number of aligned bases to the total length of the alignment, including both matches and mismatches, by 100.

[0102] The term "complementarity" refers to the ability of polynucleotides to base pair with each other. Base pairs are usually formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G) or in any other manner that allows for the formation of a duplex, including wobble base pairs formed between U and G. As those skilled in the art will be aware, when using RNA rather than DNA, uracil is the base that is considered to be complementary to adenosine rather than thymine. However, when U is indicated in the context of the present invention, the ability to substitute for T is implied unless otherwise specified.

[0103] Full complementarity, or 100% complementarity, refers to the ability of each nucleotide unit of one polynucleotide strand to hydrogen bond with a nucleotide unit of a second polynucleotide strand. Partial complementarity refers to the ability of some, but not all, nucleotide units of two strands to hydrogen bond with each other. For example, two strands are at least partially complementary if at least 6-7 base pairs can form over a strand of approximately 19-25 nucleotides. Sequences are said to be "complementary" to each other if each sequence is the (partial or complete) reverse complement (RC) of the other. For example, the sequence 5'GATC3' is perfectly complementary to its reverse complementary sequence 3'CTAG5'. Sequences may also have wobble base pairing.

[0104] The term "expression cassette", as used herein, includes a polynucleotide sequence encoding a polypeptide or RNA to be expressed and sequences controlling its expression, including any combination of cis-acting transcriptional control elements, e.g., a promoter and optionally enhancer sequences.

[0105] The term "eukaryotic cell," as used herein, refers to any cell of a eukaryotic organism. Eukaryotes include unicellular and multicellular organisms. Unicellular eukaryotes include, but are not limited to, yeast, protozoa, slime molds, and algae. Multicellular eukaryotes include, but are not limited to, animals (e.g., mammals, insects, invertebrates, nematodes, birds, fish, reptiles, and crustaceans), plants, fungi, and algae (e.g., brown algae, red algae, green algae).

[0106] As used herein, the term "stem cell" refers to a cell that can remain undifferentiated in culture for extended periods while capable of self-renewal, but also retains the ability to differentiate into one or more distinct cell types (e.g., fully differentiated cells) with specific, specialized functions. Stem cells can be totipotent, pluripotent, multipotent, or unipotent until induced to differentiate into other cell types. Totipotent cells, e.g., embryonic cells within the first few cell divisions after fertilization, can differentiate into embryonic and extraembryonic cells and are the only cells that can develop into viable humans. Preferably, the term "pluripotent stem cells" refers to cells that can differentiate into all three definitive embryonic germ layers, i.e., ectoderm, endoderm, and mesoderm, or remain undifferentiated. Pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Multipotent stem cells include adult stem cells and hematopoietic stem cells.

[0107] "Induced pluripotent stem cells" (iPSCs; embryonic-like stem cells) refer to cells obtained by dedifferentiating adult somatic cells to endow the cells with pluripotent properties, i.e., cells that can differentiate into the three embryonic germ cell layers, i.e., endoderm, ectoderm, and mesoderm. Such cells can be obtained from differentiated tissues (e.g., somatic tissues, e.g., skin) and dedifferentiated by genetic manipulation to reprogram the cells to acquire embryonic stem cell characteristics. Induced pluripotent stem cells can be formed by inducing expression of Oct-4, Sox2, Kfl4, and c-Myc in somatic cells. Induced pluripotent stem cells (iPSCs) (lung-like stem cells) can be generated from somatic cells by genetic manipulation of the somatic cells, e.g., by retroviral transduction of somatic cells, e.g., fibroblasts, hepatocytes, gastric epithelial cells, with transcription factors such as Oct-3 / 4, Sox2, c-Myc, and KLF4, such as those described in Park et al., "Reprogramming of human somatic cells to pluripotency with defined factors." Nature (2008) 451:141-146. iPSCs can be human or non-human.

[0108] The term "embryonic stem cells" refers to embryonic cells that can differentiate into cells of all three embryonic germ layers (i.e., endoderm, ectoderm, and mesoderm) or remain in an undifferentiated state. The term "embryonic stem cells" can include embryonic tissues (e.g., blastocysts) formed after early pregnancy before implantation of the embryo (i.e., pre-implantation blastocysts), expanded blastocyst cells (EBCs) obtained from post-implantation / pre-gastrulation stage blastocysts (see WO2006 / 040763), embryonic germ (EG) cells obtained from fetal reproductive tissue at any time during pregnancy, preferably before 10 weeks of gestation, and cells derived from unfertilized eggs stimulated by parthenogenesis (parthenotes).

[0109] Embryonic stem cells in some embodiments of the present invention can be obtained using well-known cell culture methods. For example, human embryonic stem cells can be isolated from human preimplantation blastocysts. Human blastocysts are usually obtained from human in vivo preimplantation embryos or from in vitro fertilized (IVF) embryos. Alternatively, single-cell human embryos can be expanded to the blastocyst stage.

[0110] It will be appreciated that commercially available stem cells can also be used in accordance with some embodiments of the present invention. Human ES cells can be purchased from the NTH Human Embryonic Stem Cell Registry [www.grants.nih.gov / stem_cells / registry / current.htm].

[0111] Furthermore, embryonic stem cells have been shown to be useful in the development of embryonic stem cells in mice (Mills and Bradley, 2001), golden hamsters [Doetschman et al., 1988, Dev Biol. 127: 224-227], rats [Iannaccone et al., 1994, Dev Biol. 163: 288-292], rabbits [Giles et al. 1993, Mol Reprod Dev. 36: 130-138; Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424-423], and several domestic animal species [Notarianni et al., 1991, J Reprod Fertil Suppl. 43: 255-260; Wheeler 1994, Reprod Fertil Dev. 6: 563-568; Mitalipova et al., 2001, Cloning. 3: 59-67] and non-human primate species (rhesus monkeys and marmosets) [Thomson et al., 1995, Proc Natl Acad Sci U S A. 92: 7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9].

[0112] The phrase "adult stem cells" (also called "tissue stem cells" or stem cells from somatic tissue) refers to any stem cell derived from somatic tissue (either from a newborn or prenatal animal, particularly a human). Adult stem cells are generally considered to be multipotent stem cells that can differentiate into multiple cell types. Adult stem cells can be derived from any adult, neonatal, or fetal tissue, such as adipose tissue, skin, kidney, liver, prostate, pancreas, intestine, bone marrow, and placenta.

[0113] According to one embodiment, the stem cells utilized by some embodiments of the present invention are bone marrow (BM)-derived stem cells, including hematopoietic, stromal, or mesenchymal stem cells (Dominici, M et al. (2001) J. Biol. Regul. Homeost. Agents. 15: 28-37). BM-derived stem cells can be obtained from the iliac crest, femur, tibia, spine, rib, or other medullary cavities.

[0114] Hematopoietic stem cells (HSCs), sometimes referred to as adult tissue stem cells, include stem cells obtained from the blood or bone marrow tissue of individuals of any age, or from the umbilical cord blood of newborn individuals. Preferred stem cells according to this aspect of some embodiments of the invention are embryonic stem cells, preferably of human or primate (e.g., monkey) origin.

[0115] Mesenchymal stem cells (MSCs), plastic pluripotent blast cells, give rise to one or more mesenchymal tissues (e.g., fat, bone, cartilage, elastic and fibrous connective tissue, myoblasts) and tissues other than those derived from the embryonic mesoderm (e.g., neural cells) in response to various influences from bioactive factors, e.g., cytokines. Such cells can be isolated from embryonic yolk sac, placenta, umbilical cord, fetal and adolescent skin, blood, and other tissues; their abundance in BM far exceeds their abundance in other tissues; therefore, isolation from BM is currently preferred. Adult tissue stem cells can be isolated using various methods known in the art, such as those disclosed by Alison, MR [J Pathol. (2003) 200(5): 547-50]. Fetal stem cells can be isolated using various methods known in the art, such as those disclosed by Eventov-Friedman S et al. [PLoS Med. (2006) 3: e215].

[0116] Hematopoietic stem cells can be isolated using various methods known in the art, such as those disclosed in "Isolation and Characterization of Hematopoietic Stem Cells" by Gerald J Spangrude and William B Stayton, "Handbook of Stem Cells," edited by Robert Lanze, Elsevier Academic Press, 2004, Chapter 54, pages 609-614.

[0117] Methods for isolating, purifying, and expanding mesenchymal stem cells (MSCs) are known in the art and include, for example, those disclosed by Caplan and Haynesworth in U.S. Pat. No. 5,486,359 and Jones EA et al., 2002, Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells, Arthritis Rheum. 46(12): 3349-60.

[0118] "Artificial" or "synthetic" when used in reference to a polynucleotide sequence means a sequence not found in nature, e.g., that is a synthetic modification of a naturally occurring sequence or that contains non-naturally occurring sequences.

[0119] The terms "polynucleotide," "nucleotide," or "nucleic acid" are used interchangeably herein and refer to a polymer composed of nucleotide monomers, particularly deoxyribonucleotide or ribonucleotide monomer chains of nucleotides, regardless of length. Nucleotides include purine-containing species such as adenine, hypoxanthine, and guanine, and their derivatives and analogs, and pyrimidines such as cytosine, uracil, and thymine, and their derivatives and analogs. These terms also encompass naturally occurring and non-naturally occurring nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar properties to the reference nucleic acid and are intended to be metabolized in a similar manner to the reference nucleotide or to have an extended half-life in the system. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Where appropriate, the term "polynucleotide" refers to a naturally occurring polymer of deoxyribonucleotide or ribonucleotide monomers. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), complementary DNA (cDNA), non-coding RNA (ncRNA), microRNA (miRNA), small RNA (sRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides of the present invention may be provided in isolated or substantially isolated form. Substantially isolated means that there may be substantial, but not complete, isolation of the polypeptide from any surrounding medium. Polynucleotides may be mixed with carriers or diluents that do not interfere with their intended use and still be considered substantially isolated. Optionally, polynucleotides of the present invention are recombinant.Recombinant means that the polynucleotide is the product of at least one cloning, restriction, or ligation step, or other procedure that results in a nucleic acid molecule that is distinct from that found in nature (e.g., in the case of cDNA).

[0120] As used herein, the term "operably linked" refers to the arrangement of various nucleic acid elements relative to one another so that the elements are functionally connected and can interact with one another in their intended manner. When nucleic acid sequence elements are operably linked, they act together to modulate each other's activity. Modulating means increasing, decreasing, or maintaining the level of activity of a particular element.

[0121] As used herein, a "polycistronic mRNA" is an mRNA that encodes two or more proteins.

[0122] As used herein, a "constitutive promoter" is a promoter that allows for the continuous transcription of an operably linked gene or genes. A constitutive promoter can be active at call under all circumstances.

[0123] The term "fluorescent protein" refers to a polypeptide that emits fluorescence and is typically detectable by flow cytometry, microscopy, or any fluorescent imaging system, and can therefore be used as a basis for selection of cells expressing such a protein. Examples of fluorescent proteins that can be used as reporters include, but are not limited to, green fluorescent protein (GFP), blue fluorescent protein (BFP), and red fluorescent protein (e.g., dsRed, mCherry, RFP).

[0124] The term "reporter nucleic acid" refers to a nucleic acid comprising a constitutive promoter operably linked to a gene encoding a fluorescent protein and a target gene (or a portion of a target gene), wherein the fluorescent protein and the target gene are transcribed as a polycistronic mRNA. A schematic diagram of a reported nucleic acid according to some embodiments is shown in Figure 31B.

[0125] The term "expressed nucleic acid" refers to a nucleic acid comprising a constitutive promoter operably linked to a nucleic acid sequence encoding an inhibitory RNA. A schematic diagram of an expressed nucleic acid according to some embodiments is shown in Figure 31A.

[0126] As used herein, the term "pool of test inhibitory RNAs" refers to two or more inhibitory RNAs to be experimentally tested for their activity (i.e., their ability to inhibit or silence a target gene).

[0127] As used herein, "transfecting" or "transfection" refers to the introduction of nucleic acid into one or more eukaryotic cells.

[0128] As used herein, "a method for detecting the inhibitory or silencing activity of one or more inhibitory RNAs against a target gene" refers to a method for assessing or experimentally assessing the extent to which an inhibitory RNA inhibits or silences its target gene.

[0129] As used herein, "a method for selecting an inhibitory RNA that exhibits silencing or inhibition of a target gene from a pool of test inhibitory RNAs" refers to a method in which inhibitory RNAs are screened for their ability to silence or inhibit the expression of a target gene, and inhibitory RNAs that exhibit silencing or inhibition of the target gene are selected.

[0130] Inhibitory RNAs that are active in cells in a context-specific manner In this disclosure, "active" means that the inhibitory RNA is transcribed and processed (if necessary) in such a way that it is active to inhibit expression of the target gene. Typically, inhibition by an inhibitory RNA is by targeting an RNA, usually an mRNA transcribed from the target gene, although other inhibitory mechanisms are possible.

[0131] "Inactive" or "reduced activity" means that the inhibitory RNA is inactive or less active in a cell, such that it is unable to inhibit, or is less able to inhibit, expression of the target gene. Inactivity or reduced activity of an inhibitory RNA can result from a lack of transcription or reduced transcription of the inhibitory RNA in the cell, reduced processing of the inhibitory RNA to its active form in the cell, or both.

[0132] In some embodiments, the inhibitory RNA is adapted to be transcribed and / or processed in a context-specific manner such that it is active at an inhibitory level in a first context (where inhibition of the target gene is desired), where the inhibitory RNA substantially reduces expression of the target gene, and is transcribed and / or processed such that it is active at a lower (preferably sub-inhibitory) level or is substantially inactive in at least a second context (where inhibition of the desired target gene is not desired or where reduced inhibition is desired).

[0133] Thus, in some embodiments, the present invention exploits the context- or cell context-specific activity of inhibitory RNAs (e.g., through context-specific transcription and / or post-transcriptional processing of the inhibitory RNA) to enable context-specific regulation of target gene expression. A variety of inhibitory RNAs that can be used in the present invention are discussed herein.

[0134] In some preferred embodiments, modulation of target gene expression is cellular context-specific, and the context-specific modulation of target gene expression occurs in a single cell between a first context (e.g., a differentiated state) and a second context (e.g., an undifferentiated state). Such cellular context-specific target gene expression can be distinguished from differential modulation of target gene expression between different cells, for example, between a first cell in a first tissue and a second cell in a second tissue. Optionally, an inhibitory RNA suitable for inhibiting target gene expression is active in a cell in a cellular context-specific manner, such that inhibition of target gene expression in the cell occurs specifically in a first context in which the inhibitory RNA is active in the cell. Optionally, inhibition does not occur, or occurs to a reduced extent, in the cell in at least a second context in which the inhibitory RNA is inactive or less active.

[0135] The activity of endogenous inhibitory RNAs in cells varies depending on the cell type and cellular context (e.g., cell type, tissue type, developmental stage, stage of differentiation, stress, microenvironment, activation, etc.). In some embodiments, the present invention utilizes modified forms of one or more endogenous inhibitory RNA(s) that are active in a context-specific or cellular context-specific manner to regulate gene expression in situations in which the one or more endogenous inhibitory RNA(s) are active, e.g., to silence or partially silence one or more target genes. In particular, in some embodiments, the present invention utilizes modified forms of endogenous miRNAs that exhibit context-specific activity (usually via context-specific transcription and / or processing) to regulate, typically partially or completely silence, target genes.

[0136] In some embodiments of the invention, expression of the target gene in the first situation is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 95%, at least 97%, at least 98%, at least 99% or 100% compared to control cells (e.g., cells not having a construct for expressing an inhibitory RNA).

[0137] In some embodiments of the invention, expression of the target gene in the first situation is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% compared to at least the second situation.

[0138] In some embodiments of the present invention, a GEiGS approach is used to modify a sequence encoding an endogenous non-coding RNA, e.g., a sequence encoding an endogenous inhibitory RNA, where the inhibitory RNA exhibits context-specific activity to retarget the endogenous inhibitory RNA and silence or partially silence a target gene in situations where the endogenous inhibitory RNA is active. In some embodiments, a GEiGS approach is used to modify a sequence encoding two or more endogenous non-coding RNAs, e.g., a sequence encoding two or more endogenous inhibitory RNAs, where the inhibitory RNA exhibits context-specific activity to retarget the endogenous inhibitory RNA and silence or partially silence one or more target genes in situations where the endogenous inhibitory RNA is active.

[0139] In some embodiments of the present invention, a nucleic acid sequence encoding a modified version of an endogenous inhibitory RNA is introduced into a cell, preferably into the cell's genome (although it may be, for example, episomal), and the inhibitory RNA exhibits context-specific activity to silence or partially silence a target gene in situations in which the endogenous inhibitory RNA is active. Thus, in some embodiments, rather than retargeting an endogenous inhibitory RNA with context-specific activity, a nucleic acid sequence encoding a modified version of an endogenous inhibitory RNA with context-specific activity can be inserted into a cell, for example, into the cell's genome. Thus, in such embodiments, the original ("in situ") endogenous inhibitory RNA can be unmodified. The modified version of the endogenous inhibitory RNA can be inserted into the cell's genome at any suitable locus for expression. In some embodiments, the modified version of the endogenous inhibitory RNA is provided in a synthetic expression cassette.

[0140] In some embodiments, two or more nucleic acid sequences encoding modified versions of two or more endogenous inhibitory RNAs are introduced into a cell, preferably into the cell's genome (although, for example, they may be episomal), and the inhibitory RNAs exhibit context-specific activity to silence or partially silence one or more target genes in situations in which at least one of the two or more endogenous inhibitory RNAs is active. In some embodiments, rather than retargeting an endogenous inhibitory RNA with a context-specific activity, two or more nucleic acid sequences encoding modified versions of an endogenous inhibitory RNA with a context-specific activity can be inserted into a cell, e.g., into the cell's genome. Thus, in such embodiments, the original ("in situ") endogenous inhibitory RNA can be unmodified. The modified versions of the endogenous inhibitory RNA can be inserted into the cell's genome at any suitable locus for expression. In some embodiments, the modified versions of the two or more endogenous inhibitory RNAs are provided in two or more synthetic expression cassettes.

[0141] In some embodiments, nucleic acid sequences encoding modified versions of endogenous inhibitory RNAs with context-specific activity can be inserted into endogenous miRNA loci. Approaches that can be adapted for the present invention are described, for example, in Senis et al. ("TALEN / CRISPR-mediated engineering of a promoterless antiviral RNAi hairpin into an endogenous miRNA locus," Nucleic Acids Research, 2017, Vol. 45, No. 1, e3 doi: 10.1093 / nar / gkw805). Thus, in some embodiments of the present invention, promoterless sequences encoding inhibitory RNAs can be inserted into endogenous miRNA loci, preferably without disrupting or inactivating any endogenous miRNAs.

[0142] inhibitory RNA Non-coding RNA molecules can be modified to provide the inhibitory RNAs described herein. According to one embodiment, non-limiting examples of non-coding RNA molecules include, but are not limited to, microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), trans-acting siRNAs (tasiRNAs), small nuclear RNAs (snRNAs or URNAs), small nucleolar RNAs (snoRNAs), small Cajal body RNAs (scaRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), extracellular RNAs (exRNAs), repeat-derived RNAs, transposable element RNAs, and long non-coding RNAs (IncRNAs). Non-coding RNAs can be modified to provide the inhibitory RNAs used in the present invention.

[0143] The term "inhibitory RNA" refers to an RNA molecule that has a degrading or silencing, e.g., partial or complete silencing, effect on its corresponding mRNA having a complementary sequence thereto. In some embodiments of the present invention, inhibitory RNA includes siRNA, shRNA, and microRNA, preferably microRNA. The term silencing RNA (sRNA) is also used herein to refer to inhibitory RNA.

[0144] According to one embodiment, non-limiting examples of inhibitory RNA molecules include, but are not limited to, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), Piwi-interacting RNA (piRNA), and trans-acting siRNA (tasiRNA).

[0145] According to some embodiments, the inhibitory RNA molecule is optionally capable of inducing RNA interference (RNAi) after cellular processing.

[0146] According to some embodiments, the inhibitory RNA molecule is processed from a precursor.

[0147] According to some embodiments, the non-coding RNA molecule or RNA silencing molecule is processed from a single-stranded RNA (ssRNA) precursor.

[0148] In some embodiments, the non-coding RNA molecule or RNA silencing molecule is processed from a double-stranded single-stranded RNA precursor.

[0149] In some embodiments, the non-coding RNA molecule or RNA silencing molecule is processed from a dsRNA precursor (eg, containing perfect and imperfect base pairing).

[0150] In some embodiments, the non-coding RNA molecule or RNA silencing molecule is processed from an unstructured RNA precursor.

[0151] According to some embodiments, the non-coding RNA molecule or RNA silencing molecule is processed from a protein-coding RNA precursor.

[0152] In some embodiments, the non-coding RNA molecule or RNA silencing molecule is processed from a non-coding RNA precursor.

[0153] According to some embodiments, the dsRNA can be derived from two different complementary RNAs or from a single RNA that folds on itself to form the dsRNA.

[0154] In some embodiments, the inhibitory RNA or the nucleic acid sequence encoding the inhibitory RNA includes flanking sequences (5' and 3' flanking sequences). In some preferred embodiments, the flanking sequences are approximately 40 bp in length. In some preferred embodiments, the flanking sequences are less than 100 bp, less than 90 bp, less than 80 bp, or less than 70 bp, preferably less than 60 bp, less than 50 bp, and more preferably less than 49 bp, less than 48 bp, less than 47 bp, less than 46 bp, less than 45 bp, less than 44 bp, less than 43 bp, less than 42 bp, or less than 41 bp in length. Flanking genomic sequences (homology arms) 40 base pairs in length exhibit superior silencing properties compared to flanking genomic sequences 350 bp or 150-350 bp in length, as shown in Figure 23.

[0155] Perfectly and imperfectly paired RNAs (i.e., double-stranded RNAs, dsRNAs), siRNAs, and shRNAs. The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme called Dicer. Dicer, also known as endoribonuclease with an RNase motif, or helicase, is an enzyme encoded by the Dicer1 gene in humans. Dicer is involved in processing dsRNA into short pieces of dsRNA known as small interfering RNAs (siRNAs). The siRNAs derived from Dicer activity are typically about 21 to about 23 nucleotides in length and contain a duplex of about 19 base pairs with two 3'-nucleotide overhangs.

[0156] Thus, some embodiments of the present invention contemplate modifying a genomic sequence encoding a dsRNA to redirect silencing specificity (including silencing activity) towards the target RNA, thereby silencing or partially silencing the target gene.

[0157] According to one embodiment, dsRNA precursors longer than 21bp are used.Various studies have demonstrated that long dsRNA can be used to silence gene expression without inducing stress response or causing significant off-target effects.See, for example, [Strat et al., Nucleic Acids Research, 2006, Vol. 34, No. 13, pp. 3803-3810; Bhargava A et al., Brain Res. Protoc. 2004; pp. 13:115-125; Diallo M. et al., Oligonucleotides. 2003; pp. 13:381-392; Paddison PJ et al., Proc. Natl. Acad. Sci. USA. 2002, 99:1443-1448; Tran N. et al., FEBS Lett. 2004, 573:127-134].

[0158] The term "siRNA" refers to small inhibitory RNA duplexes (generally between 18 and 30 base pairs) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21-mers with a central 19-bp duplex region and symmetric two-base 3'-overhangs at the termini; however, it has recently been described that chemically synthesized RNA duplexes of 25 to 30 bases in length can have as much as a 100-fold increase in potency compared to 21-mers at the same positions. The observed increase in potency obtained using longer RNAs in triggering RNAi was attributed to providing Dicer with a substrate (27-mer) instead of a product (21-mer), which was suggested to improve the rate or efficiency with which siRNA duplexes enter RISC.

[0159] The position, but not the composition, of the 3'-overhang influences the potency of siRNA, with asymmetric duplexes with a 3'-overhang on the antisense strand generally being more potent than those with a 3'-overhang on the sense strand (Rose et al., 2005).

[0160] The strands of a double-stranded interfering RNA (e.g., siRNA) can be joined to form a hairpin or stem-loop structure (e.g., shRNA). Thus, as noted, the inhibitory RNA of some embodiments of the invention can also be a short hairpin RNA (shRNA).

[0161] The term "shRNA," or short hairpin RNA, as used herein refers to an RNA molecule having a stem-loop structure, comprising first and second regions of complementary sequence, the degree and orientation of which are sufficient to allow base pairing between the regions, the first and second regions connected by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop may be between 3 and 23, 5 and 15, 7 and 13, 4 and 9, or 9 and 11, inclusive. Some of the nucleotides in the loop may participate in base pairing interactions with other nucleotides in the loop. Examples of oligonucleotide sequences that can be used to form the loop include 5'-CAAGAGA-3' and 5'-UUACAA-3' (International Patent Application Nos. WO2013126963 and WO2014107763). Those skilled in the art will recognize that the resulting single-stranded oligonucleotide will form a stem-loop or hairpin structure containing a double-stranded region capable of interacting with the RNAi machinery.

[0162] The inhibitory RNA molecules of some embodiments of the present invention need not be limited to molecules containing only RNA, but also encompass chemically modified nucleotides and non-nucleotides.

[0163] Various types of siRNAs are contemplated by the present invention, including trans-acting siRNAs (Ta-siRNAs), repeat-associated siRNAs (Ra-siRNAs), and naturally occurring antisense transcript-derived siRNAs (Nat-siRNAs).

[0164] According to one embodiment, the inhibitory RNA comprises "piRNA," a class of Piwi-interacting RNAs approximately 26 and 31 nucleotides in length. piRNAs typically form RNA-protein complexes through interactions with Piwi proteins, i.e., antisense piRNAs are typically loaded into Piwi proteins (e.g., Piwi, Ago3, and Aubergine (Aub)).

