Novel polynucleotides, cells and methods

Abstract

In particular, novel inhibitory RNAs or polynucleotide sequences encoding inhibitory RNAs, as well as expression cassettes, vectors, virions, synthetic microRNAs, synthetic pre-microRNA molecules and cells, and related medical uses and methods for altering protein expression, in particular B2M protein expression, are disclosed.

Description

[Technical Field]

[0001] The present invention relates to novel inhibitory RNAs or polynucleotide sequences encoding inhibitory RNAs, as well as expression cassettes, vectors, virions, synthetic microRNAs and synthetic pre-microRNAs, cells comprising inhibitory RNAs or polynucleotide sequences encoding inhibitory RNAs, and compositions comprising cells. The present invention also relates to novel inhibitory RNAs or polynucleotide sequences encoding inhibitory RNAs, expression cassettes, vectors, virions, synthetic microRNAs and synthetic pre-microRNAs, and cells for use in therapy and in methods of treating, for example, autoimmune diseases or cancer, and related methods of degrading messenger RNA transcribed from the beta-2 microglobulin (B2M) gene, for example, in cells, among other things. The present invention further relates to methods of altering protein expression or messenger RNA translation in cells, among other things. [Background technology]

[0002] Cell therapy is a class of advanced medical therapies in which patients are treated by transplantation of cells, which may or may not have been previously engineered to enhance their functionality. Cell therapies, such as bone marrow transplants or blood transfusions, are well-known examples of cell therapies that have 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 a number of different disease indications, such as diabetes, cancer, and blindness.

[0003] 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.

[0004] 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.

[0005] 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.

[0006] 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.

[0007] 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).

[0008] The present invention aims to address this problem by creating hypoimmunogenic cells through 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.

[0009] One approach to generating hypoimmunogenic cells is to use inhibitory RNA via the gene editing-induced gene silencing (GEiGS®) system to down-modulate MHC-I expression to a level that prevents significant adaptive immune responses but is still sufficient to inhibit NK cell-mediated responses. GEiGS is a gene silencing technology that works by redirecting the silencing specificity of endogenous non-coding RNAs (ncRNAs) with RNAi-mediated silencing activity to a desired target sequence. In humans, the redirected ncRNAs are often microRNAs (miRNAs). Because these target their targets through sequence complementarity, altering their sequence can shift their silencing specificity toward the optimal desired target. 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.

[0010] GEiGS therefore works by hijacking silencing non-coding RNAs (e.g., 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 administering double-stranded oligos or transgenes, respectively, GEiGS utilizes endogenous ncRNAs as vectors to silence desired targets.

[0011] While modifying the sequence of endogenous ncRNA to redirect its specificity 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, programmable, and specific gene silencing.

[0012] GEiGS is particularly suitable for partial silencing of MHC-I because it can provide stable, tunable silencing of a wide range of target expression. Using GEiGS to modulate B2M expression, it is possible to achieve optimal levels of MHC-I expression to evade both T cell- and NK cell-mediated responses (Figure 3). Previous studies have validated some aspects of this approach in research settings using oligo-based transgenic RNAi (Karabekian et al., 2015 - Tissue Eng Part A. 2015 Oct. 21(19-20):2559-71. doi: 10.1089 / ten.TEA.2015.0105), but its implementation in human cells for therapeutics has been hindered by limitations of existing RNAi technologies. GEiGS overcomes some of these limitations and paves the way for this approach to generate low-immunogenic cells to be applied in clinical settings.

[0013] Other approaches for partially or completely silencing gene expression using inhibitory RNA are also known in the art, for example, by inserting a suitable expression construct into the genome of the cell to drive expression of the inhibitory RNA, by providing a suitable expression construct in the cell to drive expression of the inhibitory RNA episomally (e.g., in a plasmid or other vector as appropriate), or by direct delivery of the inhibitory RNA to the cell.

[0014] 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 then transplanted into NOD mice, demonstrating improved graft survival (Wang et al., 2012). While this study demonstrates the efficacy 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, may be perfectly suited to this approach, as it has the potential to provide stable, tunable silencing.

[0015] Further advantages of the GEiGS approach include the simplicity in generating hypoimmunogenic cells, whereby editing is of a non-coding gene and does not require additional transgene expression.

[0016] The generation of hypoimmunogenic cells does not solely rely on the GEiGS approach, and other approaches to generating these cells can also be used. Other such alternative approaches include episomal methods, such as direct insertion of DNA into the genome using techniques such as homology directed repair, and transduction techniques that use viral vectors to deliver DNA or RNA sequences of interest into cells.

[0017] Overall, there remains a need to generate ready-to-use cell therapies that overcome the significant and complex challenges of transplant rejection. It is an object of the present invention to provide a novel approach to generate hypoimmunogenic cells that may be suitable for cell therapy due to their ability to successfully evade both innate and adaptive immunity. Such cells can be generated using GEiGS technology or an alternative approach in which inhibitory RNA is expressed in cells to reduce B2M expression. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] WO2012 / 145384 [Patent Document 2] WO2019 / 058253 [Patent Document 3] WO2020 / 183414 [Patent Document 4] WO2020 / 183419 [Patent Document 5] WO2006 / 040763 [Patent Document 6] U.S. Patent No. 5,486,359 [Patent Document 7] WO2013126963 [Patent Document 8] WO2014107763 [Patent Document 9] WO91 / 10470 [Patent Document 10] WO91 / 10425 [Patent Document 11] U.S. Patent No. 5,837,234 [Patent Document 12] U.S. Patent No. 5,011,472 [Patent Document 13] U.S. Patent No. 4,892,538 [Non-patent literature]

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Direct Entries51

Outdoor Track52

[0020] SUMMARY OF THE INVENTION The present invention is based in part on the surprising discovery that beta-2 microglobulin (B2M) expression can be modified, for example, using inhibitory RNA provided genomically or episomally (e.g., by GEiGS or vector transduction), to allow tunable expression of cell surface B2M, enabling cells to evade the innate and adaptive immune systems. This has the particular advantage of facilitating the generation of "ready-to-ship" allogeneic transplantable cells that are less susceptible to rejection by the recipient host's immune system.

[0021] According to a first aspect of the present invention, there is provided herein an inhibitory RNA, or a polynucleotide sequence encoding an inhibitory RNA, preferably a microRNA, wherein the inhibitory RNA is (i) SEQ ID NOs: 1 to 17, 64 to 127, (ii) variants of SEQ ID NOs: 1 to 17, 64 to 127; (iii) fragments of SEQ ID NOs: 1 to 17, 64 to 127, and (iv) a DNA sequence complementary to any one of (i), (ii), or (iii); An inhibitory RNA comprising or consisting of a sequence selected from:

[0022] According to a second aspect of the present invention, there is provided an expression cassette comprising a polynucleotide sequence encoding an inhibitory RNA in accordance with the invention operably linked to one or more regulatory elements, preferably a promoter, optionally a constitutive or inducible promoter, suitable for enabling transcription of the inhibitory RNA in a cell.

[0023] According to a third aspect of the invention there is provided a vector comprising a polynucleotide sequence encoding an inhibitory RNA according to the invention or an expression cassette according to the invention.

[0024] According to a fourth aspect of the present invention there is provided a virion comprising a vector of the present invention.

[0025] According to a fifth aspect of the present invention, there is provided a synthetic microRNA comprising or consisting of a sequence selected from any one of SEQ ID NOs: 1 to 17, 64 to 127, or a functional variant or fragment thereof.

[0026] According to a sixth aspect of the present invention, there is provided a synthetic pre-microRNA comprising or consisting of a sequence selected from any one of SEQ ID NOs: 18 to 34, 128 to 191, or a functional variant or fragment thereof.

[0027] According to a seventh aspect of the present invention there is provided a synthetic microRNA adapted to target messenger RNA transcribed from the B2M gene, the synthetic microRNA comprising a sequence according to any one of SEQ ID NOs: 1-17, 64-127 or a functional variant or fragment thereof, optionally the synthetic microRNA comprising a sequence according to any one of SEQ ID NOs: 18-34, 128-191 or a functional variant or fragment thereof.

[0028] According to an eighth aspect of the present invention there is provided a cell comprising or expressing a synthetic microRNA according to the present invention, wherein the cell has been modified to express the synthetic microRNA such that it targets messenger RNA transcribed from the B2M gene.

[0029] According to a ninth aspect of the present invention there is provided a cell comprising or expressing a sequence encoding a synthetic inhibitory RNA, preferably a synthetic microRNA, wherein the synthetic inhibitory RNA is adapted to target B2M.

[0030] According to a further aspect of the present invention there is provided a cell comprising the expression cassette, vector, virion, synthetic microRNA or pre-microRNA of the present invention.

[0031] According to a further aspect of the invention there is provided a composition comprising the cells of the invention.

[0032] In a further aspect of the invention, there is provided an inhibitory RNA or a polynucleotide encoding an inhibitory RNA, expression cassette, vector, virion, synthetic microRNA or pre-microRNA, cell or composition of the invention for use in therapy.

[0033] In a further aspect of the invention, provided herein is an inhibitory RNA or a polynucleotide encoding an inhibitory RNA, expression cassette, vector, virion, synthetic microRNA or pre-microRNA, cell or composition of the invention for use in a method of treating an autoimmune disease or cancer in a subject, wherein the autoimmune disease is preferably type 1 diabetes.

[0034] In a further aspect of the invention, provided herein is an inhibitory RNA of the invention or a polynucleotide, expression cassette, vector, virion, or synthetic microRNA or pre-microRNA encoding the inhibitory RNA for use in a method of degrading messenger RNA transcribed from the B2M gene in a cell, wherein preferably the messenger RNA is partially silenced and B2M protein expression is altered by 1% to 90%, optionally 5% to 90%, optionally 10% to 90% of expression compared to control cells. Various preferred levels of expression are discussed herein.

[0035] In yet another aspect of the invention, there is provided an inhibitory RNA or a polynucleotide, expression cassette, vector, virion or synthetic microRNA or pre-microRNA encoding the inhibitory RNA of the invention for use in a method for degrading messenger RNA transcribed from the B2M gene in a cell, wherein preferably the messenger RNA is partially silenced and B2M protein expression is altered by between 1% and 90%, optionally between 5% and 90%, optionally between 10% and 90% of expression compared to a control cell.

[0036] In some embodiments, the expression level of B2M in cells according to the present invention is, for example, 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 5% to 10% compared to control cells. The expression level of B2M in cells is within the range of 0%, 10% to 80%, 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%. In some embodiments, the expression level of B2M in cells is determined by the amount of B2M expressed on the surface of the cells. The amount of B2M present on the surface of the cells can be determined by various conventional techniques, such as flow cytometry.

[0037] In a further aspect of the invention, there is provided a cell of the invention or a composition of the invention for use in a method of treating an autoimmune disease or cancer in a subject, the method comprising administering to the subject a therapeutic amount of the cell or composition.

[0038] In a further aspect of the invention there is provided a method of treating an autoimmune disease or cancer in a subject, preferably wherein the autoimmune disease is type I diabetes, the method comprising administering to the subject a therapeutic amount of the cells or composition.

[0039] In 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 an autoimmune disease or cancer in a subject, preferably wherein the autoimmune disease is type 1 diabetes.

[0040] In a further aspect of the invention, there is provided a method for modifying protein expression in a cell, the method comprising introducing into the cell an inhibitory RNA or a polynucleotide encoding an inhibitory RNA, an expression cassette, a vector, a virion, or a synthetic microRNA or pre-microRNA of the invention.

[0041] In a further aspect of the invention, there is provided a method for modifying the translation of messenger RNA transcripts derived from protein-coding genes and the subsequent expression of proteins in a cell, comprising introducing into the cell an inhibitory RNA or a polynucleotide encoding an inhibitory RNA, an expression cassette, a vector, a virion, or a synthetic microRNA or pre-microRNA of the invention.

[0042] In a further aspect of the present invention, there is provided a method for modifying the translation of a messenger RNA transcript derived from a protein-coding gene and the subsequent expression of the protein in a cell, comprising the step of introducing into the cell a DNA editing agent that confers silencing specificity of a microRNA for a target RNA of interest, wherein the sequence of the microRNA comprises any one of SEQ ID NOs: 1-17, 64-127, whereby the microRNA is modified such that translation of the messenger RNA transcript into a protein molecule is modified.

[0043] In a further aspect of the invention there is provided a cell or cell population obtained from the method of the invention.

[0044] In a further aspect of the invention, there is provided a composition comprising a cell or cell population of the invention.

[0045] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations thereof mean "including but not limited to" and are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0046] 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.

[0047] 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.

[0048] Various aspects of the invention are described in further detail below.