[0165] miRNA - According to another, generally preferred embodiment, the inhibitory RNA molecule can be a miRNA. The terms "microRNA," "miRNA," and "miR" are synonymous and refer to a group of non-coding, single-stranded RNA molecules approximately 19-28 nucleotides in length that regulate gene expression. miRNAs are found in a wide range of organisms, including viruses, and have been shown to play roles in development, homeostasis, and disease pathogenesis.

[0166] Initially, the pre-miRNA exists as a long, non-perfect double-stranded stem-loop RNA, which is further processed by Dicer into an siRNA-like duplex containing a mature guide strand (miRNA) and a similarly sized fragment known as the passenger strand (miRNA*). The miRNA and miRNA* can be derived from opposite arms of the pri-miRNA and pre-miRNA. miRNA* sequences can be found in libraries of cloned miRNAs, but usually less frequently than miRNAs.

[0167] miRNAs initially exist as double-stranded species along with miRNA* but eventually become incorporated as single-stranded RNA into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Various proteins can form RISC, which can lead to variability in the specificity of the miRNA / miRNA* duplex, the binding site of the target gene, the activity of the miRNA (repression or activation), and which strand of the miRNA / miRNA* duplex is loaded into RISC. When the miRNA strand of the miRNA:miRNA* duplex is loaded into RISC, the miRNA* is removed and degraded. The strand of the miRNA:miRNA* duplex that loads into RISC is the one with the less tightly paired 5' end. When both ends of the miRNA:miRNA* have roughly equivalent 5' pairing, both the miRNA and miRNA* can have gene silencing activity.

[0168] RISC identifies target nucleic acids based on the high level of complementarity between miRNA and mRNA, specifically nucleotides 2-8 of the miRNA (termed the "seed sequence").

[0169] Several studies have examined the requirement for base pairing between miRNAs and their mRNA targets to achieve efficient translation inhibition (reviewed by Bartel 2004, Cell 116-281). Computational studies analyzing genome-wide miRNA binding have suggested a specific role for bases 2-8 of the 5' end of the miRNA (also called the "seed sequence") in target binding, but also recognized the role of the first nucleotide, usually found to be "A" (Lewis et al. 2005, Cell 120-15). Similarly, nucleotides 1-7 or 2-8 were used to identify and validate targets by Krek et al. (2005, Nat Genet 37-495). Target sites in mRNAs can be in the 5' UTR, 3' UTR, or coding region. Interestingly, multiple miRNAs may regulate the same mRNA target by recognizing the same or multiple sites. The presence of multiple miRNA binding sites in most genetically identified targets may indicate that the cooperative action of multiple RISCs provides the most efficient translation inhibition.

[0170] miRNAs can instruct RISC to downregulate gene expression by either of two mechanisms: mRNA cleavage or translational repression. miRNAs can specify mRNA cleavage if the mRNA has a certain degree of complementarity to the miRNA. If the miRNA directs cleavage, the cleavage is usually between nucleotides that pair with residues 10 and 11 of the miRNA. Alternatively, miRNAs can repress translation if the miRNA does not have the required degree of complementarity to the miRNA. Translational repression may be more prevalent in animals because animals may have a lower degree of complementarity between the miRNA and the binding site.

[0171] It is noteworthy that there may be variability in the 5' and 3' ends of any pair of miRNA and miRNA*. This variability may be due to variability in Drosha and Dicer enzymatic processing of the cleavage site. Variability in the 5' and 3' ends of miRNA and miRNA* may also be due to mismatches in the stem structure of the pri-miRNA and pre-miRNA. Mismatches in the stem strands may lead to a population of different hairpin structures. Variability in stem structure may also lead to variability in the products of Drosha and Dicer cleavage. It will be appreciated that pre-miRNA sequences may consist of 45-90, 60-80, or 60-70 nucleotides, and pri-miRNA sequences may consist of 45-30,000, 50-25,000, 100-20,000, 1,000-1,500, or 80-100 nucleotides.

[0172] As noted above, the methods of some embodiments of the present invention are utilized to redirect the silencing activity and / or specificity of an inhibitory RNA molecule toward a target RNA of interest (or to generate silencing activity and / or specificity in cases where the non-coding RNA molecule does not have the intrinsic ability to silence RNA molecules).

[0173] According to one embodiment, the target RNA is different from the original target RNA of the endogenous inhibitory RNA. According to various embodiments, the methods of the invention involve introducing into a eukaryotic cell a DNA editing agent that redirects the silencing activity and / or specificity of the inhibitory RNA toward the desired target RNA.

[0174] As used herein, the term "redirect silencing specificity" refers to reprogramming the original specificity of a non-coding RNA (usually an inhibitory RNA) toward a non-natural target of the non-coding RNA. Thus, the original specificity of the inhibitory RNA is destroyed (i.e., loss of function), and the new specificity is toward an RNA target other than the natural target (i.e., the RNA of interest), i.e., gain of function. It should be noted that if the original non-coding RNA does not have silencing activity, or if a construct for expressing an inhibitory RNA is introduced into a cell, only gain of function will occur.

[0175] As used herein, the term "target RNA" refers to an RNA sequence to which an inhibitory RNA molecule binds. Thus, a target RNA is considered by those skilled in the art to be a substrate for an inhibitory RNA.

[0176] The silencing results can be confirmed by examining the external characteristics of the eukaryotic cell or organism or by biochemical techniques. It will be appreciated that the inhibitory RNA molecules of some embodiments of the present invention may have some off-target specific effects, so long as they do not unduly affect the intended growth, differentiation, or function of the cell or organism.

[0177] Previous silencing methods utilizing shRNA or siRNA are known to have significant off-target effects (Rao DD, Senzer N, Cleary MA, Nemunaitis J. Cancer Gene Ther. 2009 Nov. 16(11):807-9. Doi: 10.1038 / cgt.2009.53. Epub 2009 Aug. 28 PMID: 19713999). Advantageously, in some preferred embodiments, the inhibitory RNA or nucleic acid sequence encoding the inhibitory RNA according to the present invention does not have off-target effects. In some preferred embodiments, the inhibitory RNA or nucleic acid sequence encoding the inhibitory RNA does not silence unintended targets (targets that it was designed to target, e.g., any targets other than B2M).

[0178] In some preferred embodiments, the inhibitory RNA may be an miRNA that does not have off-target effects. In some preferred embodiments, the inhibitory RNA may be an miRNA that does not silence unintended targets (targets that it is designed to target, for example, any targets other than B2M).

[0179] In some preferred embodiments, the inhibitory RNA or the nucleic acid sequence encoding the inhibitory RNA does not exhibit a loss-of-function effect after redirection of the miRNA gene used as a scaffold.

[0180] According to one embodiment, the target RNA is endogenous to the cell, e.g., derived from an endogenous gene. Various genes that express suitable target RNAs are discussed herein.

[0181] According to one embodiment, the target RNA or target RNA of interest is exogenous to the cell (also referred to herein as heterologous). In such cases, the target RNA is the product of a gene that is not naturally part of the eukaryotic cell genome (i.e., expresses a non-coding RNA). Exemplary exogenous target RNAs include, but are not limited to, the products of genes associated with infectious diseases, e.g., genes of pathogens (e.g., insects, viruses, bacteria, fungi, nematodes). Exogenous target RNAs (coding or non-coding) can include nucleic acid sequences that share sequence identity with endogenous RNA sequences of the cell or organism (e.g., can be partially homologous to the endogenous nucleic acid sequence).

[0182] Specific binding of an endogenous non-coding RNA molecule to a target RNA can be determined by a computer-based algorithm (e.g., BLAST) and verified by methods including, for example, Northern blot, in situ hybridization, QuantiGene Plex assay, etc.

[0183] The use of the term "complementary" or "complementary" means that an inhibitory RNA molecule (or at least a portion thereof present in processed form, or at least one strand of a double-stranded polynucleotide or portion thereof, or a portion of a single-stranded polynucleotide) hybridizes with a target RNA or a fragment thereof under physiological conditions to achieve regulation or function or repression of a target gene. For example, in some embodiments, the inhibitory RNA molecule inhibits 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23, 24, 25, 26, 27, 28, When compared to a sequence of 57, 58, 59, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500 or more consecutive nucleotides, the sequence has 100 percent sequence identity or at least about 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent sequence identity.

[0184] As used herein, an inhibitory RNA molecule or its processed small RNA form is said to exhibit "perfect complementarity" when every nucleotide of one of the sequences, read 5' to 3', is complementary to every nucleotide of the other sequence, read 3' to 5'. A nucleotide sequence that is perfectly complementary to a reference nucleotide sequence exhibits an identical sequence to the reverse complement of the reference nucleotide sequence.

[0185] Methods for determining sequence complementarity are well known in the art and include, but are not limited to, bioinformatics tools well known in the art (e.g., BLAST, multiple sequence alignment).

[0186] According to one embodiment, when the inhibitory RNA molecule is an siRNA or is processed into an siRNA, the complementarity is in the range of 90-100% (eg, 100%) to its target sequence.

[0187] According to one embodiment, when the inhibitory RNA molecule is an miRNA or piRNA or is processed into an miRNA or piRNA, the complementarity is in the range of 33-100% to its target sequence.

[0188] According to one embodiment, when the inhibitory RNA molecule is an miRNA, the seed sequence complementarity (ie, nucleotides 2-8 from the 5') is in the range of 85-100% (eg, 100%) to its target sequence.

[0189] According to one embodiment, the inhibitory RNA may be further processed into a small RNA form (e.g., a pre-miRNA is processed into a mature miRNA). In such cases, homology is measured based on the processed small RNA form (e.g., the mature miRNA sequence).

[0190] As used herein, the term "small RNA form" refers to a mature small RNA that is capable of hybridizing to a target RNA (or a fragment thereof). According to one embodiment, the small RNA form has silencing activity.

[0191] According to one embodiment, complementarity to the target sequence is at least about 33% of the processed small RNA form (e.g., 33% of 21-24 nt). Thus, for example, if the inhibitory RNA molecule is an miRNA, 33% of the mature miRNA sequence (e.g., 21 nt) contains seed complementarity (e.g., 7 nt of 21 nt).

[0192] According to one embodiment, the complementarity to the target sequence is at least about 45% (e.g., 45% of 21-28 nt) of the processed small RNA form. Thus, for example, if the inhibitory RNA molecule is an miRNA, 45% of the mature miRNA sequence (e.g., 21 nt) contains seed complementarity (e.g., 9-10 nt of 21 nt).

[0193] According to one embodiment, the endogenous inhibitory RNA (i.e., before modification) is typically selected to have about 10%, 20%, 30%, 33%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or up to 99% complementarity to the sequence of the desired target RNA of interest.

[0194] According to certain embodiments, the inhibitory RNA molecule (i.e., before modification) is typically selected to have 99% or less complementarity to the sequence of the target RNA of interest. According to certain embodiments, the RNA molecule of interest (i.e., before modification) is typically selected to have 98% or less complementarity to the sequence of the target RNA of interest. According to certain embodiments, the inhibitory RNA molecule (i.e., before modification) is typically selected to have 97% or less complementarity to the sequence of the target RNA of interest. According to certain embodiments, the inhibitory RNA molecule (i.e., before modification) is typically selected to have 96% or less complementarity to the sequence of the target RNA of interest. According to certain embodiments, the inhibitory RNA molecule (i.e., before modification) is typically selected to have 95% or less complementarity to the sequence of the target RNA of interest. According to certain embodiments, the inhibitory RNA molecule (i.e., before modification) is typically selected to have 90% or less complementarity to the sequence of the target RNA of interest. According to certain embodiments, the inhibitory RNA molecule (i.e., before modification) is typically selected to have 85% or less complementarity to the sequence of the target RNA of interest. According to certain embodiments, the inhibitory RNA molecule (i.e., before modification) is typically selected to have 50% or less complementarity to the sequence of the target RNA of interest. According to certain embodiments, the RNA molecule (i.e., before modification) is typically selected to have 33% or less complementarity to the sequence of the target RNA of interest.

[0195] According to one embodiment, the inhibitory RNA molecule is designed to contain at least about 33%, 40%, 45%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% complementarity to the sequence of the target RNA of interest.

[0196] According to certain embodiments, inhibitory RNA molecules are designed to contain a minimum of 33% complementarity to the target RNA of interest (eg, 85-100% seed match).

[0197] According to certain embodiments, inhibitory RNA molecules (e.g., RNA silencing molecules) are designed to contain a minimum of 40%, 45%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to the target RNA of interest. High levels of complementarity are generally preferred when high levels of inhibition / silencing are desired.

[0198] To generate silencing activity and / or specificity of an inhibitory RNA molecule, or to redirect the silencing activity and / or specificity of an inhibitory RNA molecule toward a target RNA, the sequence of an endogenous non-coding RNA molecule (e.g., an inhibitory RNA) is preferably modified using a DNA editing agent.

[0199] According to one embodiment, the inhibitory RNAs are hsa-mir-191, hsa-mir-302a, hsa-mir-302c, hsa-mir-93, hsa-mir-106a, hsa-mir-106b, hsa-mir-20a, hsa-mir-363, hsa-mir-518b, hsa-mir-744, hsa-mir-99b, hsa-mir-320a, hsa-mir-520f, hsa-mir-652, hsa-mir-1180, hsa-mir-15b, hsa-mir-23a, hsa-mir-26b, hsa-mir-335, hsa-mir-361, hsa-mir-1307, hsa-mir-205, hsa-mir-221, hsa-mir-222, hsa-mir-30e, hsa-mir-423, hsa-mir-519c, hsa-mir-92b, hsa-mir-30, hsa-mir-302b, hsa-let-7b, hsa-mir-98, hsa-let-7g, hsa-mir-126, hsa-mir-17, hsa-mir-378a, hsa-mir-671, hsa-mir-155, hsa-mir-20b, hsa-mir-34c, hsa-mir-146a, hsa-mir-29a, hsa-mir-342, hsa-miR-375, hsa-miR-143, hsa-miR-21, hsa-miR-30d, hsa-miR-192, hsa-miR-30a, hsa-miR-182, hsa-miR-148a, hsa-miR-127, hsa-miR-200c, hsa-miR-143, hsa-miR-10a, hsa-miR-122, hsa-miR-22, hsa-miR-192, hsa-miR-146b, hsa-miR-26a, hsa-miR-125a, hsa-miR-150, hsa-miR-10b, hsa-miR-181a, hsa-miR-26a, hsa-miR-92a, hsa-miR-143, hsa-miR-10a, hsa-miR-21, MIR146A (hsa-miR-146a-5p), MIR20A (hsa-miR-20a-5p), LET7C (hsa-miR-let-7c-5p), LET7i (hsa-let-7i-5p), miR-375, miR-340-5p, miR-29a-3p, miR-222-3p, miR-940,The present invention includes modified forms of miRNAs selected from the group consisting of miR-203, miR-145, miR-103a, miR-21-3p, miR-181-5p, miR-125b-5p, miR-301a-3p, miR-132, miR-29b-1, miR-27a, miR-146a, miR-222, miR-let7a, miR-320a, miR-146a, miR-let7c, miR-1246, miR-let7d-5p, miR-451, miR-21, miR-23a, miR-27a, miR-24-2, miR-155, miR125b-2, and miR-16.

[0200] According to one embodiment, the inhibitory RNA includes a modified form of miRNA selected from the group consisting of hsa-miR-375, hsa-miR-143, hsa-miR-21, hsa-miR-30d, hsa-miR-192, hsa-miR-30a, hsa-miR-182, hsa-miR-148a, hsa-miR-127, hsa-miR-200c, hsa-miR-143, hsa-miR-10a, hsa-miR-122, hsa-miR-22, hsa-miR-192, hsa-miR-146b, hsa-miR-26a, hsa-miR-125a, hsa-miR-150, hsa-miR-10b, hsa-miR-181a, hsa-miR-26a, hsa-miR-92a, hsa-miR-143, hsa-miR-10a, hsa-miR-21, MIR146A (hsa-miR-146a-5p), MIR20A (hsa-miR-20a-5p), LET7C (hsa-miR-let-7c-5p), LET7i (hsa-let-7i-5p), miR-375, miR-340-5p, miR-29a-3p, miR-222-3p, miR-940, miR-203, miR-145, miR-103a, miR-21-3p, miR-181-5p, miR-125b-5p, miR-301a-3p, miR-132, miR-29b-1, miR-27a, miR-146a, miR-222, miR-let7a, miR-320a, miR-146a, miR-let7c, miR-1246, miR-let7d-5p, miR-451, miR-21, miR-23a, miR-27a, miR-24-2, miR-155, miR125b-2 and miR-16.

[0201] According to one embodiment, the inhibitory RNA is selected from the group consisting of hsa-mir-191, hsa-mir-302a, hsa-mir-302c, hsa-mir-93, hsa-mir-106a, hsa-mir-106b, hsa-mir-200c, hsa-mir-20a, hsa-mir-21, hsa-mir-363, hsa-mir-518b, hsa-mir-744, hsa-mir-99b, hsa-mir-320a, hsa-mir-520f, hsa-mir-652, hsa-mir-1180, hsa-mir-15b, hsa-mir-160c, hsa-mir-170d, hsa-mir-180e, hsa-mir-191f, hsa-mir-302a, hsa-mir-302c, hsa-mir-93, hsa-mir-106a, hsa-mir-106b, hsa-mir-200c, hsa-mir-20a, hsa-mir-21, hsa-mir-363, hsa-mir-518b, hsa-mir-744, hsa-mir-99b, hsa-mir-320a, hsa-mir-520f, hsa-mir-652, hsa-mir-1180, hsa-mir-15b, hsa-mir-160f, hsa-mir-17 ... The miRNAs include modified forms of miRNAs selected from the group consisting of r-182, hsa-mir-23a, hsa-mir-26b, hsa-mir-335, hsa-mir-361, hsa-mir-1307, hsa-mir-205, hsa-mir-22, hsa-mir-221, hsa-mir-222, hsa-mir-30e, hsa-mir-423, hsa-mir-519c, hsa-mir-92b, hsa-mir-30, hsa-mir-302b, hsa-mir-375, hsa-let-7b and hsa-mir-98. According to one embodiment, the inhibitory RNA comprises a modified form of a miRNA selected from the group consisting of hsa-let-7g, hsa-mir-126, hsa-mir-150, hsa-mir-17, hsa-mir-191, hsa-mir-30d, hsa-mir-30e, hsa-mir-363, hsa-mir-93, hsa-mir-378a, hsa-mir-671, hsa-mir-155, hsa-mir-20b and hsa-mir-34c. According to one embodiment, the inhibitory RNA comprises a modified form of a miRNA selected from the group consisting of hsa-mir-146a, hsa-mir-29a, hsa-mir-342, hsa-mir-363, hsa-mir-126, hsa-mir-146b, hsa-mir-30d, hsa-mir-20b, hsa-mir-378a, hsa-let-7g, hsa-mir-30e, hsa-mir-671, hsa-mir-17, hsa-mir-20a, hsa-mir-93, hsa-mir-34c, hsa-let-7b and hsa-mir-155.

[0202] Additional miRNAs of interest and preferred contexts for the use of these miRNAs are discussed herein below, and the sequences of these miRNAs can be found at miRbase (https: / / www.mirbase.org / ).

[0203] According to one embodiment, the inhibitory RNA comprises a sequence selected from SEQ ID NO:1 to SEQ ID NO:17, or selected from SEQ ID NO:18 to SEQ ID NO:34. These sequences correspond to miRNAs (mature sRNA and pre-miRNA hairpin forms) that target B2M. In some preferred embodiments, the inhibitory RNA comprises SEQ ID NO:1 or 18 (Solution 8), SEQ ID NO:5 or 22 (Solution 20), SEQ ID NO:9 or 26 (Solution 29), or SEQ ID NO:10 or 27 (Solution 30). In some embodiments, the inhibitory RNA does not comprise a sequence selected from SEQ ID NO:1 to SEQ ID NO:17, or selected from SEQ ID NO:18 to SEQ ID NO:34.

[0204] According to one embodiment, the inhibitory RNA comprises a sequence selected from SEQ ID NO: 66 to SEQ ID NO: 87 or SEQ ID NO: 88 to SEQ ID NO: 109. These sequences correspond to miRNAs (mature sRNA and pre-miRNA hairpin forms) that target PPARG.

[0205] According to one embodiment, the inhibitory RNA comprises a sequence selected from SEQ ID NO: 110 to SEQ ID NO: 133 or SEQ ID NO: 134 to SEQ ID NO: 157. These sequences correspond to miRNAs (mature sRNA and pre-miRNA hairpin forms) that target IRF4. Solution 219 (SEQ ID NO: 127 and SEQ ID NO: 151) is a particularly preferred inhibitory RNA that targets IRF4.

[0206] According to one embodiment, the inhibitory RNA comprises a sequence selected from SEQ ID NO: 158 to SEQ ID NO: 181 or SEQ ID NO: 182 to SEQ ID NO: 205. These sequences correspond to miRNAs (mature sRNA and pre-miRNA hairpin forms) that target KDM6B. Solution 245 (SEQ ID NO: 177 and SEQ ID NO: 201) is a particularly preferred inhibitory RNA that targets KDM6B.

[0207] According to one embodiment, the inhibitory RNA comprises a sequence selected from SEQ ID NO: 206 to SEQ ID NO: 229 or SEQ ID NO: 230 to SEQ ID NO: 253. These sequences correspond to miRNAs (mature sRNA and pre-miRNA hairpin forms) that target STAT6. Solution 260 (SEQ ID NO: 216 and SEQ ID NO: 240) and Solution 266 (SEQ ID NO: 222 and SEQ ID NO: 246) are particularly preferred inhibitory RNAs that target STAT6.

[0208] According to one embodiment, the inhibitory RNA comprises a sequence selected from SEQ ID NO: 348 to SEQ ID NO: 411 or SEQ ID NO: 412 to SEQ ID NO: 475. These sequences correspond to miRNAs (mature sRNA and pre-miRNA hairpin forms) that target B2M. In some embodiments, the inhibitory RNA does not comprise a sequence selected from SEQ ID NO: 348 to SEQ ID NO: 411 or SEQ ID NO: 412 to SEQ ID NO: 475.

[0209] According to one embodiment, the inhibitory RNA comprises a sequence selected from SEQ ID NO: 476 to SEQ ID NO: 525 or SEQ ID NO: 526 to SEQ ID NO: 575. These sequences correspond to miRNAs (mature sRNA and pre-miRNA hairpin forms) that target FOXP3.

[0210] According to one embodiment, the inhibitory RNA comprises a sequence selected from SEQ ID NO: 576 to SEQ ID NO: 624 or SEQ ID NO: 625 to SEQ ID NO: 673. These sequences correspond to miRNAs (mature sRNA and pre-miRNA hairpin forms) that target PDCD-1.

[0211] In some embodiments, the inhibitory RNA is encoded by a nucleic acid sequence that encodes the inhibitory RNA. In some embodiments, the nucleic acid sequence that encodes the inhibitory RNA comprises a sequence selected from SEQ ID NOs: 254-347.

[0212] In some embodiments, the nucleic acid sequence encoding the inhibitory RNA comprises an HDR template sequence, hi some embodiments, the HDR template sequence is selected from SEQ ID NOs: 674-679, 59, 61, 63, and 65.

[0213] Two or more inhibitory RNAs suitable for inhibiting the expression of one or more target genes A method according to the first aspect of the invention may comprise providing a cell with a nucleic acid construct suitable for inhibiting expression of a target gene or adapted to express two or more inhibitory RNAs suitable for inhibiting expression of two or more target genes. A cell according to the second or third embodiment of the invention may be provided with or may comprise a nucleic acid construct suitable for inhibiting expression of a target gene or adapted to express two or more inhibitory RNAs suitable for inhibiting expression of two or more target genes.

[0214] In some embodiments according to the first, second or third aspect of the invention, a cell is provided with two or more nucleic acid constructs adapted to express inhibitory RNAs suitable for inhibiting expression of one or more target genes. In some embodiments according to the first, second or third aspect of the invention, a single nucleic acid construct is adapted to express two or more inhibitory RNAs suitable for inhibiting expression of one or more target genes. Optionally, the inhibition of expression is in a cell context or context specific manner.

[0215] In some embodiments, the methods optionally include providing a cell with one or two or more nucleic acid constructs adapted to express two or more inhibitory RNAs suitable for inhibiting expression of a single target gene (i.e., two or more inhibitory RNAs with the same target). In some embodiments, the cell is provided with, or the cell contains, one or two or more nucleic acid constructs adapted to express two or more inhibitory RNAs suitable for inhibiting expression of a single target gene (i.e., two or more inhibitory RNAs with the same target).

[0216] In some embodiments, the methods optionally include providing a cell with a nucleic acid construct adapted to express two or more inhibitory RNAs suitable for inhibiting expression of a single target gene (i.e., two or more inhibitory RNAs having the same target). In some embodiments, the cell is provided with, or the cell contains, a nucleic acid construct adapted to express two or more inhibitory RNAs suitable for inhibiting expression of a single target gene (i.e., two or more inhibitory RNAs having the same target). In some preferred embodiments, the cell is a pancreatic cell. In some preferred embodiments, the cell is a T cell. In some preferred embodiments, the cell is a macrophage. In some embodiments, the target gene is selected from B2M, PPARG, IRF4, KDM6B, FOXP3, PDCD-1, and STAT6. In some preferred embodiments, the target gene is B2M.

[0217] In some embodiments, two or more inhibitory RNAs are selected from SEQ ID NO:1 or 18 (Solution 8), SEQ ID NO:5 or 22 (Solution 20), SEQ ID NO:9 or 26 (Solution 29), SEQ ID NO:10 or 27 (Solution 30), SEQ ID NO:348-411, or SEQ ID NO:412-475 (targeting B2M). In some embodiments, two or more inhibitory RNAs are selected from SEQ ID NO:66-87 or SEQ ID NO:88-109 (targeting PPARG). In some embodiments, two or more inhibitory RNAs are selected from SEQ ID NO:110-133 or SEQ ID NO:134-157 (targeting IRF4). In some embodiments, two or more inhibitory RNAs are selected from SEQ ID NO:158-181 or SEQ ID NO:182-205 (targeting KDM6B). In some embodiments, two or more inhibitory RNAs are selected from SEQ ID NO:476-525 or SEQ ID NO:526-575 (targeting FOXP3). In some embodiments, two or more inhibitory RNAs are selected from SEQ ID NO:576-624 or SEQ ID NO:625-673 (targeting PDCD-1). In some embodiments, two or more inhibitory RNAs are selected from SEQ ID NO:206-229 or SEQ ID NO:230-253 (targeting STAT6).

[0218] In some embodiments, the methods optionally include providing a cell with one or two or more nucleic acid constructs adapted to express two or more inhibitory RNAs (i.e., two or more inhibitory RNAs with different targets) suitable for inhibiting expression of two or more target genes. In some embodiments, the cell is provided with, or the cell contains, one or two or more nucleic acid constructs adapted to express two or more inhibitory RNAs (i.e., two or more inhibitory RNAs with different targets) suitable for inhibiting expression of two or more target genes.

[0219] In some embodiments, the methods optionally include providing a cell with a nucleic acid construct adapted to express two or more inhibitory RNAs (i.e., two or more inhibitory RNAs with different targets) suitable for inhibiting expression of two or more target genes. In some embodiments, the cell is provided with, or the cell contains, a nucleic acid construct adapted to express two or more inhibitory RNAs (i.e., two or more inhibitory RNAs with different targets) suitable for inhibiting expression of two or more target genes. In some preferred embodiments, the cell is a pancreatic cell. In some preferred embodiments, the cell is a T cell. In some preferred embodiments, the cell is a macrophage. In some embodiments, at least one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348-411, or SEQ ID NO: 412-475 (targeting B2M). In some embodiments, at least one of the two or more inhibitory RNAs is selected from SEQ ID NO: 66-87 or SEQ ID NO: 88-109 (targeting PPARG). In some embodiments, at least one of the two or more inhibitory RNAs is selected from SEQ ID NO: 110-133 or SEQ ID NO: 134-157 (targeting IRF4). In some embodiments, at least one of the two or more inhibitory RNAs is selected from SEQ ID NO: 158-181 or SEQ ID NO: 182-205 (targeting KDM6B). In some embodiments, at least one of the two or more inhibitory RNAs is selected from SEQ ID NO: 476 to SEQ ID NO: 525 or SEQ ID NO: 526 to SEQ ID NO: 575 (targeting FOXP3).In some embodiments, at least one of the two or more inhibitory RNAs is selected from SEQ ID NO:576-624 or SEQ ID NO:625-673 (targeting PDCD-1). In some embodiments, at least one of the two or more inhibitory RNAs is selected from SEQ ID NO:206-229 or SEQ ID NO:230-253 (targeting STAT6).

[0220] In some embodiments, the method optionally includes providing a cell with a nucleic acid construct adapted to express two or more inhibitory RNAs (i.e., two or more inhibitory RNAs with different targets) suitable for inhibiting expression of two or more target genes. In some embodiments, the cell is provided with, or the cell contains, a nucleic acid construct adapted to express two or more inhibitory RNAs (i.e., two or more inhibitory RNAs with different targets) suitable for inhibiting expression of two or more target genes.

[0221] In some preferred embodiments, the cell is a pancreatic cell. In some preferred embodiments, the cell is a T cell. In some preferred embodiments, the cell is a macrophage. In some embodiments, two or more target genes are selected from B2M, PPARG, IRF4, KDM6B, FOXP3, PDCD-1, and STAT6. In some embodiments, one of the target genes is B2M and another of the target genes is selected from STAT6, IRF4, DRP1, PPARG, KDM6B, Clever-1 (also known as STAB1 or FEEL-1), LAIR1 / 2, P-selective glycoprotein ligand (PSGL-1), signal-regulatory protein alpha (SIRPa), triggering receptor expressed on myeloid cells 2 (TREM2), proton-sensing GPCR (GPR65), inhibitory leukocyte immunoglobulin-like receptor subfamily B4 (LILRB4), RASA-2, CTLA4, and PD-1 (PDCD-1).

[0222] In some embodiments, one of the target genes is B2M and another of the target genes is selected from PPARG, IRF4, KDM6B, FOXP3, PDCD-1, and STAT6. In some embodiments, the two or more inhibitory RNAs are selected from the following: - SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411 or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), - SEQ ID NO: 66 to SEQ ID NO: 87 or SEQ ID NO: 88 to SEQ ID NO: 109 (targeting PPARG), - SEQ ID NO: 110 to SEQ ID NO: 133 or SEQ ID NO: 134 to SEQ ID NO: 157 (targeting IRF4), - SEQ ID NO: 158 to SEQ ID NO: 181 or SEQ ID NO: 182 to SEQ ID NO: 205 (targeting KDM6B), - SEQ ID NO: 476 to SEQ ID NO: 525 or SEQ ID NO: 526 to SEQ ID NO: 575 (targeting FOXP3), - SEQ ID NO: 576 to SEQ ID NO: 624 or SEQ ID NO: 625 to SEQ ID NO: 673 (targeting PDCD-1) or - SEQ ID NO: 206 to SEQ ID NO: 229 or SEQ ID NO: 230 to SEQ ID NO: 253 (targeting STAT6).

[0223] In some embodiments, one of the two or more inhibitory RNAs is selected from the following: SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M).

[0224] In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs is suitable for inhibiting a target gene selected from PPARG, IRF4, KDM6B, FOXP3, PDCD-1, and STAT6.

[0225] In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs is selected from: - SEQ ID NO: 66 to SEQ ID NO: 87 or SEQ ID NO: 88 to SEQ ID NO: 109 (targeting PPARG), - SEQ ID NO: 110 to SEQ ID NO: 133 or SEQ ID NO: 134 to SEQ ID NO: 157 (targeting IRF4), - SEQ ID NO: 158 to SEQ ID NO: 181 or SEQ ID NO: 182 to SEQ ID NO: 205 (targeting KDM6B), - SEQ ID NO: 476 to SEQ ID NO: 525 or SEQ ID NO: 526 to SEQ ID NO: 575 (targeting FOXP3), - SEQ ID NO: 576 to SEQ ID NO: 624 or SEQ ID NO: 625 to SEQ ID NO: 673 (targeting PDCD-1) or - SEQ ID NO: 206 to SEQ ID NO: 229 or SEQ ID NO: 230 to SEQ ID NO: 253 (targeting STAT6).

[0226] In some embodiments, one of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene B2M, and another of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene PPARG. In some embodiments, one of the two or more inhibitory RNAs inhibits expression of the target gene B2M, and another of the two or more inhibitory RNAs inhibits expression of the target gene PPARG. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene PPARG. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs inhibits expression of the target gene PPARG. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs is selected from SEQ ID NO: 66 to SEQ ID NO: 87 or SEQ ID NO: 88 to SEQ ID NO: 109 (targeting PPARG).

[0227] In some embodiments, one of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene B2M, and another of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene IRF4. In some embodiments, one of the two or more inhibitory RNAs inhibits expression of the target gene B2M, and another of the two or more inhibitory RNAs inhibits expression of the target gene IRF4. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene IRF4. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs inhibits expression of the target gene IRF4. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs is selected from SEQ ID NO: 110 to SEQ ID NO: 133, or SEQ ID NO: 134 to SEQ ID NO: 157 (targeting IRF4).

[0228] In some embodiments, one of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene B2M, and another of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene KDM6B. In some embodiments, one of the two or more inhibitory RNAs inhibits expression of the target gene B2M, and another of the two or more inhibitory RNAs inhibits expression of the target gene KDM6B. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene KDM6B. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs inhibits expression of the target gene KDM6B. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs is selected from SEQ ID NO: 158 to SEQ ID NO: 181, or SEQ ID NO: 182 to SEQ ID NO: 205 (targeting KDM6B).

[0229] In some embodiments, one of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene B2M, and another of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene FOXP3. In some embodiments, one of the two or more inhibitory RNAs inhibits expression of the target gene B2M, and another of the two or more inhibitory RNAs inhibits expression of the target gene FOXP3. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NOs: 348 to 411, or SEQ ID NOs: 412 to 475 (targeting B2M), and another of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene FOXP3. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs inhibits expression of the target gene FOXP3. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs is selected from SEQ ID NO: 476 to SEQ ID NO: 525, or SEQ ID NO: 526 to SEQ ID NO: 575 (targeting FOXP3).

[0230] In some embodiments, one of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene B2M, and another of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene PDCD-1. In some embodiments, one of the two or more inhibitory RNAs inhibits expression of the target gene B2M, and another of the two or more inhibitory RNAs inhibits expression of the target gene PDCD-1. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NOs: 348 to 411, or SEQ ID NOs: 412 to 475 (targeting B2M), and another of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene PDCD-1. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs inhibits expression of the target gene PDCD-1. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs is selected from SEQ ID NO: 576 to SEQ ID NO: 624, or SEQ ID NO: 625 to SEQ ID NO: 673 (targeting PDCD-1).

[0231] In some embodiments, one of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene B2M, and another of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene STAT6. In some embodiments, one of the two or more inhibitory RNAs inhibits expression of the target gene B2M, and another of the two or more inhibitory RNAs inhibits expression of the target gene STAT6. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NOs: 348 to 411, or SEQ ID NOs: 412 to 475 (targeting B2M), and another of the two or more inhibitory RNAs is suitable for inhibiting expression of the target gene STAT6. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO:1 or 18 (Solution 8), SEQ ID NO:5 or 22 (Solution 20), SEQ ID NO:9 or 26 (Solution 29), SEQ ID NO:10 or 27 (Solution 30), SEQ ID NO:348 to SEQ ID NO:411, or SEQ ID NO:412 to SEQ ID NO:475 (targeting B2M), and another of the two or more inhibitory RNAs inhibits expression of the target gene STAT6. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO:1 or 18 (Solution 8), SEQ ID NO:5 or 22 (Solution 20), SEQ ID NO:9 or 26 (Solution 29), SEQ ID NO:10 or 27 (Solution 30), SEQ ID NO:348 to SEQ ID NO:411, or SEQ ID NO:412 to SEQ ID NO:475 (targeting B2M), and another of the two or more inhibitory RNAs is selected from SEQ ID NO:206 to SEQ ID NO:229 or SEQ ID NO:230 to SEQ ID NO:253 (targeting STAT6).

[0232] In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411, or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another of the two or more inhibitory RNAs is selected from SEQ ID NO: 216 and / or SEQ ID NO: 240 (targeting STAT6), SEQ ID NO: 222 and / or SEQ ID NO: 246 (targeting STAT6), SEQ ID NO: 127 and / or SEQ ID NO: 151 (targeting IRF4), and SEQ ID NO: 177 and / or SEQ ID NO: 201 (targeting KDM6B).

[0233] In some embodiments, one of the target genes is PPARG and another of the target genes is selected from IRF4, KDM6B, and STAT6. In some embodiments, one of the target genes is PPARG and another of the target genes is IRF4. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO:66-87 or SEQ ID NO:88-109 (targeting PPARG) and another of the two or more inhibitory RNAs is selected from SEQ ID NO:110-133 or SEQ ID NO:134-157 (targeting IRF4). In some embodiments, one of the target genes is PPARG and another of the target genes is KDM6B. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO:66-SEQ ID NO:87 or SEQ ID NO:88-SEQ ID NO:109 (targeting PPARG), and another of the two or more inhibitory RNAs is selected from SEQ ID NO:158-SEQ ID NO:181 or SEQ ID NO:182-SEQ ID NO:205 (targeting KDM6B).

[0234] In some embodiments, one of the target genes is PPARG and another of the target genes is STAT6. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO:66-87 or SEQ ID NO:88-109 (targets PPARG), and another of the two or more inhibitory RNAs is selected from SEQ ID NO:206-229 or SEQ ID NO:230-253 (targets STAT6).

[0235] In some embodiments, one of the target genes is IRF4 and another of the target genes is selected from PPARG, KDM6B, and STAT6. In some embodiments, one of the target genes is IRF4 and another of the target genes is KDM6B. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO:110-133 or SEQ ID NO:134-157 (targets IRF4) and another of the two or more inhibitory RNAs is selected from SEQ ID NO:158-181 or SEQ ID NO:182-205 (targets KDM6B). In some embodiments, one of the target genes is IRF4 and another of the target genes is STAT6. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO:110 to SEQ ID NO:133 or SEQ ID NO:134 to SEQ ID NO:157 (targeting IRF4), and another of the two or more inhibitory RNAs is selected from SEQ ID NO:206 to SEQ ID NO:229 or SEQ ID NO:230 to SEQ ID NO:253 (targeting STAT6).

[0236] In some embodiments, one of the target genes is KDM6B and another of the target genes is selected from PPARG, IRF4, and STAT6. In some embodiments, one of the target genes is KDM6B and another of the target genes is STAT6. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO: 158-181 or SEQ ID NO: 182-205 (targeting KDM6B) and another of the two or more inhibitory RNAs is selected from SEQ ID NO: 206-229 or SEQ ID NO: 230-253 (targeting STAT6).

[0237] In some embodiments, one of the target genes is FOXP3 and another of the target genes is PDCD-1. In some embodiments, one of the two or more inhibitory RNAs is selected from SEQ ID NO:476-525 or SEQ ID NO:526-575 (targeting FOXP3), and another of the two or more inhibitory RNAs is selected from SEQ ID NO:576-624 or SEQ ID NO:625-673 (targeting PDCD-1). In some embodiments, one of the target genes is STAT6 and another of the target genes is selected from PPARG, IRF4, and KDM6B.

[0238] In some embodiments, the methods include providing a pancreatic cell with a nucleic acid construct adapted to express two or more inhibitory RNAs suitable for inhibiting expression of two or more target genes (i.e., two or more inhibitory RNAs with different targets), as appropriate.

[0239] In some embodiments, the methods include providing a T cell with a nucleic acid construct adapted to express two or more inhibitory RNAs suitable for inhibiting expression of two or more target genes (i.e., two or more inhibitory RNAs with different targets), as appropriate.

[0240] In some embodiments, the methods include providing macrophages with a nucleic acid construct adapted to express two or more inhibitory RNAs suitable for inhibiting expression of two or more target genes (i.e., two or more inhibitory RNAs with different targets), as appropriate.

[0241] Cells and cell status As will be apparent from the above, the present invention relates to inhibitory RNAs that are active in cells in a particular (first) context. In a particular embodiment, the inhibitory RNA is a modified form of an endogenous non-coding RNA, e.g., an inhibitory RNA with modified target specificity to target a desired target gene. It will be apparent that the target gene is different from the (original) gene normally targeted by the endogenous non-coding RNA.

[0242] In some embodiments, the modified cell may be in a given context in which the inhibitory RNA is not active, and the inhibitory RNA becomes active when the cell is in a different context. As a non-limiting example, a stem cell may be modified to contain a sequence encoding an inhibitory RNA that becomes active when the cell differentiates into a desired cell type. Alternatively, an immune cell or its progenitor cell may be modified to contain a sequence encoding an inhibitory RNA that becomes active when the immune cell is activated or polarized in a particular way, or enters a particular tissue. In such cases, the inhibitory RNA is active in a desired (first) context but is substantially inactive in another context.

[0243] In some embodiments of the invention, an inhibitory RNA may be selectively expressed or selectively processed in a particular cell or tissue type, or in cells under certain conditions or in a certain state. As a non-limiting example, the inhibitory RNA may be an inhibitory RNA that is expressed in a cell type of interest (e.g., a pancreatic endocrine hormone-expressing cell or an immune cell, such as a T cell or tumor-associated macrophage), as appropriate, but not expressed in an alternative state of the pancreatic endocrine hormone-expressing cell or immune cell (e.g., an inactivated, unresponsive, dysfunctional, or degenerated state), or in another cell type (e.g., a precursor / progenitor cell of a pancreatic cell or immune cell).

[0244] In some embodiments of the present invention, the method comprises modifying a cell to contain a nucleic acid construct adapted to express an inhibitory RNA suitable for inhibiting expression of a target gene, wherein the inhibitory RNA becomes active in the cell after a change in the cellular context. Thus, the inhibitory RNA is initially inactive in the cell but becomes active once the cell is in a different context (after activation, polarization, when the cell is in a particular tissue, etc.).

[0245] In some embodiments of the present invention, the method comprises modifying progenitor cells to contain a nucleic acid construct adapted to express an inhibitory RNA suitable for inhibiting the expression of a target gene, wherein the inhibitory RNA becomes active in the cells after differentiation, and preferably, the inhibitory RNA is active in fully differentiated cells. Thus, the inhibitory RNA is initially inactive in progenitor cells, but becomes active at a later stage of differentiation.

[0246] In some embodiments of the invention, the methods include modifying a progenitor cell (e.g., a stem cell such as an iPSC) such that an inhibitory RNA is active in the cell when the progenitor cell differentiates into a desired cell type (e.g., fully differentiated).

[0247] In some embodiments of the invention, the methods involve modifying progenitor cells (e.g., stem cells such as iPSCs) such that an inhibitory RNA is active in the cell when the cell has differentiated to a desired state (e.g., fully differentiated) and when the cell is in a particular context (e.g., after activation, polarization, when the cell is in a particular tissue, etc.).

[0248] In some embodiments, an inhibitory RNA may be selectively, specifically, or preferentially active in a particular cell type. Optionally, the inhibitory RNA is inactive, or active only at low levels, in other cell types.

[0249] In some embodiments, the first context is a cell type in which the inhibitory RNA is active, and the second context is another cell type in which target gene inhibition is not desired. In some embodiments, the first context is a cell type in which the inhibitory RNA is active, and the second context is any other cell type.

[0250] In some embodiments, the inhibitory RNA is selectively active (e.g., selectively expressed or selectively processed) in a particular cell or tissue type, or in a particular cell type in a particular context. By way of example, the inhibitory RNA can be an inhibitory RNA that is expressed in a cell type of interest (e.g., an enteroendocrine cell, e.g., a pancreatic beta cell, or an immune cell, e.g., a macrophage or T cell), as appropriate, but not in another type of cell (e.g., a precursor / progenitor cell of a pancreatic beta cell or an immune cell).

[0251] In some embodiments, the inhibitory RNA may be selectively, specifically, or preferentially active in cells at a particular stage of differentiation. Optionally, the inhibitory RNA is inactive or active only at low levels in cells of the same lineage but at a different stage of differentiation. In such embodiments, the first situation is a cell at a stage of differentiation at which the inhibitory RNA is active, and the second situation can be any other stage of differentiation. In some embodiments, the first situation is when the cell is fully differentiated, e.g., into a therapeutic cell, and the second situation is when the cell is undifferentiated or partially differentiated. In some embodiments, the first situation is IPSC-derived pancreatic progenitor cells, IPSC-derived monocytes, or IPSC-derived macrophages, and the second situation is undifferentiated iPSCs. In some embodiments, the first situation is IPSC-derived pancreatic progenitor cells, and the second situation is undifferentiated iPSCs. In some embodiments, the first situation is IPSC-derived monocytes or IPSC-derived macrophages, and the second situation is undifferentiated iPSCs. The ability to conditionally silence genes in iPSC-derived differentiated (progenitor) cells but not in undifferentiated iPSCs is beneficial in cell therapy. Conventional constitutive knockout of genes intended to modulate the recipient's immune response to therapeutic cells (e.g., MHC-I, see above) in iPSCs and their differentiated progenitor cells used for cell therapy carries risks. Contaminating trace amounts of constitutively knocked-out undifferentiated iPSCs (e.g., MHC-I) in therapeutic preparations may not be detected by the recipient's immune system after transplantation and may begin to proliferate and form teratomas. On the other hand, silencing such genes only in iPSC-derived differentiated (progenitor) cells means that any contaminating undifferentiated iPSCs will be recognized as "non-self" after transplantation (e.g., without MHC-I inhibition) and will be eliminated by the recipient's immune system.

[0252] In some embodiments, the inhibitory RNA may be active in a cell only when the cell is in a specific state. For example, the inhibitory RNA may be selectively, specifically, or preferentially active in cells that are activated in a specific manner, subjected to stress, present in a specific tissue, present in a tumor microenvironment, infected with a virus, or the like. In such embodiments, the first state is the cellular state in which the inhibitory RNA is active, and the second state may be any other cellular state. In some embodiments, the first state is a Treg cell state, and the second state is a T cell effector state. In some embodiments, the first state is a cell in the tumor microenvironment (TME).

[0253] In preferred embodiments of the present invention, the cell is a eukaryotic cell. According to some embodiments, the eukaryotic cell is derived from a eukaryotic organism selected from the group consisting of mammals, insects, nematodes, birds, reptiles, fish, crustaceans, fungi, and algae. According to preferred embodiments, the eukaryotic cell is a mammalian cell. According to even more preferred embodiments, the mammalian cell is a human cell.

[0254] In certain embodiments, the cells are differentiated cells, including, but not limited to, dendritic cells, lymphocytes, erythrocytes, platelets, hematopoietic cells, pancreatic islet cells (e.g., beta cells), liver cells, muscle cells, keratinocytes, cardiomyocytes, neurons, skeletal muscle cells, eye cells, mesenchymal cells, fibroblasts, lung cells, gastrointestinal cells, vascular cells, endocrine cells, and adipocytes. In particularly preferred embodiments, the cells are therapeutic forms of any of the foregoing cells. In some preferred embodiments, the inhibitory RNA is active in the relevant differentiated cells but is inactive or less active in precursors of the foregoing cells.

[0255] In certain embodiments, the cells are stem cells, e.g., therapeutic stem cells. In some embodiments, the stem cells are suitable for differentiation into differentiated cells, including, but not limited to, dendritic cells, lymphocytes, erythrocytes, platelets, hematopoietic cells, pancreatic islet cells (e.g., beta cells), liver cells, muscle cells, keratinocytes, cardiomyocytes, neurons, skeletal muscle cells, eye cells, mesenchymal cells, fibroblasts, lung cells, gastrointestinal cells, vascular cells, endocrine cells, or adipocytes. In some preferred embodiments, the inhibitory RNA is active in the relevant stem cells, but is inactive or less active when the stem cells are differentiated.

[0256] In some preferred embodiments, the cells are pluripotent cells, such as embryonic stem cells or iPSCs.

[0257] In some preferred embodiments, the cells are iPSCs or cells derived from iPSCs.

[0258] In some preferred embodiments, the cells are hematopoietic stem cells (HSCs).

[0259] According to some particularly preferred embodiments, the cells are therapeutic cells. Therapeutic cells are cells suitable for administration to a subject with the intention or expectation of providing some therapeutic benefit to the subject. In some embodiments, the cells are therapeutic endocrine cells. In some embodiments, the cells are therapeutic immune cells.

[0260] In some preferred embodiments of the invention, the cells are context-specific hypoimmunogenic cells. More preferably, in some embodiments, the cells are context-specific hypoimmunogenic therapeutic cells. Even more preferably, the cells are hypoimmunogenic allogeneic therapeutic cells. In such embodiments, the target gene is optionally a gene associated with the MHC-I system.

[0261] In some embodiments, the inhibitory RNA is: - cells in a particular state of differentiation, such as fully differentiated cells, cells at an intermediate stage of differentiation or undifferentiated cells, - totipotent, pluripotent or multipotent cells, - cells present in specific tissues, - cancerous or precancerous cells, - tumor-associated cells, such as tumor-associated immune cells or immune cells in the tumor microenvironment, - activated cells, e.g., macrophages polarized to a specific functional state, - immune cells that are in a memory or quiescent stem cell state rather than an effector state, - cells that are hypoxic, or - Cells subjected to the unfolded protein response The present invention is specifically active in cells in a condition selected from the group consisting of:

[0262] In some preferred embodiments, the cells are cells of the endocrine system, preferably therapeutic cells of the endocrine system. In some embodiments, the cells are enteroendocrine cells, preferably therapeutic enteroendocrine cells. Endocrine cells are found in a range of endocrine glands in the human and animal body. In some embodiments, the cells are derived from the pancreas, thyroid gland, parathyroid gland, pituitary gland, pineal gland, testis, ovary, adrenal gland, thymus, or hypothalamus. Thus, the inhibitory RNA may be selectively, specifically, or preferentially active in endocrine cells derived from one of the endocrine glands.

[0263] In some preferred embodiments, the cells are pancreatic cells, more preferably pancreatic endocrine hormone-expressing cells (e.g., beta, alpha, delta, or epsilon cells, most preferably beta cells). In such cases, it is generally preferred that the inhibitory RNA is selectively, specifically, or preferentially active in such pancreatic cells. In some preferred embodiments, the inhibitory RNA is expressed, but at a reduced level, in precursor / progenitor cells of said endocrine hormone-expressing pancreatic cells and is not expressed in stem cells, e.g., iPSCs.

[0264] In some preferred embodiments, the cells are pancreatic beta cells. Thus, the inhibitory RNA is selectively, specifically, or preferentially active in pancreatic beta cells. In some preferred embodiments, the inhibitory RNA is not active, or is active at a significantly lower level, in precursor / progenitor cells of pancreatic beta cells, such as stem cells, e.g., iPSCs.