[0049] Embodiments of the present invention are further described herein below with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0050] [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] Figure 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 the GEiGS 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). [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 11a) shows results demonstrating a reduction in surface B2M expression. Approximately 24% of cells in a CD3+, mixed T cell population exhibited silencing of B2M, with an 80% reduction in mean fluorescence intensity compared to control, unedited cells. Figure 11b) shows quantification of B2M silencing in helper (CD4+) and cytotoxic (CD8+) T cells, demonstrating efficient silencing of B2M (approximately 90%) in both compartments. [Figure 12]Figure 12 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 leads to the complete absence of MHC-I (a "loss of self" phenotype), 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 13] Figure 13 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 14] Figure 14 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 15a] Figure 15A shows the reduction of B2M expression in CXCR4-expressing cells generated by in vitro differentiation of the Solution 30 heterozygous iPSC line. The iPSC miR-21S30 / +1 line showed approximately 80% reduction in B2M expression compared to the control (unmodified) iPSC line. As expected, B2M- / - iPSCs do not express B2M protein. [Figure 15b] Figure 15B 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 are 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 16a] Figure 16a 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 16b] Figure 16b 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 17] Figure 17 shows the effect of homology arm length on the efficiency of homology-dependent repair (HDR). Genomic sequences flanking 40 base pairs in length (homology arms) exhibit a dose-dependent superior effect on HDR efficiency (% HDR) compared with genomic sequences flanking 350 bp or 150-350 bp in length, which are only slightly better than the donor-less template alone. [Figure 18] Figure 18 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 19a] Figure 19a shows that multiple solutions can silence their target genes when ectopically expressed from a single construct with a constitutive promoter in iPSCs. Figure 19a 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 14). [Figure 19b] Figure 19b shows that multiple solutions can silence their target genes when ectopically expressed in iPSCs from a single construct with a constitutive promoter. Figure 19b 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 20]Figure 20 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. [Figure 21a] Figure 21a 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 21a 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 21b]Figure 21b 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 with no significant change in expression level are shown in black. Figure 21b shows transcriptome data from cells with hsa-mir-20a knockout miRNA 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 21c] Figure 21c 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 21c 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 22a]FIG. 22a shows that B2M silencing in myeloid cells leads to loss of cell surface HLA-A / B / C (and therefore MHC-I) in monocytes and macrophages. [Figure 22b] FIG. 22b shows that B2M silencing in myeloid cells leads to loss of cell surface HLA-A / B / C (and therefore MHC-I) in monocytes and macrophages. [Figure 23a] FIG. 23a shows the constructs used to validate the use of lentiviral transduction and pooled screening strategies. [Figure 23b] FIG. 23b illustrates the use of lentiviral transduction and pooled screening strategies to identify "hits" from GEiGS solutions engineered using primary human T cells. [Figure 24a] FIG. 24a shows validation of the lentiviral-based pooled screening strategy in primary human T cells using validated solutions for both eGFP and B2M. [Figure 24b] FIG. 24b shows DsRed and B2M knockdown (GFP loss) corresponding to individual solutions in combined solution lentivirus in Jurkat. DETAILED DESCRIPTION OF THE INVENTION

[0051] The patents, scientific and technical literature referred to in this specification establishes the knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of issued patents, published and pending patent applications, and other publications cited herein are incorporated by reference herein to the same extent as if each were specifically and individually indicated to be incorporated by reference. In the case of any conflict, the present disclosure will control.

[0052] Various aspects of the invention are described in further detail below.

[0053] The present invention, in some embodiments thereof, relates to inhibitory RNAs, including microRNAs, and more particularly, but not exclusively, to their use to degrade endogenous target RNAs of interest in eukaryotic cells. The invention relates in some aspects to the use of synthetic inhibitory RNAs in cells to reduce, but not eliminate, the expression of B2M, for example, for therapeutic purposes, allowing such cells to evade the innate and adaptive immune systems of the animal (usually a human) into which the cells are introduced.

[0054] The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

[0055] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by the examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein are for the purpose of description and should not be regarded as limiting.

[0056] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the specification in its entirety. Also, as used herein, the singular terms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless otherwise specified, nucleic acids are written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively. It should be understood that the present invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending on the context in which they are used by those of ordinary skill in the art.

[0057] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0058] definition The terms "RNA interference" and "RNAi" are synonymous and refer to the process in which a polynucleotide (miRNA or siRNA) containing at least one polyribonucleotide unit exerts an effect on biological processes. 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. Therefore, this term includes, inter alia, RNA precursors that are processed to form miRNA, siRNA, or other RNAs that can degrade target mRNA.

[0059] The term "gene silencing" refers to the process by which the expression of a specific gene product is reduced or attenuated by RNA interference. 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 level of expression 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.

[0060] 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 flanking 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.

[0061] 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.

[0062] 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.

[0063] 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).

[0064] Terms such as "identity" and "identical" refer to similar sequences between two polymer molecules, e.g., between two nucleic acid molecules, e.g., 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, described by Tatusova and Madden, 1999 (FEMS Microbiol Lett 174: 247-250).

[0065] 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.

[0066] The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™; Altschulr 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] The term "expression cassette", as used herein, includes a polynucleotide sequence encoding a polypeptide 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.

[0071] 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).

[0072] 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.

[0073] "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, e.g., 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.

[0074] 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).

[0075] 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.

[0076] 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].

[0077] Furthermore, embryonic stem cells have been shown to be useful in the production of embryonic stem cells from 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] and rabbits [Giles et al., 1993, Mol Reprod Dev. 36: 130-138; Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424-423], as well as 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].

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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 the 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].

[0082] 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.

[0083] 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.

[0084] "Artificial," when used in reference to a polynucleotide sequence, means a sequence not found in nature, for example, that is a synthetic modification of a naturally occurring sequence or that contains non-naturally occurring sequences.

[0085] Inhibitory RNA and Polynucleotides The term "inhibitory RNA" refers to an RNA molecule that has a degrading or silencing, e.g., partial or complete, effect on its corresponding messenger RNA that has a complementary sequence thereto. In some embodiments of the present invention, inhibitory RNA includes siRNA, shRNA, and microRNA, preferably microRNA.

[0086] According to one embodiment, the inhibitory RNA molecule is capable of RNA interference (RNAi), optionally after cellular processing.

[0087] According to some embodiments, the inhibitory RNA molecule is processed from a precursor.

[0088] According to one embodiment, the non-coding RNA molecule or RNA silencing molecule is processed from a single-stranded RNA (ssRNA) precursor.

[0089] According to one embodiment, the non-coding RNA molecule or RNA silencing molecule is processed from a double-stranded single-stranded RNA precursor.

[0090] According to one embodiment, the non-coding RNA molecule or RNA silencing molecule is processed from a dsRNA precursor (eg, containing perfect and imperfect base pairing).

[0091] According to one embodiment, the non-coding RNA molecule or RNA silencing molecule is processed from an unstructured RNA precursor.

[0092] According to one embodiment, the non-coding RNA molecule or RNA silencing molecule is processed from a protein-coding RNA precursor.

[0093] According to one embodiment, the non-coding RNA molecule or RNA silencing molecule is processed from a non-coding RNA precursor.

[0094] According to one embodiment, the dsRNA can be derived from two different complementary RNAs or from a single RNA that folds on itself to form the dsRNA.

[0095] 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.

[0096] Thus, some embodiments of the present invention contemplate modifying the genomic sequence encoding the dsRNA to redirect silencing specificity (including silencing activity) towards B2M mRNA.

[0097] 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].

[0098] 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.

[0099] 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).

[0100] 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).

[0101] 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.

[0102] 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.

[0103] 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).

[0104] According to one embodiment, the silencing 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)).

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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").

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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).

[0113] The present invention thus relates in some aspects to polynucleotide sequences encoding inhibitory RNAs, preferably microRNAs, that are composed of an altered sequence of an endogenous pre-miRNA (hairpin), also called an endogenous miRNA scaffold, i.e., a sequence altered to encode a novel silencing RNAi trigger in place of its natural guide strand. The novel polynucleotide matches a corresponding messenger RNA (mRNA) (e.g., B2M mRNA) by sequence complementarity, leading to the silencing of B2M through the RNA interference pathway.

[0114] Thus, the present invention provides an inhibitory RNA, or a polynucleotide sequence encoding an inhibitory RNA, preferably a microRNA, wherein the inhibitory RNA is: (i) SEQ ID NOs: 1 to 17, 64 to 127, (ii) variants of SEQ ID NOs: 1 to 17, 64 to 127; (iii) fragments of SEQ ID NOs: 1 to 17, 64 to 127, and (iv) a DNA sequence complementary to any one of (i), (ii), or (iii); The sequence may comprise or consist of a sequence selected from:

[0115] The present invention also provides an inhibitory RNA, or a polynucleotide sequence encoding an inhibitory RNA, preferably a microRNA, wherein the inhibitory RNA is: (i) any one of SEQ ID NOs: 18 to 34, 128 to 191; (ii) a variant of any one of SEQ ID NOs: 18 to 34, 128 to 191; (iii) a fragment of any one of SEQ ID NOs: 18 to 34, 128 to 191, and (iv) a DNA sequence complementary to any one of (i), (ii), or (iii); The sequence may comprise or consist of a sequence selected from:

[0116] The present invention further provides the following: (i) any one of SEQ ID NOs: 35 to 51; (ii) a variant of any one of SEQ ID NOs: 35 to 51, and (iii) a fragment of any one of SEQ ID NOs: 35 to 51 The present invention provides a polynucleotide sequence encoding an inhibitory RNA comprising or consisting of a sequence selected from:

[0117] The inhibitory RNAs of the invention, or polynucleotides encoding inhibitory RNAs, can silence, degrade, reduce, or alter the functional activity of a target RNA or mRNA, and optionally, the target mRNA can be translated into B2M. The inhibitory RNA can be perfectly complementary to the target or can have intentionally designed mismatches.

[0118] In some embodiments, an inhibitory RNA or a polynucleotide encoding an inhibitory RNA of the invention silences, reduces, or modifies the functional activity of a target RNA or target mRNA by at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, or at least 95%, compared to the functional activity of the target RNA or target mRNA in the absence of the inhibitory RNA or a polynucleotide encoding an inhibitory RNA of the invention. More preferably, an inhibitory RNA or a polynucleotide encoding an inhibitory RNA of the invention silences, degrades, reduces, or modifies the functional activity of a target RNA or target mRNA by at least 1%, at least 5%, at least 15%, at least 20%, at least 80%, at least 85%, at least 95%, or at least 99%, compared to the functional activity of the target RNA or target mRNA in the absence of the inhibitory RNA or a polynucleotide encoding an inhibitory RNA of the invention.

[0119] In some preferred embodiments, the inhibitory RNAs or polynucleotides encoding the inhibitory RNAs of the present invention silence, reduce, or modify the functional activity of B2M by at least 30% (e.g., Solution 30, SEQ ID NO: 10, and SEQ ID NO: 27). In some preferred embodiments, the inhibitory RNAs or polynucleotides encoding the inhibitory RNAs of the present invention silence, reduce, or modify the functional activity of B2M by at least 65% (e.g., Solution 20, SEQ ID NO: 5, and SEQ ID NO: 22). In some preferred embodiments, the inhibitory RNAs or polynucleotides encoding the inhibitory RNAs of the present invention silence, reduce, or modify the functional activity of B2M by at least 85%, preferably at least 95% (e.g., Solution 29, SEQ ID NO: 9, and SEQ ID NO: 26). In some preferred embodiments, the inhibitory RNAs or polynucleotides encoding the inhibitory RNAs of the present invention silence, reduce, or modify the functional activity of B2M by at least 80% (e.g., Solution 30, SEQ ID NO: 10, and SEQ ID NO: 27). In some embodiments, the reduction or modification of functional activity is observed in myeloid cells. In some embodiments, the reduced or altered functional activity is observed in pancreatic cells, hi some embodiments, the reduced or altered functional activity is observed in CD4+ and CD8+ T cells.

[0120] The present invention also relates to variants of the inhibitory RNAs of the invention or polynucleotides encoding the inhibitory RNAs. Generally, variants of the inhibitory RNAs of the invention or polynucleotides encoding the inhibitory RNAs comprise sequences with a high degree of sequence identity thereto. For example, variants have at least about 50%, at least about 80% sequence identity, more preferably at least about 85% sequence identity, and most preferably at least about 90% sequence identity (e.g., at least about 95%, at least about 98%, or at least about 99%) over their entire length, i.e., SEQ ID NOS: 1-51, 64-191, as appropriate, to the relevant reference sequence. A variant may preferably have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a reference sequence over its entire length, i.e., SEQ ID NOs: 1-51, 64-191. A variant may be identical to a reference sequence, i.e., SEQ ID NOs: 1-51, 64-191, except for containing one or more specific alterations. Identity to a sequence of SEQ ID NOs: 1-51, 64-191 may be measured over a region of at least 5, at least 10, at least 20, at least 50 or more consecutive nucleotides of the sequence of SEQ ID NOs: 1-51, 64-191, or more preferably over the entire length of SEQ ID NOs: 1-51, 64-191.