[0265] In some embodiments of the invention, the inhibitory RNA is selectively active (e.g., selectively expressed or selectively processed) in pancreatic endocrine hormone-expressing cells. Suitable inhibitory RNAs are selectively active when these cells are properly vascularized and responding within predicted normal fasting blood glucose concentrations (between 70 mg / dL (3.9 mmol / L) and 100 mg / dL (5.6 mmol / L)). Optionally, the inhibitory RNA is inactive or less active when the cells are in an alternative state that is inactivated, glucose unresponsive, glucose responsive but electrically silent, hormone unresponsive, dysfunctional (does not secrete insulin, glucagon, somatostatin, etc.), degenerated, or hyperproliferative, experiencing oxidative stress as a result of exposure to hypoxia.

[0266] In another preferred embodiment of the present invention, the cell is an immune cell. Immune cells of interest include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes (B cells and T cells). Particularly interesting cells include T cells, preferably cytotoxic T cells, such as T cells and macrophages adapted for T cell therapy, e.g., CAR-T cell therapy. Optionally, the inhibitory RNA is selectively active in such immune cells.

[0267] In some embodiments, the immune cells are macrophages, e.g., M1 macrophages, M2 macrophages (e.g., M2a, M2b, M2c, and M2d macrophages), Treg macrophages, or tumor-associated macrophages (TAMs). In some preferred embodiments, the inhibitory RNA is selectively, specifically, or preferentially active in macrophages, as appropriate, in M1 macrophages, M2 macrophages, Treg macrophages, or tumor-associated macrophages (TAMs). In some preferred embodiments, the inhibitory RNA is selectively, specifically, or preferentially active in macrophages that do not have a specified function, preferably tumor-associated macrophages, e.g., macrophages adapted for macrophage therapy, e.g., CAR-M cell therapy, or macrophages adapted with a CAR that targets a tumor antigen.

[0268] In some preferred embodiments, the inhibitory RNA is selectively, specifically, or preferentially active in monocytes and macrophages. Optionally, the inhibitory RNA is inactive or less active in cells other than monocytes and macrophages. Optionally, the inhibitory RNA is inactive in precursor cells of monocytes and macrophages, such as stem cells (e.g., hematopoietic stem cells, monoblasts, or iPSCs). By way of non-limiting example, in such embodiments, the inhibitory RNA can be used to direct monocytes or macrophages toward a pro-inflammatory or anti-inflammatory phenotype, for example, by silencing one or more anti-inflammatory or pro-inflammatory genes, respectively.

[0269] In some preferred embodiments, the inhibitory RNA is selectively, specifically, or preferentially active in M2 macrophages (also known as alternatively activated macrophages). Optionally, the inhibitory RNA is inactive or less active in M1 macrophages (also known as classically activated macrophages) and / or unpolarized macrophages. By way of non-limiting example, in such embodiments, the inhibitory RNA can be used to direct M2 macrophages toward a pro-inflammatory phenotype, e.g., by silencing one or more anti-inflammatory genes in M1 macrophages. Optionally, the M2 macrophages can be M2a, M2b, M2c, or M2d macrophages.

[0270] In some preferred embodiments, the inhibitory RNA is selectively, specifically, or preferentially active in M1 macrophages. Optionally, the inhibitory RNA is inactive or less active in M2 macrophages and / or unpolarized macrophages. By way of non-limiting example, in such embodiments, the inhibitory RNA can be used to direct M1 macrophages toward an anti-inflammatory phenotype, e.g., by silencing one or more pro-inflammatory genes in M1 macrophages.

[0271] In some preferred embodiments, the inhibitory RNA is selectively, specifically, or preferentially active in unpolarized macrophages. Optionally, the inhibitory RNA is inactive or less active in polarized macrophages, e.g., M1 or M2 macrophages. By way of non-limiting example, in such embodiments, the inhibitory RNA can be used to direct monocytes or macrophages toward a pro-inflammatory or anti-inflammatory phenotype, e.g., by silencing one or more anti-inflammatory or pro-inflammatory genes, respectively.

[0272] In some preferred embodiments, the inhibitory RNA is selectively, specifically, or preferentially active in tumor-associated macrophages (TAMs) or in macrophages exposed to the tumor microenvironment (TME). Optionally, the inhibitory RNA is inactive or less active in M1 macrophages (also known as classically activated macrophages) and / or unpolarized macrophages, or when the macrophages are not exposed to the TME. By way of non-limiting example, in such embodiments, the inhibitory RNA can be used to direct macrophages exposed to the TAMs or TME toward a pro-inflammatory (anti-tumor) phenotype, for example, by silencing one or more anti-inflammatory genes in M1 macrophages.

[0273] Thus, in some preferred embodiments, the inhibitory RNA is selectively, specifically, or preferentially active in T cells, hi some preferred embodiments, the inhibitory RNA is selectively, specifically, or preferentially active in tumor-infiltrating cytotoxic T cells, e.g., T cells adapted for T cell therapy, e.g., CAR-T cell therapy.

[0274] In some preferred embodiments, the inhibitory RNA is not expressed, or is expressed at significantly lower levels, in precursor / progenitor cells of these immune cells, e.g., stem cells, e.g., iPSCs, or partially differentiated immune cell progenitor cells.

[0275] In some preferred embodiments, the inhibitory RNA is selectively, specifically, or preferentially active in immune cells in a particular state of activation. In some embodiments, the inhibitory RNA is not expressed, or is expressed at significantly lower levels, in immune cells not in said state of activation. In some preferred embodiments, the inhibitory RNA is inactive, or is active at significantly lower levels, in precursor / progenitor cells of immune cells, e.g., stem cells, e.g., iPSCs. As a non-limiting example, inhibitory RNA can be used to promote T cell activity in the tumor microenvironment, e.g., by targeting genes associated with immunosuppressive states (e.g., PD-1 (PDCD-1), CTLA4, etc.) in a context-specific manner (see Figure 13).

[0276] In some embodiments of the present invention, the inhibitory RNA may be selectively active in immune cells in the tumor microenvironment (TME). For example, the inhibitory RNA may be selectively active when these cells are responsive to tumor-derived chemokines (e.g., CXCL9 / 10 / 11 / 12, CCL2 / 5, CCL17 / 22), hypoxia (ppO2 below 8-10 mmHg), tumor-associated immunosuppressive cytokines (e.g., IL10, TGF-beta, IL4, IL13), immune cell checkpoint ligands (e.g., receptors PD-1 (PDCD-1), LAG3, TIM3, CTLA-4, Clever-1), cancer-associated fibroblast (CAF) signals (e.g., IL-11, CXCL12, GDF), tumor matrix (e.g., fibronectin, versican, collagen-11A1, SFRP2), or metabolites (e.g., lactate). Optionally, the inhibitory RNA is inactive or active at low levels when cells are in an alternative state, or have migrated out of the tumor microenvironment, or are polarized to a state in which they remain inactivated, are inactivatable, are exhausted, are rendered anergic, or are immunosuppressive.

[0277] In some embodiments of the invention, inhibitory RNAs are inactive or less active in immune cells when they migrate out of the tumor microenvironment or are altered as described, and as a result can no longer silence expression of receptor genes, their signaling components, transcription factors controlling immune checkpoints (e.g., PD-1 (PDCD-1), LAG3, TIM3, CTLA-4, Clever-1), tumor-derived checkpoints, immunosuppressive cytokines, CAF ligands, tumor matrix molecules fibronectin, versican, collagen-11A1, SFRP2, metabolite transporters (e.g., lactate, monocarboxylate transporter-1 (SLC16A1), L-kynurenine receptor (aryl hydrocarbon receptor (RP85; bHLHe76)) or mitochondrial fission regulator DRP1 (dynamin-related protein 1)), and as a result, the immune cells become inactive, exhausted, immunosuppressive or anti-inflammatory.

[0278] In some preferred embodiments, the inhibitory RNA is selectively, specifically, or preferentially active in: - M2 macrophages compared to M0 macrophages, or vice versa, - M2 macrophages compared to M1 macrophages, or vice versa; - M1 macrophages compared to M0 macrophages, or vice versa, - M2a macrophages compared to M0 macrophages, or vice versa, - M2a macrophages compared to M1 macrophages, or vice versa, - M2c macrophages compared to M0 macrophages, or vice versa, - M2c macrophages compared to M1 macrophages, or vice versa, M2c macrophages compared to M2a macrophages, or vice versa, - M0, M1 or M2 macrophages compared to monocytes, or vice versa; - immune cells (e.g., T cells or TAMs) in the TME compared to immune cells not in the TME, or - stimulated T cells compared to naive T cells, or vice versa.

[0279] In some embodiments of the invention, an inhibitory RNA is expressed in a cell as a modified form of an endogenous non-coding, usually inhibitory, RNA that is selectively, specifically, or preferentially expressed in a first context, e.g., a cell or tissue type of interest, such as when the cell is in a particular state of activation or differentiation. This can be achieved by modifying the endogenous inhibitory RNA in the cell or by introducing a modified form of the endogenous inhibitory RNA. Preferably, this is achieved by modifying the endogenous inhibitory RNA in situ, e.g., via GEiGS.

[0280] Various databases providing details of the tissue and cellular expression profiles of inhibitory RNAs are well known. Those skilled in the art can readily use these to identify endogenous inhibitory RNAs suitable for modification to provide the context-specific activity of the inhibitory RNAs contemplated in the present invention. See, for example, the miRmine human miRNA expression database, https: / / guanfiles.dcmb.med.umich.edu / mirmine / ; Panwar et al., Bioinformatics. 2017 May 15; 33(10): 1554-1560; and the DIANA-miTED microRNA tissue expression database—Kavakiotis et al., "DIANA-miTED: a microRNA tissue expression database," Nucleic Acids Research, Volume 50, Issue D1, January 7, 2022, Pages D1055-D1061, http: / / www.microrna.gr / mited).

[0281] In addition, the technique of experimentally analyzing the expression profile of inhibitory RNA is well known in the art.Therefore, for any given situation (for example, cell type or condition), those skilled in the art can easily determine whether endogenous RNA is expressed at high level or low level, and therefore select one or more suitable endogenous RNAs for modification to target desired RNA, thereby achieving desired target gene silence.

[0282] Suitable methods for analyzing miRNA expression in a given cell are known in the art, and one approach suitable for use in the present invention is as follows: Small RNA and miRNA isolation—Small RNAs, including miRNAs, are isolated using the miRvana RNA isolation kit (Ambion, Austin, TX, USA) according to the manufacturer's protocol. RNA is quantified using a Qubit or Nanodrop spectrophotometer (Thermo Fisher, Wilmington, DE, USA), and quality is determined using an Agilent 6000 Nano chip (Agilent Technologies, Palo Alto, CA, USA). miRNA measurement: Quantitative real-time PCR analysis is performed as follows: RNA is reverse transcribed and PCR amplified using the miScript reverse transcription kit and miScript SYBR PCR kit (Qiagen, Valencia, CA, USA) on an ABI 7500 real-time PCR system according to the manufacturer's protocol. Values ​​from duplicate reactions are averaged and normalized to U6 SnoRNA levels. Relative expression levels are calculated according to the comparative Ct method as previously described (Schmittgen and Livak. Nat Protoc (2008) 3: 1101-1108). Alternatively, miRNAs are detected and relatively quantified using small RNA sequencing (as described in www.illumina.com / techniques / sequencing / RNA-sequencing / small-RNA-seq.html or Wake et al., BMC Genomics (2016) 17(1): 1).

[0283] In some embodiments, the inhibitory RNA is expressed at a level of at least 1000 RPM (reads per million mapped reads) in cells in the first situation, at least 2000 RPM in cells in the first situation, at least 3000 RPM in cells in the first situation, at least 4000 RPM in cells in the first situation, at least 5000 RPM in cells in the first situation, at least 10000 RPM in cells in the first situation, at least 20000 RPM in cells in the first situation, or at least 50000 RM in cells in the first situation, or at least 100000 RPM in cells in the first situation.

[0284] In some embodiments, in a second situation where it is desired that the inhibitory RNA be inactive or less active, the inhibitory RNA is expressed at a level less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of that in cells in the first situation. In some embodiments, the inhibitory RNA is expressed at a level less than 5000 RPM in cells in the second situation, less than 4000 RPM in cells in the second situation, less than 3000 RPM in cells in the second situation, less than 2000 RPM in cells in the second situation, less than 1000 RPM in cells in the second situation, less than 500 RPM in cells in the second situation, less than 300 RPM in cells in the second situation, less than 200 RPM in cells in the second situation, less than 100 RPM in cells in the second situation, or less than 50 RPM in cells in the second situation.

[0285] In some embodiments, the endogenous inhibitory RNA that has been modified to provide an inhibitory RNA of the invention is expressed at a level of at least 1000 RPM (reads per million mapped reads) in cells in the first situation, at least 2000 RPM in cells in the first situation, at least 3000 RPM in cells in the first situation, at least 4000 RPM in cells in the first situation, at least 5000 RPM in cells in the first situation, at least 10000 RPM in cells in the first situation, at least 20000 RPM in cells in the first situation, or at least 50000 RM in cells in the first situation, or at least 100000 RPM in cells in the first situation.

[0286] In some embodiments, in the second situation, the endogenous inhibitory RNA that has been modified to provide an inhibitory RNA of the invention is expressed at a level that is less than 25%, 20%, 15%, 10%, 5%, or 1% of the level of the cell in the first situation.

[0287] In some embodiments, the endogenous inhibitory RNA that has been modified to provide an inhibitory RNA of the invention is expressed at a level of less than 5000 RPM in a cell in the second situation, less than 4000 RPM in a cell in the second situation, less than 3000 RPM in a cell in the second situation, less than 2000 RPM in a cell in the second situation, less than 1000 RPM in a cell in the second situation, less than 500 RPM in a cell in the second situation, less than 300 RPM in a cell in the second situation, less than 200 RPM in a cell in the second situation, less than 100 RPM in a cell in the second situation, or less than 50 RPM in a cell in the second situation.

[0288] In pancreatic tissue, for example, hsa-miR-375, hsa-miR-143, and hsa-miR-21 are expressed at particularly high levels (189993.6, 186579.2, and 65231.3 RPM, respectively, according to the miRmine database), and various other miRNAs are also expressed at significant, but more moderate, levels (e.g., hsa-miR-30d, hsa-miR-192, hsa-miR-30a, hsa-miR-182, hsa-miR-148a, hsa-miR-127, hsa-miR-200c, etc.).

[0289] In liver tissue, for example, hsa-miR-143, hsa-miR-10a, and hsa-miR-122 are expressed at particularly high levels (93940.6, 92139.6, and 80063.1 RPM, respectively, according to the miRmine database), and various other miRNAs are also expressed at significant, but more moderate, levels (e.g., hsa-miR-22, hsa-miR-192, hsa-miR-146b, hsa-miR-26a, hsa-miR-125a, hsa-miR-150, etc.).

[0290] In brain tissue, for example, hsa-miR-10b and hsa-miR-181a are expressed at particularly high levels (198835.2 and 51936.2 RPM, respectively, according to the miRmine database), and various other miRNAs are also expressed at significant, but moderate levels (e.g., hsa-miR-26a, hsa-miR-92a, hsa-miR-143, hsa-miR-10a, hsa-miR-21, etc.).

[0291] For T cells, for example, miRNAs are known to be critical regulators of tumor-associated T lymphocytes (Xing Y et al. Tumor Immune Microenvironment and Its Related miRNAs in Tumor Progression. Front Immunol. 2021 May 18;12:624725. doi: 10.3389 / fimmu.2021.624725. PMID: 34084160; PMCID: PMC8167795).

[0292] For macrophages and monocytes, for example, in some embodiments, the inhibitory RNA is a modified version of MIR146A (e.g., hsa-miR-146a-5p), MIR20A (e.g., hsa-miR-20a-5p), LET7C (hsa-miR-let-7c-5p), or LET7i (hsa-let-7i-5p). Such miRNAs can be used, for example, to induce silencing when monocytes differentiate into macrophages, and the silencing is maintained to block the initiation of the M2-specific program.

[0293] NGS analysis shows that miR146A (hsa-miR-146a-5p) is highly expressed in M0, M1, and M2 macrophages, and qPCR also shows a strong induction of proliferative THP-1 monocytes into an M0-like state. Therefore, miR146A (hsa-miR-146a-5p) is of particular interest as an inhibitory RNA of differentiated macrophages (e.g., M0, M1, and M2 macrophages) as opposed to monocyte precursors.

[0294] For MIR20A (hsa-miR-20a-5p), a combination of NGS and qPCR showed high expression in proliferating THP-1 monocytes and M0-, M1-, and M2-like cells, making MIR20A (hsa-miR-20a-5p) particularly interesting as an inhibitory RNA for monocytes and differentiated macrophages.

[0295] For LET7C (hsa-miR-let-7c-5p), NGS shows M2a-specific induction (to be confirmed by qPCR). Therefore, LET7C (hsa-miR-let-7c-5p) is of particular interest as an inhibitory RNA of differentiated macrophages, especially M2 and / or M2a macrophages.

[0296] For LET7i(hsa-let-7i-5p), NGS showed very strong expression in M0, M1, M2a, and M2c macrophages, with moderate levels of induction compared to proliferating THP-1 monocytes. Therefore, LET7i(hsa-let-7i-5p) may be particularly interesting as an inhibitory RNA for differentiated macrophages, but may exhibit reduced levels of expression in monocytes. LET7i(hsa-let-7i-5p) may therefore be interesting as an inhibitory RNA for differentiated macrophages, but with reduced target gene inhibition (i.e., less silencing) in monocytes compared to macrophages.

[0297] Further examples of differentially expressed miRNAs in macrophages are shown in Tables 15-20 (Tables 18-23). ​​Tables 15-20 below (Tables 18-23) provide a summary of the results of differentially expressed miRNAs identified in different macrophage cell states, as follows: - Table 15 - M1 macrophages compared to M0 macrophages. - Table 16 - M2a macrophages compared to M0 macrophages. - Table 17 - M2a macrophages compared to M1 macrophages. - Table 18 - M2c macrophages compared to M0 macrophages. - Table 19 - M2c macrophages compared to M1 macrophages. - Table 20 - M2c macrophages compared to M2a macrophages.

[0298] Suitable miRNAs for use in the present invention can be selected from these tables. Note that a positive fold change represents the increase in the first designated cell type compared to the second designated cell type, and a negative fold change represents the increase in the second designated cell type compared to the first. Thus, in Table 15 (Table 18), a positive fold change represents the increase in expression in M1 compared to M0, and a negative fold change represents the increase in expression in M0 compared to M1. Generally, miRNAs with high levels of differential expression are preferred to provide more specific silencing in a given cell state (i.e., high log FC value). MiRNAs with higher expression levels may also be preferred in some cases.

[0299] Further examples of differentially expressed miRNAs in stimulated versus naive T cells are shown in Table 21. Table 21 summarizes the results of NGS differential analyses comparable to those performed to determine the results shown in Tables 15-20. Again, in general, miRNAs with higher levels of differential expression are preferred for providing more specific silencing in a given cellular state (i.e., higher logFC values). MiRNAs with higher expression levels may also be preferred in some cases.

[0300] More generally, miRNAs are known to be critical regulators of tumor-associated macrophages (Chatterjee, et al., MicroRNAs: As Critical Regulators of Tumor-Associated Macrophages. Int J Mol Sci. 2020 Sep 27;21(19):7117. doi: 10.3390 / ijms21197117. PMID: 32992449; PMCID: PMC7582892). This suggests additional miRNAs that could be adapted for context-specific gene silencing in TAMs, for example, to silence genes that promote an M2-like immunosuppressive or quiescent phenotype.

[0301] Additional examples from the literature of miRNAs associated with M2-like macrophages include (Table 1):

[0302] [Table 1A]

[0303] [Table 1B]

[0304] [Table 1C]

[0305] Therefore, these miRNA variants can be interesting for context-specific inhibition in M2 macrophages.For example, in some embodiments, these miRNA variants can be used to silence suitable target genes, and shift macrophages to a pro-inflammatory state (e.g., M1-like state).The miRNAs that are selectively expressed at high levels in M2 macrophages can be particularly interesting for context-specific silencing of target genes in therapeutic macrophages for cancer treatment.

[0306] Additional examples from the literature of miRNAs associated with M1-like macrophages include the following (Table 2):

[0307] [Table 2]

[0308] Therefore, these miRNA variants may be of interest for context-specific inhibition in M1 macrophages. For example, in some embodiments, these miRNA variants can be used to silence appropriate target genes and transition macrophages into an anti-inflammatory or pro-regenerative (tissue repair) state (e.g., an M2-like state). miRNAs selectively expressed at high levels in M1 macrophages may be particularly interesting for context-specific silencing of target genes in therapeutic macrophages for the treatment of chronic inflammatory and fibrotic conditions.

[0309] By way of non-limiting example, the present invention provides, in some preferred embodiments, cell type conditional mRNA targeted silencing, for example: (1) Generation of hypoimmunogenic cells for therapy via partial silencing of either B2M or classical HLA-(A / B / C) molecules; (2) Identifying mRNA targets (and mechanisms) that define the immunosuppressive state by silencing these cells in therapeutic macrophages targeted to the solid tumor microenvironment; or (3) Enhanced persistence of therapeutic T cells by silencing immune checkpoints Regarding.

[0310] In Table 3, further non-limiting exemplary cellular contexts are provided in which context-specific gene silencing may be provided in accordance with the present invention.

[0311] [Table 3]

[0312] miRNAs selectively expressed at high levels in M2 macrophages may be of particular interest for context-specific silencing of target genes in therapeutic macrophages for the treatment of cancer.

[0313] References from Table 3: (1)Liu Y et al., LAIR-1 suppresses cell growth of ovarian cancer cell via the PI3K-AKT-mTOR pathway. Aging (Albany NY). 2020 Sep 5;12(16):16142~16154. doi: 10.18632 / aging.103589. PMID: 32628130; PMCID: PMC7485720. (2) Zhou M et al., Role of Epithelial-Mesenchymal Transition in Retinal Pigment Epithelium Dysfunction. Front Cell Dev Biol. 2020 Jun 25;8:501. doi: 10.3389 / fcell.2020.00501. PMID: 32671066; PMCID: PMC7329994. (3) Mathewson ND et al., Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis. Cell. 2021 Mar 4;184(5):1281-1298.e26. doi: 10.1016 / j.cell.2021.01.022. Epub 2021 Feb 15. PMID: 33592174; PMCID: PMC7935772. Roth P et al. Malignant glioma cells counteract antitumor immune responses through expression of lectin-like transcript-1. Cancer Res. 2007 Apr 15;67(8):3540-4. doi: 10.1158 / 0008-5472.CAN-06-4783. PMID: 17440061. Di W et al., Clinical characterization and immunosuppressive regulation of CD161 (KLRB1) in glioma through 916 samples. Cancer Sci. 2022 Feb;113(2):756~769 pages. doi: 10.1111 / cas.15236. Epub December 24, 2021. PMID: 34881489; PMCID: PMC8819299. (4) Beneto N et al. Sanfilippo Syndrome: Molecular Basis, Disease Models and Therapeutic Approaches. Int J Mol Sci. 2020;10;21(21):7819. doi: 10.3390 / ijms21217819. PMID: 33105639; PMCID: PMC7659972. (5)Liu J, Wang F. Role of Neuroinflammation in Amyotrophic Lateral Sclerosis: Cellular Mechanisms and Therapeutic Implications. Front Immunol. 2017 Aug 21;8:1005. doi: 10.3389 / fimmu.2017.01005. PMID: 28871262; PMCID: PMC5567007. (6)Chen YJ et al. Use of "MGE enhancers" for labeling and selection of embryonic stem cell-derived medial ganglionic eminence (MGE) progenitors and neurons. PLoS One. May 1, 2013;8(5):e61956. doi: 10.1371 / journal.pone.0061956. PMID: 23658702; PMCID: PMC3641041. (7)Kohno K et al. A spinal microglia population involved in remitting and relapsing neuropathic pain. Science. 2022 Apr;376(6588):86~90. doi: 10.1126 / science.abf6805. Epub 2022 Mar 31. PMID: 35357926.

[0314] Nucleic acid constructs adapted to express inhibitory RNA In some embodiments, the nucleic acid construct adapted to express an inhibitory RNA is part of the genome of the cell (ie, is a genomic sequence).

[0315] In some embodiments of the invention, the nucleic acid construct adapted to express an inhibitory RNA is episomal.

[0316] In a preferred embodiment, the nucleic acid construct is adapted to express an miRNA.

[0317] In a preferred embodiment, the nucleic acid construct expresses a modified form of an endogenous inhibitory RNA that is expressed in a context-specific manner, where the inhibitory RNA is modified relative to the endogenous RNA so as to inhibit expression of a target gene. Thus, the context-specific expression of the endogenous inhibitory RNA is maintained, but the targeting specificity of the inhibitory RNA is redirected to inhibit expression of a target gene (different from the gene targeted by the endogenous inhibitory RNA).

[0318] In some embodiments, the nucleic acid construct adapted to express an inhibitory RNA is part of the genome of the cell and is present in an endogenous locus in the genome that encodes an endogenous non-coding RNA, e.g., an endogenous inhibitory RNA.

[0319] In some embodiments, the nucleic acid construct adapted to express an inhibitory RNA is a modified endogenous nucleic acid sequence that encodes an endogenous non-coding RNA, e.g., a retargeted endogenous inhibitory RNA that has been modified in situ.

[0320] In some embodiments of the present invention, a sequence encoding an inhibitory RNA (preferably a promoterless sequence encoding an inhibitory RNA) is preferably inserted into an endogenous miRNA locus without disrupting or inactivating any endogenous miRNA. The endogenous miRNA locus may encode an endogenous miRNA that has been modified to target a new target mRNA. Thus, in some embodiments, a modified version of an endogenous miRNA is inserted into a genomic locus containing the original (unmodified) endogenous miRNA, and the original miRNA is not disrupted or inactivated. Thus, the expression or activity of the original (unmodified) endogenous miRNA may not be altered.