[0121] Optionally, the variant is an active variant. A variant is considered to be an active variant if it elicits an effect in any suitable test assay, such as those described in the Examples below, that is at least 20%, optionally at least 50%, particularly at least 75% (e.g., at least 90%, 95% or 99%) of the activity of the reference sequence (i.e., the sequence of which the variant is a variant).

[0122] The sequences of the inhibitory RNAs or polynucleotides encoding the inhibitory RNAs of the present invention may include variants of the polynucleotide sequences of SEQ ID NOS: 1-51, 64-191, in which modifications, such as nucleotide additions, deletions, or substitutions, have been made to the sequences of any one of SEQ ID NOS: 1-51, 64-191. Variants may be, for example, conservatively modified variants. "Conservatively modified variants" are those in which the change(s) result in substitutions / deletions / additions of residues that do not substantially affect the biological function of the variant. Typically, such biological function of the variant will be to induce silencing of B2M protein expression. Variants may include homologs of polynucleotides found in other species.

[0123] In some embodiments, variants of the inhibitory RNA or polynucleotides encoding the inhibitory RNA of the invention contain several substitutions, e.g., conservative substitutions (e.g., 1 to 25, e.g., 1 to 10, particularly 1 to 5, and especially 1 nucleotide may be changed), when compared to a reference sequence. The number of substitutions, e.g., conservative substitutions, may be up to 20%, e.g., up to 10%, e.g., up to 5%, e.g., up to 1%, of the number of nucleotides in the reference sequence.

[0124] Depending on the length of the polynucleotide reference sequence, a fragment according to the invention typically comprises at least 9 contiguous nucleotides (e.g., at least 9 or 10), e.g., at least 12 contiguous nucleotides (e.g., at least 15 or at least 20 contiguous nucleotides), particularly at least 50 or more contiguous nucleotides from the full-length polynucleotide sequence. In some embodiments, the fragment will be at least 10%, e.g., at least 20%, e.g., at least 50%, e.g., at least 70% or at least 80% of the length of the full-length polynucleotide sequence, as appropriate.

[0125] In one embodiment of the invention, the polynucleotide is RNA. In an alternative embodiment, the polynucleotide of the invention is DNA. In a further embodiment of the invention, the polynucleotide of the invention is a synthetic nucleic acid sequence (e.g., a polynucleotide sequence having a sequence that does not occur in nature).

[0126] DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) refer to nucleic acid molecules with backbones of sugar moieties that are deoxyribosyl and ribosyl, respectively. The sugar moieties can be linked to the four natural bases (adenine (A), guanine (G), cytosine (C), and thymine (T) in DNA and adenine (A), guanine (G), cytosine (C), and uracil (U) in RNA). A "corresponding RNA" is an RNA that has the same sequence as a reference DNA, except for the substitution of thymine (T) in DNA with uracil (U) in RNA. The sugar moieties can also be linked to unnatural bases, such as inosine, xanthosine, 7-methylguanosine, dihydrouridine, and 5-methylcytidine. The natural phosphodiester linkages between the sugar (deoxyribosyl / ribosyl) moieties can optionally be replaced with phosphorothioate linkages. Optionally, the nucleic acids of the invention comprise naturally occurring bases attached to a deoxyribosyl or ribosyl sugar backbone with phosphodiester linkages between the sugar moieties. In one embodiment, the nucleic acids of the invention are DNA. For example, the nucleic acids comprise or consist of a sequence selected from SEQ ID NOs: 1-34, 64-191.

[0127] In one embodiment of the present invention, the polynucleotide further comprises an miRNA scaffold. The miRNA scaffold may consist of at least 50, at least 100, or at least 150 nucleotide bases. The inhibitory RNAs or polynucleotides encoding the inhibitory RNAs of the present invention are composed of modified sequences of endogenous pre-miRNAs (hairpins), also referred to herein as "endogenous miRNA scaffolds," "scaffold portions," or simply "scaffolds." The nucleotide bases of the miRNA scaffold may flank both sides of the novel polynucleotide sequences of the present invention, i.e., SEQ ID NOS: 1-17, 64-127.

[0128] Therefore, in one aspect, the present invention provides miRNA scaffolds useful for generating non-naturally occurring miRNAs. The non-naturally occurring miRNAs of the present disclosure include miRNA scaffolds derived from specific endogenous miRNAs (i.e., at least 60% identical, at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and up to 100% identical thereto). A single miRNA scaffold of the present disclosure can be used to provide an almost unlimited number of different non-naturally occurring miRNAs. Endogenous miRNAs from which the miRNA scaffold sequences of the present disclosure are derived include, but are not limited to, eukaryotic miRNA scaffold sequences, preferably mammalian miRNA scaffold sequences, and more preferably human pre-miRNA scaffolds. In some embodiments of the invention, the human pre-microRNA scaffolds include 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-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, hsa-let-7g and hsa-mir-98.

[0129] In some embodiments of the present invention, an inhibitory RNA or a polynucleotide sequence encoding an inhibitory RNA encoded by any one of SEQ ID NOS: 1-17, 64-127 is inserted into a human pre-microRNA sequence. Preferably, SEQ ID NOS: 1 or 7 is inserted into hsa-mir-191 (SEQ ID NOS: 18 or 24, respectively), SEQ ID NOS: 2 or 11 is inserted into hsa-mir-302a (SEQ ID NOS: 19 or 28, respectively), SEQ ID NOS: 3 or 12 is inserted into hsa-mir-302c (SEQ ID NOS: 20 or 29, respectively), SEQ ID NOS: 4 or 16 is inserted into hsa-mir-93 (SEQ ID NOS: 21 or 33, respectively), SEQ ID NOS: 5 is inserted into hsa-mir-106a (SEQ ID NOS: 22), and SEQ ID NOS: 6 is inserted into hsa-mir-106b. (SEQ ID NO:23), SEQ ID NO:8 is inserted into hsa-mir-200c (SEQ ID NO:25), SEQ ID NO:9 is inserted into hsa-mir-20a (SEQ ID NO:26), SEQ ID NO:10 is inserted into hsa-mir-21 (SEQ ID NO:27), SEQ ID NO:13 is inserted into hsa-mir-363 (SEQ ID NO:30), SEQ ID NO:14 is inserted into hsa-mir-518b (SEQ ID NO:31), SEQ ID NO:15 is inserted into hsa-mir-744 (SEQ ID NO:32), and SEQ ID NO:17 is inserted into hsa-mir-99b (SEQ ID NO:34). Preferably, SEQ ID NO:64, SEQ ID NO:73, or SEQ ID NO:89 is inserted into hsa-mir-320a (SEQ ID NO:128, SEQ ID NO:137, or SEQ ID NO:153, respectively). Preferably, SEQ ID NO: 65, SEQ ID NO: 77, or SEQ ID NO: 94 is inserted into hsa-mir-520f (SEQ ID NO: 129, SEQ ID NO: 141, or SEQ ID NO: 158, respectively). Preferably, SEQ ID NO: 66, SEQ ID NO: 78, or SEQ ID NO: 95 is inserted into hsa-mir-652 (SEQ ID NO: 130, SEQ ID NO: 142, or SEQ ID NO: 159, respectively). Preferably, SEQ ID NO: 67 is inserted into hsa-mir-106a (SEQ ID NO: 131). Preferably, SEQ ID NO: 68 or SEQ ID NO: 79 is inserted into hsa-mir-1180 (SEQ ID NO: 132 or SEQ ID NO: 143, respectively).Preferably, SEQ ID NO:69 or SEQ ID NO:81 is inserted into hsa-mir-15b (SEQ ID NO:133 or SEQ ID NO:145, respectively). Preferably, SEQ ID NO:70, SEQ ID NO:82 or SEQ ID NO:99 is inserted into hsa-mir-182 (SEQ ID NO:134, SEQ ID NO:146 or SEQ ID NO:163, respectively). Preferably, SEQ ID NO:71 or SEQ ID NO:87 is inserted into hsa-mir-23a (SEQ ID NO:135 or SEQ ID NO:151, respectively). Preferably, SEQ ID NO:72 is inserted into hsa-mir-26b (SEQ ID NO:136). Preferably, SEQ ID NO:74 or SEQ ID NO:90 is inserted into hsa-mir-335 (SEQ ID NO:138 or SEQ ID NO:154, respectively). Preferably, SEQ ID NO:75 or SEQ ID NO:91 is inserted into hsa-mir-361 (SEQ ID NO:139 or SEQ ID NO:155, respectively). Preferably, SEQ ID NO: 76 is inserted into hsa-mir-518b (SEQ ID NO: 140). Preferably, SEQ ID NO: 80 is inserted into hsa-mir-1307 (SEQ ID NO: 144). Preferably, SEQ ID NO: 83 is inserted into hsa-mir-205 (SEQ ID NO: 147). Preferably, SEQ ID NO: 84 is inserted into hsa-mir-22 (SEQ ID NO: 148). Preferably, SEQ ID NO: 85 is inserted into hsa-mir-221 (SEQ ID NO: 149). Preferably, SEQ ID NO: 86 is inserted into hsa-mir-222 (SEQ ID NO: 150). Preferably, SEQ ID NO: 88 is inserted into hsa-mir-30e (SEQ ID NO: 152). Preferably, SEQ ID NO: 92 is inserted into hsa-mir-423 (SEQ ID NO: 156). Preferably, SEQ ID NO: 93 is inserted into hsa-mir-519c (SEQ ID NO: 157). Preferably, SEQ ID NO: 96 is inserted into hsa-mir-92b (SEQ ID NO: 160). Preferably, SEQ ID NO: 97, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 109 is inserted into hsa-mir-302a (SEQ ID NO: 161, SEQ ID NO: 167, SEQ ID NO: 168, or SEQ ID NO: 173, respectively).Preferably, SEQ ID NO:98, SEQ ID NO:107, SEQ ID NO:108, or SEQ ID NO:111 is inserted into hsa-mir-302c (SEQ ID NO:162, SEQ ID NO:171, SEQ ID NO:172, or SEQ ID NO:175, respectively). Preferably, SEQ ID NO:100 is inserted into hsa-mir-191 (SEQ ID NO:164). Preferably, SEQ ID NO:101 or SEQ ID NO:102 is inserted into hsa-mir-30 (SEQ ID NO:165 or SEQ ID NO:166, respectively). Preferably, SEQ ID NO:105, SEQ ID NO:106, or SEQ ID NO:110 is inserted into hsa-mir-302b (SEQ ID NO:169, SEQ ID NO:170, or SEQ ID NO:174, respectively). Preferably, SEQ ID NO:112, SEQ ID NO:113, or SEQ ID NO:114 is inserted into hsa-mir-375 (SEQ ID NO:176, SEQ ID NO:177, or SEQ ID NO:178, respectively). Preferably, SEQ ID NO:115, SEQ ID NO:119, SEQ ID NO:122 or SEQ ID NO:125 is inserted into hsa-let-7b (SEQ ID NO:179, SEQ ID NO:183, SEQ ID NO:186 or SEQ ID NO:189, respectively). Preferably, SEQ ID NO:116, SEQ ID NO:120, SEQ ID NO:123 or SEQ ID NO:126 is inserted into hsa-let-7g (SEQ ID NO:180, SEQ ID NO:184, SEQ ID NO:187 or SEQ ID NO:190, respectively). Preferably, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124 or SEQ ID NO:127 is inserted into hsa-mir-98 (SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:185, SEQ ID NO:188 or SEQ ID NO:191, respectively).

[0130] In some preferred embodiments, the polynucleotide sequence encoding the inhibitory RNA comprises genomic sequences flanking both sides of a human pre-microRNA. In some preferred embodiments, the genomic sequences flanking both sides of a human pre-microRNA are approximately 40 bp in length. In some preferred embodiments, the genomic sequences flanking both sides of a human pre-microRNA are less than 100 bp, less than 90 bp, less than 80 bp, or less than 70 bp, preferably less than 60 bp or less than 50 bp, 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. Genomic sequences flanking both sides of a 40-base pair length (homology arms) exhibit superior silencing efficacy compared to genomic sequences flanking both ends of a length of 350 bp or 150-350 bp, as shown in FIG. 17 .