[0321] In some embodiments, the nucleic acid construct adapted to express an inhibitory RNA is a modified endogenous nucleic acid sequence encoding an endogenous non-coding RNA, where the endogenous non-coding RNA (e.g., the endogenous inhibitory RNA) is modified to inhibit expression of a target gene (e.g., target a transcript from the target gene). In some embodiments, the nucleic acid construct adapted to express an inhibitory RNA is comprised in a synthetic expression cassette. In some embodiments, the nucleic acid construct adapted to express an inhibitory RNA is comprised in an expression vector.

[0322] In some embodiments of the invention, the nucleic acid construct adapted to express an inhibitory RNA is part of the genome of the cell and is present in a locus that does not encode an endogenous non-coding RNA.

[0323] The nucleic acid construct that is adapted to express the inhibitory RNA that is suitable for inhibiting the expression of target gene can be introduced into cell by any method known to those skilled in the art.In some embodiments, the nucleic acid construct that is adapted to express the inhibitory RNA that is suitable for inhibiting the expression of target gene is introduced by lentivirus delivery.

[0324] Target gene It will be apparent that the present invention provides for the targeting of a wide range of target genes in any given situation. Of particular interest in the present invention are target genes that can be partially or completely silenced to induce a specific phenotype, property, or behavior of a cell in a given situation. For example, it may be intended to alter the immunogenicity of a cell, to make the cell active or quiescent, or to make the cell pro-inflammatory or anti-inflammatory.

[0325] In some embodiments of the present invention, it is desirable to target genes that alter, preferably reduce, the immunogenicity of cells.This is particularly interesting for therapeutic cells, especially allogeneic therapeutic cells, such as allogeneic stem cell-derived islet cells.As discussed herein, genes of the MHC-I complex are particularly interesting for generating low immunogenic cells.

[0326] Thus, in some embodiments, the target gene is a gene of the MHC-I complex. In some preferred embodiments, the target gene is beta2-microglobulin (B2M). In other embodiments, the target gene is an HLA-A, HLA-B, and / or HLA-C gene. In some embodiments, the target gene is a combination of HLA-A and B or C, or HLA-B and C, or HLA-A / B / C genes.

[0327] In some embodiments, target genes of inflammatory pathways in allogeneic stem cell derived therapeutic cells (e.g., islet cells) to alter, preferably reduce, the immunogenicity of the cells not associated with the MHC-I complex are selected from TAP1 / 2, CD74, PSMB9, chemokine ligand 10 (CXCL10), CXCL5, CXCL9, signaling molecules STAT1, JAK1 / 2, HNRNPA1P48, GBP2, and TRIM.

[0328] In some embodiments of the invention, it may be desirable to target genes that alter immune cell activity, for example, genes that, when inhibited, lead to a pro-inflammatory phenotype or an immunosuppressive (anti-inflammatory) phenotype.

[0329] It will become apparent that there are some conditions in which it is desirable to make immune cells pro-inflammatory, for example, in tumors. In other conditions, it is desirable to make immune cells immunosuppressive or anti-inflammatory, for example, in autoimmune or other inflammatory diseases. Macrophages and T cells are of particular interest given their important roles in immune activity and signaling in many situations.

[0330] As a non-limiting example, in macrophages, it may be desirable to target one or more genes to render the cells pro-inflammatory, or at least avoid an immunosuppressive state. This may be of particular interest due to context-specific expression in the TME, where macrophages typically become tumor-associated macrophages (TAMs), key cells that create an immunosuppressive microenvironment by producing cytokines, chemokines, growth factors, and triggering the release of inhibitory immune checkpoint proteins in T cells. Thus, it would be desirable to have one or more inhibitory RNAs active in the TME targeting one or more genes, such that inhibition of said one or more genes modifies macrophages to promote a pro-inflammatory or non-immunosuppressive state.

[0331] Thus, in some embodiments, the target gene is preferably one or more genes associated with promoting an immunosuppressive state (and thus promoting a pro-inflammatory phenotype) in immune cells, particularly macrophages. For example, the target gene may be one or more selected from the group consisting of STAT6, IRF4, DRP1, PPARG, KDM6B, Clever-1 (also known as STAB1 or FEEL-1), LAIR1 / 2, P-selective glycoprotein ligand (PSGL-1), signal-regulatory protein alpha (SIRPa), triggering receptor expressed on myeloid cells 2 (TREM2), proton-sensing GPCR (GPR65), inhibitory leukocyte immunoglobulin-like receptor subfamily B4 (LILRB4), and PD-1 (PDCD-1).

[0332] In some embodiments, the target gene is preferably selected from one or more genes expressed in tumor-associated macrophages (TAMs). For example, the target gene may be one or more selected from the group consisting of TREM2, Clever-1 (also known as FEEL-1 and STAB-1), LAIR1 / 2, and inhibitory leukocyte immunoglobulin-like receptor subfamily B4 (LILRB4).

[0333] TREM2 plays an immunosuppressive role in cancer development, as shown by Katzenelenbogen et al. [Katzenelenbogen, Y., Sheban, F., Yalin, A., Yofe, I., Svetlichnyy, D., Jaitin, DA, Bornstein, C., Moshe, A., Keren-Shaul, H., Cohen, M., Wang, SY, Li, B., David, E., Salame, TM, Weiner, A., & Amit, I. (2020). Coupled scRNA-Seq and Intracellular Protein Activity Reveal an Immunosuppressive Role of TREM2 in Cancer. Cell, 182(4), pp. 872-885.e19. https: / / doi.org / 10.1016 / J.CELL.2020.06.032], which is incorporated herein by reference.Systemic blockade of Clever-1 (also known as FEEL-1 and STAB-1) using antibodies has been described by Virtakoivu et al. [Virtakoivu, R., Rannikko, JH, Viitala, M., Vaura, F., Takeda, A., Lonnberg, T., Koivunen, J., Jaakkola, P., Pasanen, A., Shetty, S., de Jonge, MJA, Robbrecht, D., Ma, YT, Skytta, T., Minchom, A., Jalkanen, S., Karvonen, MK, Mandelin, J., Bono, P., & Hollmen, M. (2021). Systemic Blockade of Clever-1 Elicits Lymphocyte Activation Alongside Checkpoint Molecule Downregulation in Patients with Solid Tumors: Results from a Phase I / II Clinical Trial. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research For Cancer Research, 27(15), pp. 4205-4220. https: / / doi.org / 10.1158 / 1078-0432.CCR-20-4862], there are numerous tissues that express molecules that can induce lymphocyte activation in patients with solid tumors but act as "sinks" that limit the "antitumor effect."LAIR-1 activation by tumor-specific isoforms of collagen has been reported by Keerthivasan et al. [Keerthivasan, S., Senbabaoglu, Y., Martinez-Martin, N., Husain, B., Verschueren, E., Wong, A., Yang, YA, Sun, Y., Pham, V., Hinkle, T., Oei, Y., Madireddi, S., Corpuz, R., Tam, L., Carlisle, S., Roose-Girma, M., Modrusan, Z., Ye, Z., Koerber, JT, & Turley, SJ (2021). Homeostatic functions of monocytes and interstitial lung macrophages are regulated via collagen domain-binding receptor LAIR1. Immunity, 54(7), pp. 1511–1526. e8], which is incorporated herein by reference. It inhibits the pro-inflammatory macrophage phenotype in vitro, as shown in [https: / / doi.org / 10.1016 / J.IMMUNI.2021.06.012]. Inhibitory leukocyte immunoglobulin-like receptor subfamily B4 (LILRB4) [Sharma, N., Atolagbe, OT, Ge, Z., & Allison, JP (2021). LILRB4 suppresses immunity in solid tumors and is a potential target for immunotherapy. The Journal of Experimental Medicine, 218(7). https: / / doi.org / 10.1084 / JEM.20201811] acts as a myeloid cell checkpoint and also suppresses the anti-tumor, pro-inflammatory state of macrophages, as shown in Sharma, et al., incorporated herein by reference.

[0334] T cells also play an important role in the TME. Tumor-infiltrating lymphocytes (TILs) and CAR-T cells are usually tolerized by the activity of immune checkpoint pathways, such as PD-1 (PDCD-1), CTLA4, LAG3, and TIM3. Therefore, in some embodiments, the target gene is preferably one or more genes associated with a tolerized state in T cells. For example, the target gene may be one or more selected from the group consisting of PD-1 (PDCD-1), CTLA4, LAG3, TIGIT (T cell immunoreceptor with Ig and ITIM domains), and TIM3. The target gene may also preferably be one or more genes upregulated in an exhausted state in T cells. For example, the target gene may be one or more selected from the group consisting of TOX subfamily (thymocyte selection-associated high mobility group box protein), NR4A1 (nuclear receptor 4A1; Nur77, TR3, NGFI-B), and CBL-B (E3 ubiquitin protein ligase).

[0335] An additional example of a target gene in T cells is RASA-2.

[0336] RASA-2 (RAS GTPase-activating protein) ablation in T cells has been reported by Carnevale et al. [Carnevale J, Shifrut E, Kale N, Nyberg WA, Blaeschke F, Chen YY, Li Z, Bapat SP, Diolaiti ME, O'Leary P, Vedova S, Belk J, Daniel B, Roth TL, Bachl S, Anido AA, Prinzing B, Ibanez-Vega J, Lange S, Haydar D, Luetke-Eversloh M, Born-Bony M, Hegde B, Kogan S, Feuchtinger T, Okada H, Satpathy AT, Shannon K, Gottschalk S, Eyquem J, Krenciute G, Ashworth A, Marson A. RASA2 ablation in T cells boosts antigen sensitivity and long-term function. Nature.], incorporated herein by reference. 2022 Sep;609(7925):174-182. doi: 10.1038 / s41586-022-05126-w. Epub 2022 Aug 24. PMID: 36002574; PMCID: PMC9433322], enhancing antigen sensitivity and long-term function.

[0337] In some embodiments of the present invention, it is desirable to target genes expressed on the cell surface. For example, it may be desirable to silence a gene encoding an antigen to prevent the cell from being recognized by an antibody that recognizes the antigen. Thus, in some embodiments, the target gene is preferably one or more genes expressed on the surface of a cell, e.g., a T cell or its progenitor (e.g., a hematopoietic stem cell (HSC) or other T cell progenitor, e.g., a common lymphoid progenitor or small lymphocyte). For example, the target gene may be a T cell-specific surface protein, e.g., CD52. Genes encoding other cell surface proteins may also be of interest, and thus the present invention contemplates the context-specific silencing of genes encoding cell surface proteins other than CD52. Optionally, the target gene encodes any marker protein specific to a given cell type, e.g., an immune cell type. In some preferred embodiments, the target gene encodes a cell surface protein that is the target of an antibody used in the depletion of immune cells or their progenitor cells (e.g., lymphodepletion), such that cells in which the target gene is silenced are protected from the antibody. By way of non-limiting example, this approach can be used to target one or more genes selected from those encoding CD117, CD27, CD45, CD90, CD110, and CD184. See, e.g., Czechowicz et al., "Selective hematopoietic stem cell ablation using CD117-antibody-drug-conjugates enables safe and effective transplantation with immunity preservation," 2019 Feb. 6;10(1):617.

[0338] In some preferred embodiments, the target gene encodes CD52. In some preferred embodiments of the present invention, CD52 is silenced in a context-specific manner in activated T cells. In some embodiments, a gene encoding both B2M and CD52 is the target gene for context-specific silencing. In some embodiments, target genes encoding both B2M and CD52 are downregulated in the same context, for example, in T cells, in activated T cells, and / or in T cell progenitor cells. Context-specific silencing of CD52 and / or B2M in activated T cells is a particularly preferred embodiment.

[0339] In some cases, it may be desirable to context-specifically silence a target gene in cells in which other genes are constitutively silenced, e.g., knocked out or knocked down (e.g., by CRISPR). Constitutive silencing can also be achieved using GEiGS, e.g., using a solution based on a miRNA scaffold with a broad expression pattern (e.g., miRNAs expressed in T cells, activated T cells, as well as resting T cells). For example, the methods of the present invention can be used to silence, e.g., activated T cells in which CD52 is constitutively silenced, e.g., knocked out (i.e., CD52- B2M can be silenced in a context-specific manner in CAR-T cells.

[0340] Silencing CD52 may be particularly desirable for use in cell therapy, e.g., cell-based cancer therapy, in which anti-CD52 antibodies are used to "condition" patients prior to administration of therapeutic cells. One particularly important example is the use of anti-CD52 antibodies prior to allogeneic T-cell therapy, but anti-CD52 antibodies are also useful in allogeneic hematopoietic stem cell transplantation (HSCT). Specifically, anti-CD52 antibodies (e.g., alemtuzumab) are administered to patients about to receive allogeneic CAR-T cell infusion or allogeneic HSCT. This treatment kills the patient's own T cells, creating a niche for the transplanted cells to expand and / or target the cancer after infusion. Silencing CD52 in patients receiving therapeutic CAR-T or HSC cells prevents their elimination by circulating anti-CD52 antibodies, allowing a window in which the therapeutic cells can survive and kill cancer cells. In some cases, knocking out CD52 can lead to the generation of dysfunctional T cells, so context-specific silencing of CD52 can be advantageous, for example, to reduce the risk of undesirable T cell phenotypes (see also Kinsella et al., "CD52 / GPI- T-Cells Are Enriched for Alloreactive Specificity and Predict Acute Graft-Versus-Host-Disease After Stem Cell Transplantation," Transplantation and Cellular Therapy, Vol. 27, No. 6, 2021, pp. 475.e1-475.e9). Therefore, as noted above, silencing CD52 in activated T cells is a particularly preferred embodiment of the present invention. However, in other cases, constitutive silencing of CD52 may be desirable, for example, by CD52 knockout or by GEiGS using a solution based on an miRNA scaffold that has a broad expression pattern in T cells (i.e., expressed in activated as well as resting T cells).For more information regarding CD52 and its silencing, see also Zhao et al., "The Immunological Function of CD52 and Its Targeting in Organ Transplantation," Inflamm. Res. (2017) 66(7):571-8, and Kamali et al., "CRISPR / Cas9-mediated knockout of clinically relevant alloantigenes in human primary T cells," BMC Biotechnology (2021) 21:9. It will be appreciated that other immune cell markers associated with cell depletion can be targeted and silenced to protect cells from other cell depletion approaches.

[0341] In other embodiments, one or more of CD117, CD27, CD45, CD90, CD110, and CD184 can be silenced in cells either constitutively or in a context-specific manner. In some embodiments, B2M is silenced in a context-specific manner and one or more of CD117, CD27, CD45, CD90, CD110, and / or CD184 are silenced in a context-specific manner. In some embodiments, B2M is silenced in a context-specific manner and one or more of CD117, CD27, CD45, CD90, CD110, and / or CD184 are silenced in a context-specific manner in the same context as B2M. In some embodiments, B2M is silenced in a context-specific manner and one or more of CD117, CD27, CD45, CD90, CD110, and / or CD184 are constitutively silenced. CD117, CD27, CD45, CD90, CD110 and / or CD184 represent other targets for immune cell depletion.

[0342] In some approaches, such as allogeneic adoptive T cell therapy, it may be desirable to silence endogenous T cell receptors (TCRs) to reduce graft-versus-host disease (GvHD) other than tumors. Silencing of endogenous TCRs is usually permanent, for example, by knocking out the TRAC gene. Thus, in some embodiments, endogenous TCRs are silenced in cells by knocking out or otherwise silencing the TRAC gene, as appropriate, for example, by GEiGS using an miRNA scaffold-based solution that has a broad expression pattern in T cells. However, silencing of endogenous TCRs can also be context-specific, for example, when the TRAC gene is a target gene according to the present invention. Thus, in some embodiments, the TRAC gene is a target gene for context-specific silencing, for example, for specific silencing in T cells or activated T cells.

[0343] In some embodiments, both CD52 and TCR are silenced in T cells. In some embodiments, CD52, TCR, and B2M are silenced in T cells. In some preferred embodiments, TCR is constitutively silenced, for example, by knocking out the TCR in T cells (e.g., by knocking out the TRAC gene) or via GEiGS using a solution based on an miRNA scaffold that has a broad expression pattern in T cells (i.e., expressed in activated as well as resting T cells). In some embodiments, TCR is knocked out in T cells, and CD52 and B2M are optionally silenced in a context-specific manner, where CD52 and B2M are silenced in activated T cells. In some embodiments, TCR and CD52 are knocked out in T cells, and B2M is optionally silenced in a context-specific manner, where B2M is silenced in activated T cells. Suitable approaches for context-specific silencing of target genes in T cells generally or in activated T cells are discussed herein.

[0344] Methods for modifying cells In some embodiments, the method comprises modifying the cell to provide a nucleic acid construct adapted to express an inhibitory RNA.

[0345] In some embodiments, the methods use gene editing techniques to provide inhibitory RNA molecules designed to target and interfere with an RNA molecule of interest (endogenous or exogenous to the eukaryotic cell, preferably endogenous to the eukaryotic cell).

[0346] The gene editing technology of some embodiments of the present invention involves genome editing of inhibitory RNA molecules (e.g., endogenous), yet is stable and heritable. The inventors use a genome editing-induced gene silencing (GEiGS) platform that can utilize endogenous non-coding RNA molecules of eukaryotic cells, including, for example, RNA silencing molecules (e.g., siRNA, miRNA, piRNA, tasiRNA, tRNA, rRNA, antisense RNA, etc.), and modify them to target any RNA target of interest. Using GEiGS, those skilled in the art can screen potential non-coding RNA molecules and edit several nucleotides in these endogenous RNA molecules, thereby redirecting their activity and / or specificity to effectively and specifically target any RNA of interest, including, for example, mRNA encoding B2M protein.

[0347] Thus, provided is a method for inhibiting translation of an RNA transcript derived from a target protein-coding gene (e.g., B2M) in a cell, comprising introducing into the cell an RNA editing agent that confers silencing specificity of a non-coding RNA molecule for a target RNA of interest, wherein the sequence of the non-coding RNA molecule is altered to target the RNA transcript of the target gene of interest, thereby modifying the non-coding RNA molecule to alter translation of the RNA transcript into a protein molecule.

[0348] Various non-limiting examples of genome editing methods and RNA editing agents used to induce nucleic acid alterations in inhibitory RNA molecules can be used in accordance with certain embodiments of the invention.

[0349] Genome editing using engineered endonucleases—This approach refers to a reverse genetics method that uses engineered nucleases to cut, typically at desired locations in the genome, creating specific double-strand breaks (DSBs), which are then repaired by cell-intrinsic processes such as homologous recombination (HR) or nonhomologous end joining (NHEJ). NHEJ directly joins the DNA ends in the double-strand break (DSB) with or without minimal end trimming, while HR utilizes a homologous donor sequence (i.e., a sister chromatid formed during S phase) as a template to regenerate / copy the missing DNA sequence at the break site. To introduce specific nucleotide modifications into genomic DNA, a donor DNA repair template (exogenously provided single-stranded or double-stranded DNA) containing the desired sequence must be present during HR.

[0350] Genome editing cannot be performed using traditional restriction endonucleases because most restriction enzymes recognize a few base pairs on DNA as their target, and these sequences are often found in numerous locations throughout the genome, resulting in multiple cuts that are not restricted to the desired location. To overcome this challenge and create site-specific single- or double-strand breaks (DSBs), several distinct classes of nucleases have been discovered and bioengineered to date. These include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR / Cas9 systems.

[0351] Meganucleases - Meganucleases are generally classified into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cys box family, and the HNH family. These families are characterized by structural motifs that affect catalytic activity and recognition sequences. For example, members of the LAGLIDADG family are characterized by having one or two copies of the conserved LAGLIDADG motif. The four families of meganucleases are widely divergent from each other with respect to conserved structural elements and, as a result, DNA recognition sequence specificity and catalytic activity. Meganucleases are generally found in microbial species and have the unique property of having extremely long recognition sequences (>14 bp), thus making them naturally highly specific for cleavage at the desired location.

[0352] This can be used to create site-specific double-strand breaks (DSBs) in genome editing. Those skilled in the art can use these naturally occurring meganucleases, but the number of such naturally occurring meganucleases is limited. To overcome this problem, mutagenesis and high-throughput screening methods have been used to create meganuclease variants that recognize unique sequences. For example, various meganucleases have been fused to create hybrid enzymes that recognize novel sequences.

[0353] Alternatively, the DNA-interacting amino acids of the meganuclease can be altered to design sequence-specific meganucleases. Alternatively, commercially available technologies, such as Precision Biosciences' Directed Nuclease Editor™ genome editing technology, can be used to obtain meganucleases with site-specific cleavage characteristics.

[0354] ZFNs and TALENs - Two distinct classes of engineered nucleases, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have both been shown to be effective in generating targeted double-strand breaks (DSBs).

[0355] ZFN and TALEN restriction endonuclease technologies utilize a nonspecific DNA-cleaving enzyme linked to a specific DNA-binding domain (either a series of zinc finger domains or TALE repeats, respectively). Restriction enzymes are typically selected whose DNA recognition and cleavage sites are distant from each other. The cleavage moiety is then separated and linked to a DNA-binding domain, thereby generating an endonuclease with extremely high specificity for the desired sequence. An exemplary restriction enzyme with such properties is Fokl. Furthermore, Fokl has the advantage of requiring dimerization for nuclease activity, which means that specificity is greatly increased because each nuclease partner recognizes a unique DNA sequence. To enhance this effect, Fokl nucleases that can function only as heterodimers and have increased catalytic activity have been engineered. Nucleases that function as heterodimers avoid the potential for unwanted homodimer activity and therefore increase the specificity of double-strand breaks (DSBs).

[0356] Therefore, for example, to target a specific site, ZFN and TALEN are constructed as a nuclease pair, and each member of the pair is designed to bind to the adjacent sequence of the targeted site.When transiently expressed in cells, the nuclease binds to its target site, and the Fokl domain heterodimerizes to create double-strand breaks (DSBs).The repair of these double-strand breaks (DSBs) through the non-homologous end joining (NHEJ) pathway often results in small deletions or small sequence insertions (indels).Because each repair performed by NHEJ is unique, a single nuclease pair can be used to generate a series of alleles with a variety of different insertions or deletions at the target site.

[0357] Generally, NHEJ in gene editing is relatively accurate (approximately 75-85% of DSBs in human cells are repaired by NHEJ within approximately 30 minutes of detection). However, even if repair is accurate, erroneous NHEJ is relied upon because the repair product is mutagenic, and the recognition / cleavage site / PAM motif is lost / mutated, or the nuclease continues to cleave until the transiently introduced nuclease is no longer present. Deletions typically range anywhere from a few base pairs to hundreds of base pairs in length, but larger deletions have been successfully generated in cell culture by simultaneously using two pairs of nucleases. Furthermore, if a fragment of DNA with homology to the targeted region is introduced along with the nuclease pair, the double-strand break (DSB) can be repaired by homologous recombination (HR) (e.g., in the presence of a donor template) to generate a specific modification.

[0358] Although the nuclease moieties of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases lies in their DNA recognition peptides. ZFNs rely on Cys2-His2 zinc fingers, while TALENs rely on TALEs. Both of these DNA recognition peptide domains have the characteristic of naturally occurring in combination in proteins. Cys2-His2 zinc fingers are usually found in repeats spaced 3 bp apart and are found in various combinations in various nucleic acid-interacting proteins. On the other hand, TALEs are found in repeats with a one-to-one recognition ratio between amino acids and recognized nucleotide pairs. Because both zinc fingers and TALEs occur in repetitive patterns, various combinations can be tried to create a wide variety of sequence specificities. Approaches for generating site-specific zinc finger endonucleases include, among others, modular assembly (zinc fingers correlating with triplet sequences are attached in a row to cover the required sequence), OPEN (low stringency selection of peptide domains versus triplet nucleotides in a bacterial system, followed by high stringency selection of peptide combinations versus the final target), and one-hybrid screening of bacterial zinc finger libraries. ZFNs can also be designed and are commercially available, for example, from Sangamo Biosciences™ (Richmond, Calif.).

[0359] The T-GEE system (TargetGene's Genome Editing Engine) provides a programmable nucleoprotein molecular complex that assembles in vivo in a target cell, contains a polypeptide moiety, and a specificity-conferring nucleic acid (SCNA), and is capable of interacting with a predetermined target nucleic acid sequence. The programmable nucleoprotein molecular complex is capable of specifically modifying and / or editing a target site within the target nucleic acid sequence and / or altering the function of the target nucleic acid sequence. The nucleoprotein composition includes (a) a polynucleotide molecule encoding a chimeric polypeptide and including (i) a functional domain capable of modifying the target site and (ii) a linking domain capable of interacting with the specificity-conferring nucleic acid, and (b) a specificity-conferring nucleic acid (SCNA) that includes (i) a nucleotide sequence complementary to a region of the target nucleic acid adjacent to the target site and (ii) a recognition region capable of specifically attaching to the linking domain of the polypeptide. The composition enables precise, reliable, and cost-effective modification of a predetermined nucleic acid sequence target through base pairing between the specificity-conferring nucleic acid and the target nucleic acid, with high specificity and binding capacity of the molecular complex for the target nucleic acid. The composition is less genotoxic, modular in assembly, utilizes a single platform without customization, is practical for standalone use outside of specialized core facilities, and has a short development timeframe and reduced cost.