[0131] In another embodiment of the invention, the polynucleotide sequence encoding the inhibitory RNA comprises genomic sequences flanking a human pre-microRNA, and optionally the polynucleotide sequence comprises the following: (i) SEQ ID NO: 35 (including SEQ ID NO: 2 or SEQ ID NO: 19), (ii) SEQ ID NO: 36 (including SEQ ID NO: 11 or SEQ ID NO: 28); (iii) SEQ ID NO: 37 (including SEQ ID NO: 3 or SEQ ID NO: 20); (iv) SEQ ID NO: 38 (including SEQ ID NO: 12 or SEQ ID NO: 29), (v) SEQ ID NO: 39 (including SEQ ID NO: 9 or SEQ ID NO: 26), (vi) SEQ ID NO: 40 (including SEQ ID NO: 8 or SEQ ID NO: 25), (vii) SEQ ID NO: 41 (including SEQ ID NO: 10 or SEQ ID NO: 27), (viii) SEQ ID NO: 42 (including SEQ ID NO: 13 or SEQ ID NO: 30); (iv) SEQ ID NO: 43 (including SEQ ID NO: 4 or SEQ ID NO: 21); (x) SEQ ID NO: 44 (including SEQ ID NO: 16 or SEQ ID NO: 33), (xi) SEQ ID NO: 45 (including SEQ ID NO: 5 or SEQ ID NO: 22); (xii) SEQ ID NO: 46 (including SEQ ID NO: 6 or SEQ ID NO: 23), (xiii) SEQ ID NO: 47 (including SEQ ID NO: 1 or SEQ ID NO: 18), (xvi) SEQ ID NO: 48 (including SEQ ID NO: 7 or SEQ ID NO: 24), (xv) SEQ ID NO: 49 (including SEQ ID NO: 15 or SEQ ID NO: 32; (xvi) SEQ ID NO: 50 (including SEQ ID NO: 14 or SEQ ID NO: 31, or (xvii) SEQ ID NO: 51 (including SEQ ID NO: 17 or SEQ ID NO: 34) Includes:

[0132] In a still further embodiment of the invention there is provided an inhibitory RNA or a polynucleotide sequence encoding an inhibitory RNA according to the invention, wherein the inhibitory RNA targets an exon sequence contained in the messenger RNA from the B2M gene, optionally exon 2-1, 2-2 or exon 4, and preferably the inhibitory RNA is (i) SEQ ID NO: 2 or SEQ ID NO: 19, which targets B2M gene exon 2-2; (ii) SEQ ID NO: 11 or SEQ ID NO: 28, which targets B2M gene exon 4; (iii) SEQ ID NO: 3 or SEQ ID NO: 20, which targets B2M gene exon 2-2; (iv) SEQ ID NO: 12 or SEQ ID NO: 29, which targets B2M gene exon 4; (v) SEQ ID NO: 9 or SEQ ID NO: 26, which targets B2M gene exon 4; (vi) SEQ ID NO: 8 or SEQ ID NO: 25, which targets B2M gene exon 4; (vii) SEQ ID NO: 10 or SEQ ID NO: 27, which targets B2M gene exon 4; (viii) SEQ ID NO: 13 or SEQ ID NO: 30, which targets B2M gene exon 4; (iv) SEQ ID NO: 4 or SEQ ID NO: 21, which targets B2M gene exon 2-2; (x) SEQ ID NO: 16 or SEQ ID NO: 33, targeting B2M gene exon 4; (xi) SEQ ID NO: 5 or SEQ ID NO: 22, which targets B2M gene exon 4; (xii) SEQ ID NO: 6 or SEQ ID NO: 23, which targets B2M gene exon 4; (xiii) SEQ ID NO: 1 or SEQ ID NO: 18, which targets B2M gene exon 2-2; (xvi) SEQ ID NO: 7 or SEQ ID NO: 24, which targets B2M gene exon 4; (xv) SEQ ID NO: 15 or SEQ ID NO: 32, which targets B2M gene exon 4; (xvi) SEQ ID NO: 14 or SEQ ID NO: 31, which targets B2M gene exon 4; (xvii) SEQ ID NO: 17 or SEQ ID NO: 34, which targets B2M gene exon 4; (xviii) SEQ ID NOs: 64 to 66 or 128 to 130, which target B2M exon 2-1; (xix) SEQ ID NOs: 67 to 78 or 129 to 142 targeting B2M exon 2-2, and / or (xx) SEQ ID NOs: 79 to 96 or 143 to 160 targeting B2M exon 4 Includes:

[0133] The polynucleotide sequence encoding the inhibitory RNA can be introduced into cells by any method known to those skilled in the art.In some embodiments, the nucleic acid construct adapted to express the inhibitory RNA suitable for inhibiting the expression of target gene is introduced via lentiviral delivery.

[0134] The inhibitory RNAs or polynucleotide sequences encoding the inhibitory RNAs according to the present invention are particularly suitable for treating chronic inflammatory conditions, as they can buffer MHC-I induction by inflammatory stimuli, as shown in Figure 18 and Example 13 below.

[0135] Previous methods utilizing shRNA or siRNA are known to have significant off-target effects, as shown by Rao et al. [Comparative assessment of siRNA and shRNA off-target effects: what is slowing clinical development. 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], which is incorporated herein by reference. Advantageously, in some preferred embodiments, the inhibitory RNA or polynucleotide sequence encoding the inhibitory RNA according to the present invention does not have off-target effects. In some preferred embodiments, the inhibitory RNA or polynucleotide sequence encoding the inhibitory RNA does not silence unintended targets (targets that it was designed to target, e.g., any targets other than B2M). In some preferred embodiments, the inhibitory RNA or the polynucleotide sequence encoding the inhibitory RNA does not exhibit a loss-of-function effect after redirection of the miRNA gene used as a scaffold.

[0136] Expression cassettes and vectors Polynucleotides can be synthesized according to methods well known in the art. Polynucleotide sequences encoding the inhibitory RNA of the present invention can be provided in the form of expression cassettes that contain regulatory sequences operably linked to the inserted sequence, thus allowing for expression of the polypeptide of the present invention in vivo. These expression cassettes are, in turn, usually provided within vectors (e.g., plasmids or recombinant viral vectors). Such expression cassettes may be administered directly to a host subject. Alternatively, a vector containing a polynucleotide of the present invention may be administered to a host subject. Preferably, the polynucleotide is prepared and / or administered using a genetic vector. A suitable vector can be any vector that is capable of carrying a sufficient amount of genetic information and allows for expression of the polynucleotide of the present invention.