[0360] CRISPR-Cas systems and all their variants (also referred to herein as "CRISPR") - Many bacteria and archaea contain endogenous RNA-based adaptive immune systems that can degrade the nucleic acids of invading phages and plasmids. These systems consist of clustered regularly interspaced short palindromic repeats (CRISPR) nucleotide sequences that generate RNA components and CRISPR-associated (Cas) genes that encode protein components. CRISPR RNA (crRNA) contains short stretches of homology to specific viral and plasmid DNA and acts as a guide, directing Cas nucleases to degrade complementary nucleic acids in the corresponding pathogens. Studies of the type II CRISPR / Cas system of Streptococcus pyogenes have shown that three components form an RNA / protein complex that together are sufficient for sequence-specific nuclease activity: the Cas9 nuclease, a crRNA containing 20 base pairs of homology to the target sequence, and a trans-activating crRNA (tracrRNA) (Jinek et al. Science (2012) 337: 816-821).

[0361] It was further demonstrated that a synthetic chimeric guide RNA (sgRNA) composed of a fusion between a crRNA and a tracrRNA can direct Cas9 to cleave a DNA target complementary to the crRNA in vitro. It has also been demonstrated that transient expression of Cas9 in conjunction with synthetic sgRNAs can be used to create targeted double-strand breaks (DSBs) in a variety of different species (Cho et al., 2013 (Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol. 2013;31(3):230-232); Cong et al., 2013 (Multiplex genome engineering using CRISPR / Cas systems. Science. 2013;339(6121):819-823); DiCarlo et al., 2013 (Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 2013;41(7):4336-4343); Hwang et al., 2013a, b (Nature Biotechnology 31, 227-229 (2013)); Heritable and Precise zebrafish genome editing using a CRISPR-Cas system. PLoS One. 2013;8(7):e68708 Jinek et al., 2013 (RNA-programmed genome editing in human cells eLife 2:e00471); Mali et al., 2013 (RNA-guided human genome engineering via Cas9. Science. 2013 Feb. 15;339(6121):823-6)).

[0362] The CRISPR / Cas system for genome editing contains two distinct components: an sgRNA and an endonuclease, e.g., Cas9. The sgRNA (also referred to herein as a short guide RNA (sgRNA)) is typically a 20-nucleotide sequence that encodes a combination of a target homologous sequence (crRNA) and an endogenous bacterial RNA (tracrRNA) that links the crRNA to the Cas9 nuclease in a single chimeric transcript. The gRNA / Cas9 complex is recruited to the target sequence through base pairing between the sgRNA sequence and complementary genomic DNA / RNA. For successful binding of Cas9, the genomic target sequence must also contain the correct protospacer adjacent motif (PAM) sequence immediately following the target sequence. Binding of the gRNA / Cas9 complex localizes Cas9 to the genomic target sequence, allowing Cas9 to cleave both strands of DNA and create a double-strand break (DSB). Double-strand breaks (DSBs) generated by CRISPR / Cas can undergo homologous recombination or NHEJ and are susceptible to specific sequence modifications during DNA repair.

[0363] The Cas9 nuclease has two functional domains: RuvC and HNH, each of which cleaves a different DNA strand. When both of these domains are active, Cas9 creates a double-strand break (DSB) in genomic DNA or RNA.

[0364] A significant advantage of CRISPR / Cas is the high efficiency of the system combined with the ability to easily generate synthetic sgRNAs. This creates a system that can be easily modified to target modifications at different genomic sites and / or to target different modifications at the same site. Thus, protocols have been established that allow for the simultaneous targeting of multiple genes. The majority of cells that carry mutations exhibit biallelic mutations in the targeted genes.

[0365] However, the apparent flexibility in the base-pairing interactions between the sgRNA sequence and the genomic DNA target sequence allows imperfect matches to the target sequence to be cleaved by Cas9.

[0366] Engineered versions of the Cas9 enzyme containing a single inactive catalytic domain, either RuvC or HNH, are called "nickases." Cas9 nickases have only one active nuclease domain and cleave only one strand of target DNA, creating single-strand breaks or "nicks." Single-strand breaks or nicks are primarily repaired by single-strand break repair mechanisms involving proteins such as, but not limited to, PARP (sensor) and the XRCC1 / LIG III complex (ligation). Single-strand breaks (SSBs) generated by topoisomerase I toxins or drugs that trap PARP1 at naturally occurring SSBs can persist, and when cells enter S phase and a replication fork encounters such SSBs, they become single-ended DSBs that can only be repaired by HR. However, nicks in two adjacent, opposing strands introduced by Cas9 nickases are treated as double-strand breaks, often referred to as "double-nick" CRISPR systems. Double nicks, which are essentially nonparallel DSBs, can be repaired like other DSBs by HR or NHEJ, depending on the desired effect on the gene target and the presence and cell cycle stage of the donor sequence (HR is of much lower abundance and can occur only in the S and G2 phases of the cell cycle). Therefore, if specificity and reduction of off-target effects are important, using Cas9 nickase to create a double nick by designing two sgRNAs with target sequences on opposite strands of genomic DNA in close proximity would reduce off-target effects, since either sgRNA alone would result in a nick that is unlikely to alter genomic DNA, although these events are not impossible.

[0367] A modified version of the Cas9 enzyme containing two inactive catalytic domains (dead Cas9 or dCas9) lacks nuclease activity but can still bind to DNA based on sgRNA specificity. dCas9 can be used as a platform for DNA transcription regulators to activate or repress gene expression by fusing the inactive enzyme to known regulatory domains. For example, dCas9 alone can be bound to a target sequence in genomic DNA to interfere with gene transcription.

[0368] Additional variants of Cas9 that may be used according to some embodiments of the present invention include, but are not limited to, CasX and Cpfl (also known as Cas12a). CasX enzymes comprise a distinct family of RNA-guided genome editors that are smaller in size than Cas9, are found in bacteria (not typically found in humans), and are therefore less likely to elicit an immune system / response in humans. CasX also utilizes a different PAM motif compared to Cas9 and can therefore be used to target sequences in which the Cas9 PAM motif is not found.

[0369] CRISPR systems can be fused to various effector domains, such as DNA cleavage domains. DNA cleavage domains can be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which DNA cleavage domains can be derived include, but are not limited to, restriction endonucleases and homing endonucleases (see, for example, the New England Biolabs Catalog or Belfort et al. (1997) Nucleic Acids Res.). In an exemplary embodiment, the cleavage domain of the CRISPR system is a Fokl endonuclease domain or a modified Fokl endonuclease domain. Furthermore, the use of homing endonucleases (HEs) is another alternative. HEs are small proteins (<300 amino acids) found in bacteria, archaea, and unicellular eukaryotes. A distinctive feature of HEs is that they recognize relatively long sequences (14-40 bp) compared to other site-specific endonucleases, such as restriction enzymes (4-8 bp). HEs have historically been classified by small, conserved amino acid motifs. At least five such families have been identified: LAGLIDADG, GIY-YIG, HNH, His-Cys Box, and PD-(D / E)xK, which are related to EDxHD enzymes and are considered by some to be distinct families. At the structural level, HNH and His-Cys Box share a common fold (termed bba-metal) with PD-(D / E)xK and EDxHD enzymes. The catalytic and DNA recognition strategies of each family are distinct, lending them to varying degrees of engineering for various applications. Exemplary homing endonucleases that can be used in accordance with some embodiments of the present invention include, but are not limited to, I-Crel, I-Tevl, I-Hmul, I-Ppol, and I-Ssp68031.

[0370] Modified versions of CRISPR, such as dead CRISPR (dCRISPR-endonuclease), can also be used to inhibit CRISPR transcription (CRISPRi) or activate CRISPR transcription (CRISPRa).

[0371] Other versions of CRISPR that can be used in accordance with some embodiments of the present invention include genome editing, which uses components from the CRISPR system together with other enzymes to place point mutations directly into cellular DNA or RNA.

[0372] Therefore, according to one embodiment, the editing substance is a DNA editing substance. Preferably, the DNA editing substance comprises a DNA editing system. More preferably, the DNA editing system comprises a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a homology directed repair (HDR), a CRISPR-endonuclease, a dCRISPR-endonuclease or a homing endonuclease.

[0373] According to one embodiment, the DNA editing agent does not comprise an endonuclease. According to another embodiment, the DNA editing agent comprises an endonuclease. Optionally, in some embodiments, the endonuclease is any one of Cas9, Cas12a, CasX, CasY, CasPhi, MAD7, or Cas13, or a functional variant thereof. According to a preferred embodiment, the endonuclease is Cas9. According to another preferred embodiment, the endonuclease comprises a catalytically inactive endonuclease.

[0374] According to a further embodiment of the present invention, the DNA editing agent is linked to a reporter for monitoring its expression in a cell, e.g., a eukaryotic cell. The reporter may be a fluorescent reporter protein. The term "fluorescent protein" refers to a polypeptide that emits fluorescence and is typically detectable by flow cytometry, microscopy, or any fluorescent imaging system, and can therefore be used as a basis for selecting cells expressing such a protein. Examples of fluorescent proteins that can be used as reporters include, but are not limited to, green fluorescent protein (GFP), blue fluorescent protein (BFP), and red fluorescent protein (e.g., dsRed, mCherry, RFP). A non-limiting list of fluorescent or other reporters includes proteins that can be detected by luminescence (e.g., luciferase) or colorimetric assays (e.g., GUS). According to certain embodiments, the fluorescent reporter is a red fluorescent protein (e.g., dsRed, mCherry, RFP) or GFP.

[0375] cell therapy The present invention is applicable to a wide range of therapeutic cells. In some preferred embodiments, the cells have reduced immunogenicity, for example, by context-specific silencing of the MHC-I system. However, silencing other genes may be relevant in many other contexts to modify the phenotype of the cells (e.g., to promote a pro-inflammatory or anti-inflammatory phenotype).

[0376] Therapeutic cells can be suitably derived from pluripotent stem cell lines, e.g., embryonic stem cell lines or induced pluripotent stem cell lines. Other therapeutic cells are suitably derived from tissue-resident, multipotent stem or progenitor cells of distinct developmental origins, e.g., hematopoietic stem cells (HSCs, hemangioblasts), bone, cartilage, and adipose stem cells (mesenchymal, lateral plate), striated muscle stem cells (skeletal (satellite), cardiac, epicardial), smooth muscle stem cells (vascular pericytes), hemangioblast endothelial stem cells, neural stem cells, glial precursors, Müller glial cells, non-myelinating Schwann cells, hair follicle stem (bulge) cells, liver stem cells, bile duct cells, basal epithelial stem cells in the epidermis (skin), proximal trachea / bronchioles and gastrointestinal (GI) tract, alveolar type II cells in the lung, and kidney mesenchyme.

[0377] These stem / progenitor cells are suitable for differentiation into differentiated cells including, but not limited to, dendritic cells, lymphocytes, bone marrow cells, erythrocytes, platelets, hematopoietic cells, pancreatic islet cells (e.g., beta, alpha cells), liver cells (hepatocytes, cholangiocytes), skeletal muscle cells (multinucleated myotubes), keratinocytes, cardiomyocytes (atrial, ventricular), conducting heart cells, vascular smooth muscle cells, neurons, glial cells, pial choroid cells, ocular (corneal) cells, photoreceptor cells, retinal interneurons, retinal ganglion cells, mesenchymal cells, fibroblasts, lung cells, gastrointestinal cells, vascular cells, endocrine cells, or adipocytes.

[0378] Therapeutic cells are cells suitable for administration to a subject with the intention or expectation of providing some therapeutic benefit to the subject.

[0379] Non-limiting examples of specific cell and tissue therapies of interest in the present invention include replacement (regeneration, restoration) of cornea, retinal cells (retinal ganglion, amacrine, photoreceptor), retinal pigment epithelium, anti-inflammatory cell therapies for acute and chronic CNS injury and degeneration, neuronal replacement such as inhibitory interneurons, white matter degeneration and multiple sclerosis, glial replacement for neuropathic pain, therapeutic endocrine cells to regenerate pancreatic, thyroid or adrenal function, therapeutic liver cells for detoxification, skeletal and cardiac muscle replacement (regeneration, restoration), and immune cells such as macrophages, (CAR) T cells, regulatory T cells, neutrophils, granulocytes, NK cells, NK / T cells, and eosinophils.

[0380] Therapeutic cells of the endocrine system preferably include enteroendocrine cells, preferably therapeutic enteroendocrine cells, such as those of the pancreas, thyroid, parathyroid, pituitary, pineal, adrenal gland, thymus, and hypothalamus.

[0381] Preferred therapeutic cells for the pancreas are endocrine hormone-expressing cells (beta, alpha, delta, epsilon cells, most preferably beta cells).

[0382] Preferred therapeutic macrophage cells are those that are anti-tumor and pro-inflammatory as cancer treatments.

[0383] Other preferred therapeutic macrophage cells are those that are anti-inflammatory, pro-regenerative, pro-angiogenic and pro-tissue remodeling for treating chronic fibrotic diseases such as idiopathic pulmonary fibrosis, chronic kidney disease, non-alcoholic steatohepatitis (NASH), scleroderma, rheumatoid arthritis, ulcerative colitis and myelofibrosis.

[0384] Preferred therapeutic T cells are those that are anti-tumor effector cells for hard-to-treat cancers or memory T cells or alternative regulatory T cells as treatments for autoimmune diseases such as psoriasis, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, and pernicious anemia.

[0385] composition The cells or cell populations of the invention may be formulated for delivery in a composition. The composition of the invention optionally comprises the cells or cell populations of the invention. The composition may be provided in the form of a kit.

[0386] Thus, in one embodiment of the present invention, there is provided a composition comprising a cell or cell population of the present invention. The composition of the present invention may also contain other compounds, which may be biologically active or inactive. Optionally, the composition of the present invention is a sterile composition suitable for parenteral administration.

[0387] Many suitable carriers known to those skilled in the art can be used in the compositions of the present invention, and the optimal type of carrier to be used will vary depending on the mode of administration. The compositions of the present invention can be formulated for any suitable mode of administration, including, for example, parenteral, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration, preferably parenteral, for example, intramuscular, subcutaneous, or intravenous administration. For parenteral administration, the carrier preferably contains water, and may contain buffers for pH control, stabilizers such as surfactants and amino acids, and tonicity modifiers such as salts and sugars. If the composition is intended to be provided in a lyophilized form for dilution at the time of use, the formulation may contain a cryoprotectant, for example, a sugar such as trehalose. For oral administration, any of the above carriers or solid carriers can be used, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate.

[0388] Thus, compositions of the invention may contain buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, or dextran), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with respect to the blood of the recipient, suspending agents, viscosity enhancing agents, and / or preservatives. Alternatively, compositions of the invention may be formulated as a lyophilizate.

[0389] The effective amount of the isolated cells of the present invention for transplantation or for treating a disease state will vary depending on several factors, such as the type of tissue, the severity of the disease state, the transplant response, the reason for transplantation, and the age and general health of the patient. The effective amount can be determined by a skilled researcher or clinician through routine practice. Due to the reduced immunogenicity of the transplanted cells, a relatively large amount of cells can be tolerated by the patient to achieve the desired therapeutic effect. Alternatively, the cells can be transplanted repeatedly at intervals until the desired therapeutic effect is achieved.

[0390] The administration route of the cells of the present invention is not limited to any particular method. Exemplary delivery routes include, but are not limited to, intravenous, intramuscular, subcutaneous, intraperitoneal, transcutaneous, intradermal, and subcutaneous routes. The cells of the present invention can also be administered locally by injection. For example, the cells can be injected into an injured joint, a fractured bone, an infarct site, an ischemic fluid, or its periphery.

[0391] In certain embodiments, cells are administered via a delivery device, including, but not limited to, a syringe. For example, cells can be suspended in a solution or pharmaceutical composition contained in such a delivery device. A "solution" or "pharmaceutical composition" includes a physiologically compatible buffer and, optionally, a pharmaceutically acceptable carrier or diluent in which the cells of the invention remain viable. The use of such carriers and diluents is well known in the art. Solutions include, but are not limited to, physiologically compatible buffers, such as Hank's solution, Ringer's solution, or physiologically buffered saline. Cells can be maintained in the solution or pharmaceutical composition for short-term storage without loss of viability. In certain embodiments, cells are frozen for long-term storage without loss of viability according to cryopreservation methods well known in the art.

[0392] Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran, but remain fluid enough to be easily delivered by syringe injection. The solutions are preferably sterile, stable under the conditions of manufacture and storage, and free of microbial contamination through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. The cells contained in the solution may be stem cells or differentiated cells as described herein, in a pharmaceutically acceptable carrier or diluent and, optionally, other ingredients as set forth above.

[0393] The cells can be administered systemically (e.g., intravenously) or locally (e.g., directly into the myocardial defect under echocardiographic guidance or by direct application to an accessible damaged tissue or organ during open surgery). For injection, the cells can be in an injectable liquid suspension preparation or in a biocompatible medium that is in liquid form and becomes semi-solid at the site of the damaged tissue. A syringe, controllable endoscopic delivery device, or other similar device can be used, as long as the needle lumen is of sufficient diameter (e.g., at least 30 gauge or longer) to avoid physical damage to the cells during delivery.

[0394] In certain other embodiments, cells can be transplanted via a solid support, such as a planar or three-dimensional matrix. The matrix or surface is surgically implanted into an appropriate site in the patient. For example, a patient requiring a pancreas transplant can have differentiated cells on a solid support surgically implanted into the peritoneum or portal vein of the liver. Exemplary solid supports include, but are not limited to, patches, gel matrices (e.g., GELFOAM® manufactured by Pharmacia-Upjohn), polyvinyl alcohol sponge (PVA) collagen gel implants (e.g., IVALON, Unipoint Industries, High Point, NC), and other similar or equivalent devices. Various other encapsulation techniques can be used with the cells of the present invention, e.g., WO91 / 10470, WO91 / 10425, U.S. Pat. No. 5,837,234, U.S. Pat. No. 5,011,472, U.S. Pat. No. 4,892,538).

[0395] Aspects of the present invention are further demonstrated by the following non-limiting examples. [Example]

[0396] introduction Advantages of GEiGS Since its discovery in the 1990s, RNAi has been utilized in various forms to silence desired target genes, primarily using oligos (siRNA) or ectopically expressed hairpins (shRNA). miRNA-based vectors for shRNA have been widely used. These mimic endogenous miRNAs, with the stem sequence replaced with a selected shRNA sequence. Several scaffolds have been used, the most common being miR-30 (Fellmann et al., 2013). miR30-based vectors are more efficient than standard hairpin constructs, are more effective at silencing targets, and have a superior off-target profile; for this reason, they are currently being used in shRNA clinical applications (Esrick et al., 2021).

[0397] In their current form, miR-based shRNAs are usually still implemented by ectopic expression as lentiviral transgenes that are integrated into the genome randomly or within safe-harbor loci, e.g., AAVS1, where they are transcribed from exogenous regulatory elements. GEiGS, on the other hand, uses modifications of endogenous loci that encode silencing molecules and exploits the programming of expression of endogenous loci to redirect the silencing specificity of silencing RNAs encoded in these loci, which offers a very distinct advantage: the ability to induce stable, tunable, programmable, and specific gene silencing.

[0398] 1. Stability. While siRNA oligos are transient in nature, shRNAs induce variable expression with transgenes inactivated by epigenetic mechanisms. In contrast, endogenous gene loci are never unexpectedly silenced, and therefore GEiGS silencing RNAs (sRNAs) are stably expressed.

[0399] 2. Tunability. GEiGS allows for the redirection of miRNA scaffolds with different levels of expression, thus allowing the abundance of GEiGS sRNAs to be tailored according to cellular engineering requirements. This is notoriously difficult to achieve with current RNAi approaches and impossible with CRISPR KO, which is binary in nature.

[0400] 3. Programmability. Importantly, GEiGS can provide programmable gene silencing. By redirecting developmentally regulated and cell state-specific miRNAs, GEiGS silencing can be programmed to be deployed only once therapeutic cells reach a specific stage in differentiation.

[0401] 4. Specificity. Importantly, ensuring that expression levels of the redirected miRNA scaffold, and therefore abundance of GEiGS® sRNA, are maintained at physiological levels in cells means that normal de novo pathways for sRNA production are not oversaturated, with the result that there is a much lower incidence of off-target gene silencing effects. This is in stark contrast to shRNA and siRNA approaches, which are notorious for generating artifactual off-target gene effects.

[0402] The GEiGS technology is deployed via the following workflow (see Figure 4): 1. GEiGS® Solutions Computational Design. 2. Experimental verification of solution silencing activity by ectopic expression. 3. Gene editing of endogenous miRNA scaffolds in desired cell types.

[0403] Experimentally validated GEiGS solutions can be used in therapeutic applications, for example, to silence B2M in primary human cells, edited into the genome as a lentiviral transgene or expressed from an endogenous miRNA locus. Further information on GEiGS can be found in WO2019 / 058253, WO2020 / 183414, and WO2020 / 183419.

[0404] As an alternative to GEiGS, a suitable inhibitory RNA construct can be inserted into a genome, for example, into a locus encoding one or more endogenous miRNAs. Such an approach, which can be adapted to the present invention, is described, for example, in Senis et al. ("TALEN / CRISPR-mediated engineering of a promoterless antiviral RNAi hairpin into an endogenous miRNA locus," Nucleic Acids Research, 2017, Vol. 45, No. 1, e3 doi: 10.1093 / nar / gkw805). Thus, in some embodiments of the present invention, a promoterless sequence encoding an inhibitory RNA can be inserted into an endogenous miRNA locus, preferably without disrupting or inactivating any endogenous miRNAs.

[0405] Conditional hypoimmunogenic therapeutic cells: Of particular interest in the present invention are conditionally hypoimmunogenic therapeutic cells. Cell therapy is a class of advanced medical therapies in which patients are treated by transplantation of cells that may or may not have been previously engineered to enhance their functionality. Cell therapy, such as bone marrow transplantation or blood transfusion, is a well-known example of cell therapy that has existed in medicine for decades. However, more recently, novel cell therapies have been developed through the use of genetic engineering techniques and have been successfully used in many different disease indications, such as diabetes, cancer, and blindness.

[0406] Cell therapy can be autologous, where the therapeutic cells originate from the patient being treated, or allogeneic, where the cells originate from an unrelated donor. The autologous approach has the benefit of maximizing the chances of engraftment after transplantation, but can be expensive to produce and may not be applicable to patients with severe or terminal illnesses who are unable to donate their cells for preparation.

[0407] In contrast, allogeneic cell therapy, in principle, can be more efficiently produced from healthy donors, which could potentially reduce costs, and theoretically could be administered immediately and used to treat acutely ill, recently diagnosed patients. However, allogeneic therapy currently requires invasive immunosuppression to reduce the risk of graft rejection after transplantation. Even then, administration of immunosuppressive doses does not guarantee long-term graft rejection. To become mainstream, allogeneic cell therapy will have to overcome the barrier of graft rejection.

[0408] Graft rejection is primarily driven by the host adaptive immune system in response to mismatched major histocompatibility complex I (MHC-I complex) expressed on the surface of transplanted cells. MHC proteins, also known as human leukocyte antigens, are encoded by a cluster of gene complexes known as the human leukocyte antigen (HLA) system. HLA genes are codominantly expressed and highly polymorphic. The expression of multiple distinct alleles favors the adaptive immune system's defense against cancerous cells or pathogens such as viruses or bacteria. However, it is this high degree of polymorphism that presents a major barrier to allogeneic transplantation, including allogeneic cell therapy.

[0409] As discussed, MHC-I is present on the surface of virtually all human cells and plays a key role in immune surveillance (Figure 1), recognizing self from nonself. MHC-I is a dimer of β2-microglobulin and HLA chains. HLA chains are highly polymorphic; that is, individuals express a wide variety of dimer combinations in the population. As a result, the immune system is trained to selectively tolerate only those MHC-I variants expressed by an individual. In the context of transplantation, unless the donor and host express identical HLA variants (which is highly unlikely unless close relatives are involved), the host immune system will recognize nonself MHC-I variants on the surface of donor cells and mount a response (also known as alloimmunity). MHC-I is therefore the primary source of immunogenicity in allogeneic transplantation.

[0410] Numerous approaches have been attempted to overcome the barriers to graft rejection. Traditionally, graft recipients undergo lifelong immunosuppression, which is not only ineffective but also renders recipients vulnerable to serious diseases such as infectious diseases or cancer. Genetic engineering approaches have also been considered. One such approach is to generate hypoimmunogenic cells by completely knocking out MHC-I expression (see, for example, WO2012 / 145384). While this strategy successfully evades responses from the host's adaptive immunity, it exposes the graft to rejection by cells of the innate immune system through the activity of natural killer (NK) cells. NK cells play an important role in detecting abnormal cells and respond potently to cells lacking MHC-I expression, which can be a hallmark of malignant cells. The complete absence of MHC-I is also referred to as a "loss of self" phenotype; these cells are efficiently detected and lysed by NK cells. At a mechanistic level, this occurs because MHC-I molecules function as inhibitory ligands for NK cells. MHC-I KO cells require additional engineering to stably express tolerizing ligands to avoid killing by innate immune cells (Figure 2).

[0411] The present invention aims to address this issue by generating conditionally hypoimmunogenic cells through context-specific partial silencing of MHC-I. As discussed, the MHC-I complex is a heterodimer composed of hypervariable HLA proteins and beta-2 microglobulin (B2M). By partially knocking down B2M expression, partial silencing of MHC-I can allow transplanted cells to escape rejection driven by both the innate (NK-mediated) and adaptive (T cell-mediated) immune systems.

[0412] One approach to generating conditionally hypoimmunogenic cells is to down-modulate MHC-I expression, for example, using inhibitory RNA via a gene editing-inducible gene silencing (GEiGS) system, to a level that prevents significant adaptive immune responses but is still sufficient to inhibit NK cell-mediated responses.

[0413] The potential for partial MHC-I silencing was demonstrated in an in vivo study in which hypoimmunogenic islet cells were generated by siRNA-mediated MHC-I knockdown and subsequently transplanted into NOD mice, demonstrating improved transplant survival (Wang et al., 2012, Diabetes. December 2012;61(12):3247-54). While this study demonstrates the efficiency of RNAi-mediated gene silencing to partially silence MHC-I, the transient nature of siRNA means that the approach may not be feasible for clinical application. On the other hand, GEiGS, for example, has the potential to provide stable, tunable silencing and would be perfectly suited for this approach.

[0414] However, a risk of hypoimmunogenic therapeutic cells is that they are well adapted to evade the immune system and will not be targeted by the immune system, e.g., if they migrate from their desired location and / or dedifferentiate. This is of particular concern if the therapeutic cells have the potential to dedifferentiate, e.g., revert to an earlier stage of differentiation where they are more likely to proliferate and / or migrate from their intended site.

[0415] Thus, low immunogenicity would be advantageous provided that the cells are in their desired state, i.e., at their desired level of differentiation and / or in their intended location.

[0416] Thus, in some embodiments, the cells are conditionally hypoimmunogenic cells, typically hypoimmunogenic therapeutic cells.

[0417] In some embodiments, inhibitory RNAs targeting B2M (eg, comprising a sequence according to any one of SEQ ID NOs: 1-17) are selectively active in therapeutic cells in desired situations.

[0418] For example, an inhibitory RNA targeting B2M is expressed in a cell in a desired context at a level of at least 1000 RPM, at least 2000 RPM (reads per million mapped reads) in a cell, at least 3000 RPM in a cell, at least 4000 RPM in a cell, at least 5000 RPM in a cell, at least 10000 RPM in a cell, at least 20000 RPM in a cell, or at least 50000 RPM in a cell, or at least 100000 RPM in a cell. Preferably, the inhibitory RNA is expressed in a cell in a desired context at a level of at least 50000 RPM or at least 100000 RPM.

[0419] In pancreatic tissue, for example, hsa-miR-375, hsa-miR-143, and hsa-miR-21 are expressed at particularly high levels (189993.6, 186579.2, and 65231.3 RPM, respectively, according to the miRmine database), and various other miRNAs are also expressed at significant, but more moderate, levels (e.g., hsa-miR-30d, hsa-miR-192, hsa-miR-30a, hsa-miR-182, hsa-miR-148a, hsa-miR-127, hsa-miR-200c, etc.).

[0420] In some embodiments, the inhibitory RNA is hsa-miR-375, hsa-miR-143, or hsa-miR-21 modified to target B2M. In some embodiments, the inhibitory RNA is hsa-miR-30d, hsa-miR-192, hsa-miR-30a, hsa-miR-182, hsa-miR-148a, hsa-miR-127, or hsa-miR-200c modified to target B2M.

[0421] In one exemplary embodiment of the invention, the target gene is B2M. In some embodiments of the invention, expression of B2M in cells in the first situation is altered in a range of 1% to 90%, optionally 5% to 90%, optionally 10% to 90%, compared to control cells.

[0422] Expression of B2M can be altered, degraded, reduced, or silenced in accordance with the present invention. In one embodiment of any aspect of the present invention, expression of B2M is altered in a range of 1% to 90%, optionally 5% to 90%, and optionally 10% to 90%. Optionally, for example, protein expression is altered by 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 1% to 10%, 1% to 5%, 5% to 80%, 5% to 70%, 5% to 60%, 5% to 50%, 5% to 40%, 5% to 30%, 5% to 20%, 5% to 10%, or 10% to 80% compared to control cells. , 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, or 80% to 90%. It is an advantage of the present invention that one skilled in the art can modulate, typically reduce, protein expression by a desired amount, e.g., to a suitable level, compared to control cells. In some embodiments, the expression level of B2M or any other cell surface protein in a cell is determined by the amount of B2M or other protein expressed on the surface of the cell. The amount of B2M present on the surface of a cell can be determined by various conventional techniques, e.g., flow cytometry.

[0423] In some embodiments of the invention, the inhibitory RNA is inactive or less active when the pancreatic endocrine hormone-expressing cells are altered as described, such that they are no longer able to silence expression of B2M or HLA-A / B / C and become immunogenic.

[0424] Context-specific gene silencing in immune cells: As discussed above, in some embodiments of the invention, the inhibitory RNA is specifically active in immune cells, e.g., in particular types of immune cells or in immune cells in a particular state of activation or polarization.

[0425] As an example, the solid tumor microenvironment (TME) poses particular challenges for developers of CAR-T and other engineered immune effector cell therapies to overcome. Solid tumors are composed of, in addition to the cancer cells themselves, stroma (cancer-associated fibroblasts (CAFs)) and a variety of other cells, such as immune cells, e.g., macrophages, T cells (helper, cytotoxic, regulatory), etc. Solid tumors create a microenvironment that allows them to recruit, regulate, and restrain immune effector cells in maintaining immune tolerance and promoting tumor growth. Evidence indicates that the TME can disrupt the activity of tumor-infiltrating T cells, even CAR-T cells engineered to be highly active against tumor-expressed specific antigens (Sterner RC, Sterner RM. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 2021 Apr 6;11(4):69. doi: 10.1038 / s41408-021-00459-7. PMID: 33824268; PMCID: PMC8024391). Tumor cells use cytokines, immune checkpoint signals, and metabolic intermediates as signals to maintain resistance.Metabolic intermediates are potent signals for cell differentiation in immune cells; for example, lactate produced by tumor cells (Colegio OR, Chu NQ, Szabo AL, Chu T, Rhebergen AM, Jairam V, Cyrus N, Brokowski CE, Eisenbarth SC, Phillips GM, Cline GW, Phillips AJ, Medzhitov R. Functional polarization of tumor-associated macrophages by tumor-derived lactic acid. Nature. 2014 Sep 25;513(7519):559-63. doi: 10.1038 / nature13490. Epub 2014 Jul 13. PMID: 25043024; PMCID: PMC4301845) have been demonstrated to polarize macrophages into an immunosuppressive state, i.e., tumor-associated macrophages (TAMs), through a HIF1α-dependent mechanism, which may play an important role in tumor growth. CAFs and extracellular matrix generated in the TME of aggressive cancers also contribute to the generation of an immunomodulatory phenotype that supports tumor growth (Puttock et al. 2022. bioRxiv. doi.org / 10.1101 / 2022.08.11.503568). Extracellular matrix (ECM) remodeling is associated with invasive cancer, the establishment of an immunosuppressive environment, and poor clinical responses to immunotherapy (Chakravarthy et al. 2018. Nature Comms. 9, 4692, doi:10.1038 / s41467-018-06654-8). These ECM compositions positively correlate with TAM infiltration.

[0426] TAMs are a key cell type in the TME, and solid tumors have a high abundance of them. This selective activation (M2-like) state correlates with poor prognosis in several malignancies, including ovarian cancer (Zhang et al. 2014. J Ovarian Res 7, 19, doi:10.1186 / 1757-2215-7-19). TAMs are widely considered to be a key cell type in establishing and maintaining an immune-tolerant TME. They are currently the target of several anti-cancer therapies (PI3K-gamma inhibitor Eganelisib, Infinity Pharm. https: / / www.infi.com / home / our-development-program / ipi-549 / ). TAMs are important in creating an immunosuppressive TME, and they do so through the production of cytokines, chemokines, and growth factors, and by inducing inhibitory immune checkpoint proteins in T cells. There are multiple pathways in the TME that contribute to macrophage polarization into TAMs.

[0427] Therapeutic CAR-T cells are subject to inhibition in the TME via similar pathways: TILs and CAR-Ts are tolerized by the activity of immune checkpoint pathways, such as PD-1 (PDCD-1), CTLA4, LAG3, and TIM3.

[0428] Different states of immune cells are associated with different metabolic programs due to their distinct energy demands, and altering cellular metabolism can influence immune cell fate (Chang CH, Curtis JD, Maggi LB Jr, Faubert B, Villarino AV, O'Sullivan D, Huang SC, van der Windt GJ, Blagih J, Qiu J, Weber JD, Pearce EJ, Jones RG, Pearce EL. Cell. 2013 Jun 6;153(6):1239-51. doi: 10.1016 / j.Cell.2013.05.016.). Controlling cellular metabolism through mitochondrial structure has been shown to influence cell fate (Buck MD, O'Sullivan D, Klein Geltink RI, Curtis JD, Chang CH, Sanin DE, Qiu J, Kretz O, Braas D, van der Windt GJ, Chen Q, Huang SC, O'Neill CM, Edelson BT, Pearce EJ, Sesaki H, Huber TB, Rambold AS, Pearce EL. Cell. 2016 Jun 30;166(1):63-76. doi: 10.1016 / j.Cell.2016.05.035. Epub 2016 Jun 9).

[0429] By promoting mitochondrial fusion, oxidative phosphorylation and fatty acid oxidation are increased in activated antitumor T cells, favoring the formation of memory T cells, which improves the durability of antitumor activity. Inhibition of the DRP1 gene favors mitochondrial fusion (blocking its fission), leading to increased oxidative phosphorylation and fatty acid oxidation.

[0430] Another metabolic target that regulates T cell fate is the mammalian target of rapamycin (mTORC1). Pharmacological inhibition of mTORC1 increased fatty acid oxidation and memory T cell formation, but complete knockout of this gene is undesirable because it reduces both memory and effector T cell formation.

[0431] Antagonism of a single pathway or combinatorial inhibition of disease pathways in solid tumors is unlikely to prevent TAM formation. Macrophage-specific deletion of Dicer completely blocks the process, macrophage polarization into TAMs varies in response to miRNAs (Baer C, Squadrito ML, Laoui D, Thompson D, Hansen SK, Kiialainen A, Hoves S, Ries CH, Ooi CH, De Palma M. Suppression of microRNA activity amplifies IFN-γ-induced macrophage activation and promotes anti-tumor immunity. Nat Cell Biol. 2016 July;18(7):790-802 doi: 10.1038 / ncb3371), and T cell activation induces the expression of specific miRNAs. Inducible and conditional target silencing in GEiGS-engineered cells enables combinatorial cellular immunotherapy.

[0432] Engineered (e.g., GEiGS-modified) macrophages or T / CAR-T cells can take advantage of changes in miRNA expression induced in response to the TME. TME-responsive (e.g., GEiGS-modified) macrophages expressing appropriate miRNAs can promote a pro-inflammatory program within the TME, promoting the activation of tumor-infiltrating lymphocytes (TILs) and other T cells (CAR-Ts). Upregulated miRNA loci normally induced in or by the TME can be redirected to silence targets, thereby preventing or reversing the TAM state and promoting a pro-inflammatory phenotype. Maintenance of the pro-inflammatory polarization state is conditional on exposure to TME cues. Once cells escape the solid TME, miRNAs driving the pro-inflammatory phenotype will be downregulated, avoiding systemic, immune-related extratumoral toxicity (e.g., cytokine release syndrome). Other approaches use constitutively induced pro-inflammatory macrophage polarization but lack the controls to prevent incidental extratumoral effects.

[0433] It would also be therapeutically beneficial to generate T / CAR-T cells that are similarly TME-responsive. Engineered (e.g., GEiGS-engineered) T cells detect the presence of TME signals and block the activity of these immune checkpoint pathways, for example, by silencing PD-1 (PDCD-1), CTLA4, LAG3, or TIM3. Other approaches rely on constitutive silencing of these checkpoints, which can lead to severe immune-related adverse effects (e.g., cytokine release syndrome) in patients. Furthermore, conditional (e.g., GEiGS-mediated) blockade of mitochondrial fission in the TME by redirecting induced miRNAs to conditionally silence DRP1 favors the differentiation and persistence of memory T cells, allowing mitochondrial dynamics to continue and contribute to mitochondrial homeostasis (e.g., mitophagy).

[0434] Such cellular modifications (e.g., GEiGS modifications) described herein will allow for specific silencing of these pathways only once immune cells (e.g., T cells or macrophages) reach the tumor.

[0435] These are just some of the potential applications of the invention described herein, and one skilled in the art may apply this approach in many other cell types and situations.

[0436] Example 1 Computational Design of GEiGS Solutions A B2M solution was generated using a computational pipeline. The pipeline inputs for GEiGS targeting B2M were the B2M mRNA sequence (GenBank accession: NM_004048.4), the human genome reference sequence (GRCh38), miRNA annotation (miRbase v22), and cell type-specific miRNA quantification.

[0437] miRNA expression in human iPSCs and ESCs was quantified using publicly available next-generation sequencing (NGS) datasets: ENCODE accession ENCSR958UOC, ENCSR430YFL.

[0438] A computational pipeline run generated a shortlist of 50 GEiGS® solutions predicted to silence B2M. GEiGS® solution designs consisted of modified sequences of endogenous pre-miRNA (hairpin), also known as endogenous miRNA scaffolds, that encoded novel silencing RNAi triggers in place of their native guide strands. The novel GEiGS® silencing RNAs were predicted to match B2M mRNA by sequence complementarity and lead to silencing of B2M through the RNA interference pathway.

[0439] The solution design process also generated a corresponding negative control construct, also referred to as a "dummy" solution, in which the same endogenous miRNA scaffold as in the corresponding GEiGS® solution was modified to express a scrambled sequence of the GEiGS® silencing RNA.

[0440] Example 2 Validation of GEiGS solution silencing activity by plasmid-based ectopic expression Nineteen designs (17 B2M GEiGS solutions and two "dummy" controls) were used to generate extended GEiGS solutions. The extended solutions consisted of the GEiGS solutions as designed using the computational pipeline and 50–150 bases of genomic sequence flanking each endogenous pre-miRNA on which each GEiGS solution was based. This was included to avoid omitting important sequences that act as signals for the enzyme Drosha.

[0441] The solutions tested were:

[0442] [Table 4A]

[0443] [Table 4B]

[0444] The sequence of the expanded solution was as follows [SEQ ID NO]: >hsa-mir-302a_11_B2M-exon2-2

[0445] [ka]

[0446] >hsa-mir-302a_35_B2M-exon4

[0447]

Chem.

[0448] >hsa-mir-302c_12_B2M-exon2-2

[0449]

Chem.

[0450] >hsa-mir-302c_36_B2M-exon4

[0451]

Chem.

[0452] >hsa-mir-20a_29_B2M-exon4

[0453]

Chem.

[0454] >hsa-mir-200c_27_B2M-exon4

[0455]

Chem.

[0456] >hsa-mir-21_30_B2M-exon4

[0457]

Chem.

[0458] >hsa-mir-363_41_B2M-exon4

[0459]

change

[0460] hsa-mir-93_19_B2M-exon2-2

[0461]

change

[0462] hsa-mir-93_49_B2M-exon4

[0463]

change

[0464] hsa-mir-106a_20_B2M-exon4

[0465]

change

[0466] hsa-mir-106b_21_B2M-exon4

[0467]

change

[0468] hsa-mir-191_8_B2M-exon2-2

[0469]

change

[0470] hsa-mir-191_26_B2M-exon4

[0471]

change

[0472] >hsa-mir-744_47_B2M-exon4

[0473] [ka]

[0474] >hsa-mir-518b_43_B2M-exon4

[0475] [ka]

[0476] >hsa-mir-99b_50_B2M-exon4

[0477] [ka]

[0478] >hsa-mir-302a_23_None (negative control)

[0479] [ka]

[0480] >hsa-mir-200c_13_None (negative control)

[0481] [ka]

[0482] In addition to the negative control constructs listed in Table 4 above, additional miR30-based constructs were purchased from Vector Builder (Neg Ctrl 0).

[0483] The extended GEiGS solution was synthesized and cloned into a custom-made expression plasmid (plasmid VB210602-1567ytv, see Figure 5). The plasmid is a mammalian expression system in which the EF1 alpha (EF1a) promoter drives transcription of the extended GEiGS solution. On the same plasmid, DsRed Express 2 (DsRed) cDNA is expressed from the human PGK (hPGK) promoter. Both the GEiGS solution and DsRed cDNA are transcribed when transfected into mammalian / human cells. DsRed can be used as a transfection marker to indicate successfully transfected cells. The level of DsRed expression (fluorescence intensity) in cells can be assayed by flow cytometry and used as a surrogate for the expression level / abundance of the GEiGS solution.

[0484] The cloned solutions were individually transfected into human iPSC line WTC-11 (Allen Institute of Cell Science) by nucleofection (Lonza). Expression of GEiGS sRNA in transfected cells from the vector was measured by qPCR and compared with that of the corresponding endogenous miRNA scaffold. This showed that GEiGS sRNA was expressed at levels comparable to those of the endogenous miRNA scaffold (Figure 6), indicating that expression from the plasmid is not supraphysiological and therefore unlikely to cause significant off-target effects or interfere with the normal function of the RNAi pathway in cells.

[0485] To evaluate the effectiveness of the solution in silencing B2M, transfected cells were harvested 72 hours after transfection, stained for surface B2M expression using an APC-conjugated antibody, and analyzed by flow cytometry. If the GEiGS solution design is effective, transcription of the extended GEiGS solution should lead to its successful sequential post-transcriptional processing by Drosha and Dicer, expression of the mature GEiGS silencing RNA, integration into RISC, and ultimately B2M mRNA degradation. We evaluated the efficiency of post-transcriptional processing of the extended GEiGS solution, the efficiency of integration into RISC, and the potential of the GEiGS sRNA to trigger B2M mRNA degradation.

[0486] By using flow cytometry to simultaneously measure the expression of DsRed (the transfection and solution-expressed reporter) and B2M (the GEiGS solution target), we were able to generate a dose-dependent curve showing the residual B2M expression in cells versus the amount present in solution (Figure 7). As expected, for the negative control construct, the level of B2M did not change, regardless of the level of GEiGS solution expression. For the effective solution, B2M levels decreased in correlation with the level of solution expression in cells. In other words, cells in which transfection led to higher levels of DsRed / solution expression showed reduced levels of B2M.

[0487] The efficiency of the GEiGS solution was quantified through flow cytometry gating, as shown in Figure 7. Debris was gated out using the FSC-A and SSC-A flow cytometry channels, and then doublets were gated out using the FSC-A and FSC-H channels. The filtered data was then plotted as B2M vs. DsRed. Quantification was performed in two different ways:

[0488] Gate on all cells expressing DsRed (when compared to untransfected controls) and then extract the mean B2M fluorescence intensity in this subpopulation.

[0489] A gate was placed that was more selective for only cells expressing high levels (top 20%) of DsRed, and then the mean fluorescence intensity of B2M was calculated in this subpopulation.

[0490] Both approaches were consistent, allowing for comparison of the efficacy of GEiGS solutions targeting different B2Ms. A wide range of B2M silencing was achieved across the 17 solutions tested experimentally, ranging from 10% to 90% (Figure 8). The most effective solutions demonstrated potent silencing activity. When expressed at moderate levels, two of the 17 solutions tested knocked down endogenous B2M by >80%, and four of the 17 knocked down endogenous B2M by >65%. When expressed at high levels, three of the 17 solutions tested knocked down endogenous B2M by >80%, and seven of the 17 solutions knocked down endogenous B2M by >65%. Solution activity was reproducible across experimental replicates (mean CV = 14%, median CV = 11%), demonstrating the robustness of this assay.

[0491] Thus, using this set of solutions, we were able to achieve distinct levels of MHC-I silencing across a broad range of MHC-I expression in engineered cells, allowing us to identify MHC-I levels that can evade both adaptive and innate immune responses after transplantation in the host.

[0492] Example 3 Gene editing of endogenous miRNA scaffolds in desired cell types Solutions 8, 20, 29, and 30 (see Table 2) were implemented as full GEiGS, i.e., by editing the endogenous locus of the respective miRNA scaffold. Gene editing was performed by CRISPR knock-in using the following gRNA and HDR template sequences:

[0493] [Table 5A]

[0494] [Table 5B]

[0495] The gRNA was obtained as synthetic sgRNA from Integrated DNA Technologies (IDT). Cas9 protein was also obtained from IDT. The sgRNA / Cas9 ribonucleoprotein (RNP) was transfected into iPSCs using nucleofection. Five days after transfection, CRISPR pools were cloned into 96-well plates using the F.SIGHT platform (Cytena), and single-cell deposition was confirmed using the NYONE imaging system (Synentec). Cell colonies were expanded for 7–10 days and then genotyped by PCR, Sanger sequencing, and NGS amplicon sequencing. Confirmed GEiGS lines were analyzed by flow cytometry to assess the level of B2M expression.

[0496] For Solution 29, both heterozygous (WT / KI) and homozygous (KI / KI) clones were isolated, expressing sRNA monoallelically and biallelically, respectively. Because MIR106A is X-linked (hemizygous), GEiGS clones isolated for Solution 20 expressed sRNA only monoallelically. For Solution 30, using Cas12a (Cpf1) protein (also from IDT) instead of Cas9 resulted in more efficient generation of homozygous (KI / KI) clones.

[0497] The GEiGS strains showed uniform silencing of B2M, with very similar variation in B2M expression between the control parent strain (WT / WT) and the GEiGS strains (Figure 9). Quantification of B2M silencing in multiple clones showed consistent B2M silencing across different clones of the same genotype, reflecting the reproducible silencing activity of GEiGS (Figure 10). On average, heterozygous WT / KI clones in solution 29 showed silencing of up to 89% ± SD 2% (n = 4) of B2M, while homozygous KI / KI clones in the same solution showed silencing of up to 96% ± SD 0.5% (n = 2) (Figure 10). For solution 20, hemizygous KI clones showed B2M silencing of up to 65% ± SD 1.2% (n = 4) (Figures 9 and 10), and for solution 30, homozygous KI clones showed approximately 31% level of B2M silencing (n = 1).

[0498] Example 4 Gene editing of endogenous miRNA scaffolds in primary human T lymphocytes Solution 30 (see Example 2 and Tables 4 and 5) was implemented as a full GEiGS, i.e., by editing the endogenous locus of each miRNA scaffold. Several stimulation-inducible miRNAs could be detected in primary T cells (see Amarel et al. 2017 - EMBO J. 2017 Feb 1;36(3):346-360. doi: 10.15252 / embj.201694335).

[0499] Gene editing was performed by CRISPR knock-in using the methodology described in the paper by Roth et al. 2018 (Nature. 2018 Jul;559(7714):405-409. doi: 10.1038 / s41586-018-0326-5).

[0500] The method used in Roth et al. (2018) is recommended for use preferably with fresh cells, bulk T cells or subpopulations sorted by fluorescence-activated cell sorting (FACS), and cells from whole blood or leukapheresis, but can also be used with cells recovered from cryopreservation.

[0501] [Table 6]

[0502] Sol-30 gRNA was obtained as synthetic sgRNA from Integrated DNA Technologies (IDT). Cas9 protein was obtained from IDT. Immediately after isolation from donor peripheral blood mononuclear cells (PBMCs), T cells were incubated with anti-human CD3 / CD28 magnetic dynabeads (ThermoFisher) at a bead-to-cell concentration of 1:1 at 200 U ml -1 IL-2, 5 ng ml -1 of IL-7 and 5ng ml -1The cells were stimulated for 2 days with a cytokine cocktail of IL-15. The beads were collected and then electroporated according to standard methods (see, e.g., Roth et al.). The sgRNA / Cas9 ribonucleoprotein (RNP) was transfected into primary human T cells using electroporation. After electroporation, T cells were stimulated with 500 U ml -1 T cells were cultured in medium containing 100 μg of IL-2. T cells were maintained at a density of approximately 1 million cells per ml of medium throughout the culture period. Three days after electroporation, cells were isolated for analysis of surface B2M expression using flow cytometry, and gDNA and RNA were extracted for PCR, Sanger sequencing, and NGS amplicon sequencing to assess gene editing efficiency.

[0503] Results show a reduction in surface B2M expression (Figure 4). Within the CD3-positive, mixed T cell population, approximately 24% of cells exhibited silencing of B2M (Figure 12a), with an 80% reduction in mean fluorescence intensity (n=1) compared to control unedited cells. Analysis confirms that both CD4- and CD8-edited T cells exhibited a similar reduction in B2M surface expression (Figure 12b).

[0504] Example 5 Demonstration of evasion from both T cell- and NK cell-mediated immune responses in culture for a clonal GEiGS engineered cell line To determine whether partial silencing of B2M allows cells to escape both adaptive and innate immune cell responses, published methods have been used to establish the reactivity of donor-derived cytotoxic T lymphocytes (CTL) or NK (natural killer or LAK, lymphokine-activated killer) cells in cell cultures against GEiGS-modified cell lines (Haga K et al. 2006, Hacke K et al. 2009, Han X et al. 2019). Donor CTL and NK cells are conveniently isolated from commercial suppliers of peripheral blood mononuclear cells (PBMCs).

[0505] T lymphocytes are first preactivated in tissue culture to render them alloreactive. This is accomplished via stimulation with cells, cell extracts, or stimulated cells presenting HLA-antigens that are recognized as foreign (because they are mismatched) by the donor T lymphocytes. Once activated, the donor cells are then mixed in culture with a GEiGS-modified cell line at a defined ratio (usually 10 donor cells:1 GEiGS-modified cell), although other ratios have also been tested. After a predetermined period (48 hours) in mixed cell culture, effector donor T lymphocyte activation by the GEiGS-modified cell line is analyzed by measuring proliferation or inflammatory cytokine secretion (measured by ELISA). Alternatively, the degree of T lymphocyte-mediated cytotoxicity of the GEiGS-modified cell line is assessed by quantifying the death of the GEiGS-modified cell line by measuring the release of the cytosolic enzyme lactate dehydrogenase into the culture medium, the release of pre-loaded calcein AM, and / or staining with cell death markers (e.g., propidium iodide), and / or visualization of GEiGS-modified cells pre-loaded with a fluorescent marker (e.g., CFSC) over time. Neither undifferentiated iPSC lines (knock-in for Solution 20 or 29) nor iPSC lines differentiated into pancreatic progenitor cells (knock-in for Solution 30) quantitatively express B2M at the same levels as the parental iPSC line.

[0506] NK cells are potent, non-HLA-restricted effectors that recognize and kill cells lacking MHC-I surface expression ("self-defective"), such as malignant cells. In related coculture assays, NK cells from the same PBMC donor are mixed in culture with GEiGS-modified cell lines at a defined ratio (typically 5:1 or 1:1). After a defined period (4-48 hours) in mixed cell culture, the degree of NK cell-mediated GEiGS-modified cell cytotoxicity is assessed by quantifying the killing of the GEiGS-modified cell line (as described above). Neither undifferentiated iPSC lines (knock-in for Solution 20 or 29) nor iPSC lines differentiated into pancreatic progenitors (knock-in for Solution 30) quantitatively express B2M at the same levels as the parental iPSC lines.

[0507] Thus, we demonstrate that efficient, dose-dependent reduction of HLA surface expression on human cells confers enhanced resistance to alloreactive T lymphocyte-mediated cytotoxicity while avoiding non-MHC-restricted killing.

[0508] Example 6 Selection of a GEiGS solution for partial silencing of B2M using miRNA scaffolds expressed in a context-specific manner for validation by plasmid-based ectopic expression and GEiGS

[0509] [Table 7]

[0510] Several expansion designs tested were based on endogenous pre-miRNAs that were more highly expressed in undifferentiated iPSCs than in their differentiated progeny. These solutions are listed as follows:

[0511] [Table 8]

[0512] >hsa-mir-191_8_B2M-exon2-2

[0513] [ka]

[0514] >hsa-mir-302c_12_B2M-exon2-2

[0515] [ka]

[0516] >hsa-mir-106a_20_B2M-exon4

[0517] [ka]

[0518] >hsa-mir-20a_29_B2M-exon4

[0519] [ka]

[0520] >hsa-mir-21_30_B2M-exon4

[0521] [ka]

[0522] >hsa-mir-518b_43_B2M-exon4

[0523] [ka]

[0524] Example 7 Demonstration of B2M silencing in pancreatic progenitors identified in culture from clonal GEiGS-modified iPSCs Solution 30 (see Table 7) was implemented as a full GEiGS to demonstrate that B2M silencing can be programmed to be deployed only once therapeutic cells reach a specific stage of differentiation, as in the case of iPSC-derived pancreatic cells. Because this miRNA scaffold is known to be developmentally regulated and expressed in a cell-state-specific manner in pancreatic cells (Jin W et al. 2019, see also Figure 11), the endogenous locus of each miRNA (hsa-mir-21) is modified (as described in Example 3).

[0525] Pancreatic progenitor cells were generated from a control parental line (WT / WT) using a commercially available set of reagents and a validated methodology (Stem Cell Technology; STEMDiff™ Pancreatic Progenitor Kit) that reproducibly generates progenitor cells through a four-step process: definitive endoderm, primitive gut tube, posterior foregut endoderm, and pancreatic progenitor cells. After 14 days in continuous culture, specific progenitor cells generated were isolated and assessed by flow cytometry for staining levels for pancreatic progenitor markers (PDX-1, NKX6.1, co-stained for B2M). Results show that a significant proportion of specific progenitor cells are positive for both pancreatic cell markers, and the majority of PDX-1-positive cells also stain for B2M.

[0526] For solution 30, both heterozygous (WT / KI) and homozygous (KI / KI) clones were isolated, thus expressing sRNA monoallelically and biallelically, respectively. GEiGS clones were evaluated for uniform, quantitative, and reproducible levels of B2M silencing at designated pancreatic progenitor stages when cells were differentiated from iPSCs using current methods, and B2M expression was compared to the control parental line (WT / WT). The level of B2M silencing was contingent on the progenitor cells remaining in a differentiated state; this was lost if the progenitor cells dedifferentiated, proliferated, or degenerated.

[0527] Example 8 Demonstration of B2M silencing in monocytes identified in culture from clonal GEiGS-modified iPSCs Solution 30, implemented as a full GEiGS (see Table 7), demonstrates that B2M silencing can be programmed to be deployed in other therapeutic cell types, including iPSC-derived monocytes. As in Example 3, the endogenous locus of each miRNA (hsa-mir-21) is modified. Homozygous (KI / KI) clones of Solution 30 were isolated, thus expressing the sRNA biallelically. GEiGS Solution 30 clones were evaluated for uniform, quantitative, and reproducible levels of B2M silencing upon differentiation of cells from iPSCs to monocytes. The has-mir-21 scaffold is developmentally regulated in monocytes and expressed in a cell state-specific manner (Sheedy FJ. Turning 21: Induction of miR-21 as a Key Switch in the Inflammatory Response. Front Immunol. 2015 Jan 29;6:19. doi: 10.3389 / fimmu.2015.00019. PMID: 25688245; PMCID: PMC4310327).

[0528] There are numerous published protocols for the generation of monocytes and macrophages from human pluripotent stem cells (e.g., Happle, C., Lachmann, N., Ackermann, M., Mirenska, A., Gohring, G., Thomay, K., Mucci, A., Hetzel, M., Glomb, T., Suzuki, T. et al. (2018). Pulmonary transplantation of human induced pluripotent stem cell-derived macrophages ameliorates pulmonary alveolar proteinosis. Am. J. Respir. Crit. Care Med. 198, pp. 350-360; Lachmann, N., Ackermann, M., Frenzel, E., Liebhaber, S., Brennig, S., Happle, C., Hoffmann, D., Klimenkova, O., Luttge, D., Buchegger, T. et al. (2015). Large-scale hematopoietic differentiation of human induced pluripotent stem cells provides granulocytes or macrophages for cell replacement therapies. Stem Cell Reports 4, pp. 282-296 and Zhang, H., Xue, C., Shah, R., Bermingham, K., Hinkle, CC, Li,W., Rodrigues, A., Tabita-Martinez, J., Millar, JS, Cuchel, M. et al. (2015). Functional analysis and transcriptomic profiling of iPSC derived macrophages and their application in modeling Mendelian disease. Circ. Res. 117, pp. 17-28.

[0529] Monocytes were generated from an isogenic control parental line (parental control WT / WT line) and homozygous (KI / KI) clones for Solution 30 over a 3-4 week period using a validated tetrad-based protocol. Undifferentiated iPSCs were first differentiated into myeloid progenitor cells, and then monocytes were generated using X-VIVO™ 15 serum-free hematopoietic cell medium (Lonza) supplemented with the cytokines IL-3 and macrophage colony-stimulating factor (M-CSF). All reagents were commercially available.

[0530] Monocytes were efficiently generated after approximately three weeks of continuous culture, isolated, and assessed by flow cytometry for staining levels for the monocyte markers CD14 and CD45 (see Figure 19a). More than 90% of the cells were double-positive for the known monocyte markers CD14 and CD45. iPSC lines (GEiGS Solution 30 (S30 / S30 genotype) and an isogenic control) were also used to generate monocytes, which were then stained for residual cell surface B2M expression using flow cytometry. When expanded as undifferentiated iPSCs, more than 93% of the cells expressed B2M for both the isogenic control and GEiGS Solution 30 clonal lines. However, after differentiation into monocytes, the GEiGS Solution 30 line showed a significant reduction in the number of cells with the same amount of cell surface-expressed B2M compared to the isogenic control line (77% compared to 16% of cells with residual B2M) (see Figure 19b).

[0531] Some solutions identified for silencing B2M (e.g., solution 30) were implemented as complete GEiGS, i.e., by editing the endogenous locus of the respective miRNA scaffold in iPSCs and differentiating the stem cells into myeloid cells.

[0532] GEiGS was performed using individual solutions of gRNAs obtained as synthetic sgRNAs from Integrated DNA Technologies (IDT). Cas9 protein was obtained from IDT. Isolated iPSCs were electroporated using a Lonza 4D-Nucleofector device (4D-25 Nucleofector Core Unit: Lonza, AAF-1002B; 4D-Nucleofector X Unit: AAF-1002X) (Buffer P3, device setting CM-137) and then contacted with the sgRNA / Cas9 ribonucleoprotein complex (RNP) and the appropriate dsDNA HDR template, either generated in-house or commercially available (IDT or Genewiz).

[0533] After electroporation, clonal selection, and characterization, clonal iPSC cell lines with either a homozygous knockout of the miRNA-21 gene or a homozygous knock-in of Solution 30 into the miR-21 locus were differentiated into monocytes and then monocyte-derived macrophages in macrophage medium (DMEM high glucose supplemented with 10% FBS [Gibco], 2 mM GlutaMAX, 100 U / ml penicillin, 100 μg / ml streptomycin, and 100 ng / ml M-CSF [Peprotech]) by adding half a volume of 1x cytokine-containing medium to each well, changing the medium every 2–3 days.

[0534] After several days of differentiation in culture, cells were isolated for analysis of target MHC-I protein expression (B2M or HLA-A / B / C) and relati...

Claims

1. 1. A method for regulating expression of a target gene in a cell in a context-specific manner, comprising: - providing the cell with a nucleic acid construct adapted to express an inhibitory RNA suitable for inhibiting expression of the target gene; Includes - the inhibitory RNA is active in a cell in a context-specific manner, such that inhibition of expression of the target gene occurs specifically in the first context in which the inhibitory RNA is active in said cell; Optionally, the method wherein in at least a second situation in which the inhibitory RNA is inactive or less active, no inhibition occurs or occurs in a reduced amount.

2. 2. The method of claim 1, wherein the nucleic acid construct is a modified form of an endogenous sequence encoding an inhibitory RNA, preferably an miRNA, that has been modified to target a transcript from a target gene.

3. 3. The method of claim 1 or 2, comprising genetically modifying a sequence encoding an endogenous inhibitory RNA to alter its targeting specificity to target a transcript of the target gene.

4. 4. The method of any one of claims 1 to 3, wherein the cells are context-specific hypoimmunogenic cells, preferably context-specific hypoimmunogenic therapeutic cells.

5. 5. The method of any one of claims 1 to 4, wherein the cell is an immune cell or a precursor thereof, for example, the cell is a macrophage or a T cell or a precursor thereof.

6. 6. The method of any one of claims 1 to 5, wherein the inhibitory RNA targets a target gene associated with the MHC-I complex, optionally the target gene is a B2M, HLA-A, HLA-B and / or HLA-C gene, such that when the inhibitory RNA is active, expression of the MHC-I complex in the cell is reduced, preferably when the target gene is B2M.

7. The inhibitory RNA may be: - cells in a particular state of differentiation, such as fully differentiated cells, cells at an intermediate stage of differentiation or undifferentiated cells, - totipotent, pluripotent or multipotent cells, - cells present in specific tissues, - cancerous or precancerous cells, - tumor-associated cells, such as tumor-associated immune cells or immune cells in the tumor microenvironment, - polarized cells, e.g., macrophages, - activated or polarized immune cells, - cells that are hypoxic, or - Cells subjected to the unfolded protein response 7. The method of claim 1, wherein the method is specifically active in cells in a state selected from the group consisting of:

8. The cells are: - therapeutic immune cells or their precursor cells, - therapeutic endocrine cells or their precursor cells, preferably pancreatic beta cells or their precursor cells, - therapeutic CNS cells or their precursor cells, optionally neurons or glial cells (e.g. astrocytes, oligodendrocytes, ependymal cells or microglial cells) or their precursor cells, - therapeutic epithelial cells or their precursor cells, - therapeutic muscle cells or their precursor cells, such as cardiac muscle cells, skeletal muscle cells and smooth muscle cells; - Therapeutic pluripotent stem cells 8. The method of any one of claims 1 to 7, wherein the allogeneic therapeutic cells are selected from the group consisting of:

9. A cell or cell population obtainable by the method of any one of claims 1 to 8.

10. 1. A genetically modified cell comprising a nucleic acid construct adapted to express an inhibitory RNA suitable for inhibiting expression of a target gene, the inhibitory RNA is active in a cell in a context-specific manner, such that inhibition of expression of the target gene occurs specifically in the first context in which the inhibitory RNA is active in said cell; Optionally, the genetically modified cell, wherein inhibition does not occur or is reduced in at least a second situation in which the inhibitory RNA is inactive or less active.

11. The cell of claim 10, wherein the nucleic acid construct is a modified endogenous sequence encoding an inhibitory RNA, preferably an miRNA, that has been modified to target a transcript from a target gene.

12. the cells are adapted to express at least one additional inhibitory RNA that is active in the cells in a context-specific manner; Optionally, the inhibition of expression of the second or further target gene occurs specifically in a first situation in which the inhibitory RNA is active in said cell; or 12. The method of any one of claims 1 to 8 or the cell of any one of claims 10 or 11, wherein inhibition of expression of the second or further target gene occurs specifically in a second or further situation in which at least one further inhibitory RNA is active in the cell.

13. 13. The cell of any one of claims 10 to 12, comprising a sequence encoding a genetically modified endogenous inhibitory RNA with altered targeting specificity to target a transcript of a target gene.

14. 14. A cell according to any one of claims 10 to 13, comprising a nucleic acid construct adapted to express an inhibitory RNA suitable for inhibiting expression of a target gene, wherein the inhibitory RNA becomes active in the cell after a change in the cellular context.

15. 15. The cell according to any one of claims 10 to 14, which is a context-specific hypoimmunogenic cell, preferably a context-specific hypoimmunogenic therapeutic cell.

16. The inhibitory RNA targets more than one target gene, optionally one of the target genes is B2M and another of the target genes is selected from PPARG, IRF4, KDM6B, FOXP3, PDCD-1 and STAT6, and optionally one of the two or more inhibitory RNAs is selected from SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29), SEQ ID NO: 10 or 27 (Solution 30), SEQ ID NO: 348 to SEQ ID NO: 411 or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), and another one of the two or more inhibitory RNAs is selected from SEQ ID NO: 66 to SEQ ID NO: 87 or SEQ ID NO: 88 to SEQ ID NO: 109 (targeting PPARG), SEQ ID NO: 110 to SEQ ID NO: 133 or SEQ ID NO: 134 to SEQ ID NO: 157 (targeting IRF4), SEQ ID NO: 158 to SEQ ID NO: 181 or SEQ ID NO: 182 to SEQ ID NO: No. 205 (targeting KDM6B), SEQ ID NO: 476 to SEQ ID NO: 525 or SEQ ID NO: 526 to SEQ ID NO: 575 (targeting FOXP3), SEQ ID NO: 576 to SEQ ID NO: 624 or SEQ ID NO: 625 to SEQ ID NO: 673 (targeting PDCD-1), or SEQ ID NO: 206 to SEQ ID NO: 229 or SEQ ID NO: 230 to SEQ ID NO: 253 (targeting STAT6), or one of the target genes is PPARG and another of the target genes is IRF4, K 16. The method or cell of any one of claims 1 to 15, wherein one of the target genes is selected from KDM6B and STAT6, or one of the target genes is IRF4 and another of the target genes is selected from PPARG, KDM6B and STAT6, or one of the target genes is KDM6B and another of the target genes is selected from PPARG, IRF4 and STAT6, or one of the target genes is FOXP3 and another of the target genes is PDCD-1.

17. 17. The method or cell of any one of claims 1 to 16, wherein the cell is a progenitor cell that has been modified to contain a nucleic acid construct adapted to express an inhibitory RNA suitable for inhibiting expression of a target gene, and wherein the inhibitory RNA is active in the cell after differentiation, preferably the inhibitory RNA is active in the cell when the cell is fully differentiated.

18. the cell is a differentiated cell selected from the group consisting of leukocytes, dendritic cells, lymphocytes, monocytes, macrophages, erythrocytes, platelets, hematopoietic cells, pancreatic islet cells (e.g., beta cells), liver cells, muscle cells, keratinocytes, cardiac myocytes, nerve cells, skeletal muscle cells, eye cells, mesenchymal cells, fibroblasts, lung cells, gastrointestinal cells, vascular cells, endocrine cells, and adipocytes; 18. The method or cell of any one of claims 1 to 17, wherein the cell is a stem cell, optionally a stem cell suitable for differentiating into a differentiated cell selected from the group consisting of a leukocyte, a dendritic cell, a lymphocyte, an erythrocyte, a platelet, a hematopoietic cell, a pancreatic islet cell (e.g., a beta cell), a liver cell, a muscle cell, a keratinocyte, a cardiac muscle cell, a neuron, a skeletal muscle cell, an eye cell, a mesenchymal cell, a fibroblast, a lung cell, a gastrointestinal cell, a vascular cell, an endocrine cell, or an adipocyte.

19. The inhibitory RNA is a modified form of an endogenous miRNA selected from the group consisting of hsa-miR-375, hsa-miR-143, hsa-miR-21, hsa-miR-30d, hsa-miR-192, hsa-miR-30a, hsa-miR-182, hsa-miR-148a, hsa-miR-127, and hsa-miR-200c, and / or the inhibitory RNA has the following sequence: - SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29) or SEQ ID NO: 10 or 27 (Solution 30) (targeting B2M), - SEQ ID NO: 66 to SEQ ID NO: 87 or SEQ ID NO: 88 to SEQ ID NO: 109 (targeting PPARG), - SEQ ID NO: 110 to SEQ ID NO: 133 or SEQ ID NO: 134 to SEQ ID NO: 157 (targeting IRF4), - SEQ ID NO: 158 to SEQ ID NO: 181 or SEQ ID NO: 182 to SEQ ID NO: 205 (targeting KDM6B), - SEQ ID NO: 348 to SEQ ID NO: 411 or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), - SEQ ID NO: 476 to SEQ ID NO: 525 or SEQ ID NO: 526 to SEQ ID NO: 575 (targeting FOXP3), - SEQ ID NO: 576 to SEQ ID NO: 624 or SEQ ID NO: 625 to SEQ ID NO: 673 (targeting PDCD-1), or - SEQ ID NO: 206 to SEQ ID NO: 229 or SEQ ID NO: 230 to SEQ ID NO: 253 (targeting STAT6) 19. The method or cell of any one of claims 1 to 18, comprising one of:

20. The cell is a macrophage or a precursor thereof, and the inhibitory RNA is selected from the group consisting of: - situations in which the macrophages are unpolarized macrophages, - Classically activated macrophages (M1) - Macrophages are selectively activated (M2), - situations in which the macrophages are regulatory macrophages (Mregs), or - Macrophages in the TME active in macrophages in one of or the miRNA is selected from the group consisting of MIR146A (e.g., hsa-miR-146a-5p), MIR20A (e.g., hsa-miR-20a-5p), LET7C (hsa-miR-let-7c-5p), and LET7i (hsa-let-7i-5p); 20. The method or cell of any one of claims 1 to 19.

21. The cell is a T cell or a precursor thereof, and the inhibitory RNA is selected from the group consisting of: - the situation where the T cells are CD8+ T cells, - the situation where the T cells are CD4+ T cells, - the situation where the T cells are CD4+ T helper cells, optionally Th1, Th2, Th17, Th9, Tfh or Th22 cells; - the situation where the T cells are memory T cells, - the T cells are CD4+ Treg cells, optionally FOXP3+ Treg cells or FOXP3- Treg cells; - T cell exhaustion (e.g., cell status, or - The situation where T cells are in the TME 21. The method or cell of any one of claims 1 to 20, wherein the cell is specifically active in T cells in one of the following:

22. 1. A nucleic acid construct encoding an inhibitory RNA suitable for inhibiting expression of a target gene, for context-specific inhibition of expression of a target gene in a cell, wherein the inhibitory RNA is active in the cell in a context-specific manner, such that inhibition of expression of the target gene occurs in a first context in which the inhibitory RNA is active in said cell, the construct comprising a nucleic acid sequence selected from the following list: - SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29) or SEQ ID NO: 10 or 27 (Solution 30) (targeting B2M), - SEQ ID NO: 66 to SEQ ID NO: 87 or SEQ ID NO: 88 to SEQ ID NO: 109 (targeting PPARG), - SEQ ID NO: 110 to SEQ ID NO: 133 or SEQ ID NO: 134 to SEQ ID NO: 157 (targeting IRF4), - SEQ ID NO: 158 to SEQ ID NO: 181 or SEQ ID NO: 182 to SEQ ID NO: 205 (targeting KDM6B), - SEQ ID NO: 348 to SEQ ID NO: 411 or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), - SEQ ID NO: 476 to SEQ ID NO: 525 or SEQ ID NO: 526 to SEQ ID NO: 575 (targeting FOXP3), - SEQ ID NO: 576 to SEQ ID NO: 624 or SEQ ID NO: 625 to SEQ ID NO: 673 (targeting PDCD-1), or - SEQ ID NO: 206 to SEQ ID NO: 229 or SEQ ID NO: 230 to SEQ ID NO: 253 (targeting STAT6) A nucleic acid construct comprising a sequence selected from the group consisting of:

23. The following list: - SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29) or SEQ ID NO: 10 or 27 (Solution 30) (targeting B2M), - SEQ ID NO: 66 to SEQ ID NO: 87 or SEQ ID NO: 88 to SEQ ID NO: 109 (targeting PPARG), - SEQ ID NO: 110 to SEQ ID NO: 133 or SEQ ID NO: 134 to SEQ ID NO: 157 (targeting IRF4), - SEQ ID NO: 158 to SEQ ID NO: 181 or SEQ ID NO: 182 to SEQ ID NO: 205 (targeting KDM6B), - SEQ ID NO: 348 to SEQ ID NO: 411 or SEQ ID NO: 412 to SEQ ID NO: 475 (targeting B2M), - SEQ ID NO: 476 to SEQ ID NO: 525 or SEQ ID NO: 526 to SEQ ID NO: 575 (targeting FOXP3), - SEQ ID NO: 576 to SEQ ID NO: 624 or SEQ ID NO: 625 to SEQ ID NO: 673 (targeting PDCD-1), or - SEQ ID NO: 206 to SEQ ID NO: 229 or SEQ ID NO: 230 to SEQ ID NO: 253 (targeting STAT6) An inhibitory RNA comprising or consisting of a sequence selected from the following or a functional variant thereof:

24. 22. A composition comprising the cells of any one of claims 9 to 21.

25. 25. A cell according to any one of claims 9 to 21 or a composition according to claim 24 for use in therapy.

26. A reporter nucleic acid comprising, or consisting of, a constitutive promoter operably linked to a gene encoding a fluorescent protein and a target gene (or a portion of the target gene), wherein the fluorescent protein and the target gene are transcribed as a polycistronic mRNA.

27. 27. The reporter nucleic acid of claim 26, wherein the target gene is inserted into the 3' untranslated region of the fluorescent protein.

28. 28. The reporter nucleic acid of claim 26 or 27, wherein the target gene is a target gene of an inhibitory RNA, and / or the target gene is selected from PDCD-1, B2M, PPARG, IRF4, KDM6B, FOXP3, or STAT6.

29. 29. The reporter nucleic acid of any one of claims 26 to 28, wherein the constitutive promoter is EF1a and / or the fluorescent protein is GFP, optionally CopGFP.

30. - WPRE, - adjacent long terminal repeat (LTR) sequences, and / or - a gene for a cell surface protein operably linked to a constitutive promoter, optionally the constitutive promoter is PGK, and optionally the cell surface protein is tNGFR.

30. The reporter nucleic acid of any one of claims 26 to 29, further comprising:

31. 31. A reporter nucleic acid according to any one of claims 26 to 30 for use in detecting the inhibition or silencing of a target gene by one or more inhibitory RNAs.

32. 31. Use of a reporter nucleic acid according to any one of claims 26 to 30 in detecting the inhibition or silencing of a target gene by one or more inhibitory RNAs.

33. - a reporter nucleic acid according to any one of claims 26 to 31, and - an expressible nucleic acid comprising a constitutive promoter operably linked to a nucleic acid sequence encoding an inhibitory RNA; An expression system comprising:

34. - the expressed nucleic acid is flanked by long terminal repeat (LTR) sequences, - the constitutive promoter in the expressed nucleic acid is EF1a, - the inhibitory RNA is a sequence encoding an endogenous non-coding RNA, e.g., a sequence encoding an endogenous inhibitory RNA, which has been modified to retarget the endogenous inhibitory RNA to silence or partially silence the target gene via a GEiGS approach, or the inhibitory RNA is a GEiGS extension solution; and / or - the expressed nucleic acid further comprises an additional fluorescent protein operably linked to a constitutive promoter, optionally the additional fluorescent protein is dsRed; 34. The expression system of claim 33.

35. 35. An expression system according to claim 33 or 34, wherein the reporter nucleic acid and the expression nucleic acid are provided in two separate lentiviral vectors.

36. 36. An expression system according to any one of claims 33 to 35, comprising two or more expressed nucleic acids.

37. 37. The expression system of claim 36, wherein the two or more expressed nucleic acids are provided in two or more separate lentiviral vectors.

38. 1. A method for detecting the inhibitory or silencing activity of one or more inhibitory RNAs against a target gene, comprising: - transfecting a cell(s) with an expression system according to any one of claims 33 to 37, - binding the inhibitory RNA to a target gene within the polycistronic mRNA; - degrading the polycistronic mRNA; and - reduction of the fluorescent signal from the fluorescent protein in the cell(s); A method comprising:

39. 1. A method for selecting an inhibitory RNA that exhibits silencing or inhibition of a target gene from a pool of test inhibitory RNAs, comprising: - transfecting a cell or cells with an expression system according to any one of claims 33 to 37, and - selecting the inhibitory RNA transfected into the cell(s) that exhibit a reduced fluorescent signal from the fluorescent protein; A method comprising:

40. - incubating the cell(s) under conditions suitable for expression of the reporter nucleic acid and the expressed nucleic acid, and / or - detecting a reduction in the fluorescent signal, optionally by flow cytometry.

40. The method of claim 38 or 39, further comprising:

41. 41. The method of any one of claims 38 to 40, wherein the cell is a macrophage, an iPSC or a T cell, optionally a primary T cell or a T cell line.

Images