[0137] The present invention, in some embodiments, includes such expression vectors containing polynucleotide sequences encoding the inhibitory RNAs of the present invention. Such expression vectors are routinely constructed in the art of molecular biology and may include, for example, the use of plasmid DNA and appropriate initiators, promoters, enhancers, and other elements, such as, for example, polyadenylation signals, that may be necessary to enable expression of the polynucleotides of the present invention and that are positioned in the correct orientation. Other suitable vectors will be apparent to those skilled in the art.

[0138] In some embodiments, the expression cassette may be a synthetic expression cassette. In other embodiments, the expression cassettes disclosed herein comprise a polynucleotide sequence encoding an inhibitory RNA operably linked to one or more regulatory elements, preferably a promoter, optionally a constitutive or inducible promoter, suitable for enabling transcription of the inhibitory RNA in a cell. In other embodiments of the invention, the expression cassette comprises a promoter operably linked to a sequence encoding an expression product, optionally a polynucleotide sequence (e.g., an miRNA or a transgene, e.g., a therapeutic transgene). Alternatively, the expression cassettes disclosed herein may comprise an inducible promoter operably linked to a sequence encoding an expression product, optionally a polynucleotide sequence (e.g., an miRNA or a transgene, e.g., a therapeutic transgene). Preferably, the promoter is EF1 alpha.

[0139] In some embodiments, the expression product is a therapeutic expression product. In some preferred embodiments, the therapeutic expression product is suitable for use in the treatment of a disease or condition. In some embodiments, the disease or condition is an autoimmune disease, optionally type 1 diabetes or cancer. In preferred embodiments, the therapeutic expression product includes one that is useful in the treatment of a disease, preferably type 1 diabetes or cancer.

[0140] Optionally, the expression cassette includes sequences that provide or encode one or more, preferably all, of a ribosome binding site, a start codon, a stop codon, and a transcription termination sequence. Optionally, the expression cassette includes nucleic acid encoding a post-transcriptional regulatory element. Optionally, the expression cassette includes nucleic acid encoding a polyA element.

[0141] Disclosed herein is a vector comprising a polynucleotide sequence or expression cassette encoding an inhibitory RNA according to the present invention. In one embodiment of the present invention, the vector comprises DNA encoding a regulatory element that allows transcription of a non-coding RNA molecule in a cell. The regulatory element can be positioned relative to the expressible nucleotide sequence so that the element can affect its regulatory activity, i.e., the regulatory element is "operably linked." Alternatively, the regulatory element can be a transcriptional regulatory element with enhancer activity, e.g., located adjacent to the promoter and the nucleotide sequence to be transcribed, or at some distance, including upstream and downstream, from them, and still exert a detectable enhancing effect on the level of expression of the encoded reporter molecule. Regulatory elements, including promoters, terminators, enhancers, and silencers for eukaryotes and prokaryotes, are well known in the art and can be chemically synthesized, obtained from naturally occurring nucleic acid molecules, or purchased from commercial sources. In one embodiment of the present invention, the DNA encoding the regulatory element that allows transcription is that of a non-coding RNA, preferably an miRNA, in a cell, preferably a eukaryotic cell, more preferably a mammalian cell, e.g., a human cell or an iPSC cell.

[0142] The vector may be a gene or mRNA therapy vector. In some embodiments of the present invention, the vector is a viral vector, preferably a lentivirus, adenovirus, adeno-associated virus (AAV), or retrovirus vector. The AAV may be selected from the group consisting of AAV2, AAV5, AAV6, AAV7, AAV8, AAV9 BNP116, rh10, AAV2.5, AAV2i8, AAVDJ8, and AAV2G9, or derivatives thereof. The AAV vector may be used as a self-complementary, double-stranded AAV vector (scAAV) to overcome one of the limiting steps in AAV transduction (i.e., the conversion of single-stranded to double-stranded AAV), although the use of a single-stranded AAV vector (ssAAV) is also encompassed herein. The AAV vector may be chimeric, meaning that it contains components from at least two AAV serotypes, for example, the ITRs of AAV2 and the capsid protein of AAV5. In other embodiments of the invention, the vector is a viral vector, preferably an alphavirus, herpesvirus, arenavirus, measles virus, poxvirus, or paramyxovirus vector. In some embodiments of the invention, the vector is a plasmid. Such plasmids may contain a variety of other functional nucleic acid sequences, such as one or more selectable markers, one or more origins of replication, multiple cloning sites, etc. In some embodiments, the plasmid is plasmid VB210602-1567ytv from VectorBuilder.

[0143] In some embodiments of the present invention, the vector is an expression vector for expression in eukaryotic cells. Examples of eukaryotic expression vectors include, but are not limited to, pW-LNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene; pSVK3, pBPV, pMSG, and pSVL available from Amersham Pharmacia Biotech; and pCMVDsRed2-express, pIRES2-DsRed2, pDsRed2-Mito, and pCMV-EGFP available from Clontech. Many other vectors are well known and commercially available. For mammalian adenovirus vectors, the pSV and pCMV series of vectors are particularly well known, non-limiting examples. There are many well-known yeast expression vectors, including, but not limited to, yeast integrating plasmids (Yip) and yeast replicative plasmids (Yrp).

[0144] The present invention further provides a recombinant virion (virus particle) comprising the vector described above.

[0145] Expression cassettes according to aspects of the invention may include polynucleotide sequences encoding two or more inhibitory RNAs according to the invention. The inhibitory RNAs may be inhibitory RNAs disclosed herein or other inhibitory RNAs. In some embodiments, the two or more inhibitory RNAs have the same target (e.g., two or more inhibitory RNAs with the same target, such as B2M). In some embodiments, the two or more inhibitory RNAs may comprise or consist of a sequence selected from SEQ ID NOs: 1-17, 64-127, or a functional variant or fragment thereof. In some embodiments, the two or more inhibitory RNAs may comprise or consist of a sequence selected from SEQ ID NOs: 1-17, or a functional variant or fragment thereof. In some embodiments, the two or more inhibitory RNAs may comprise or consist of a sequence selected from SEQ ID NOs: 64-127. In some embodiments, two or more inhibitory RNAs may comprise or consist of a sequence selected from SEQ ID NOs: 18-34, 128-191, or a functional variant or fragment thereof. In some embodiments, two or more inhibitory RNAs may comprise or consist of a sequence selected from SEQ ID NOs: 18-34. In some embodiments, two or more inhibitory RNAs may comprise or consist of a sequence selected from SEQ ID NOs: 128-191. In some embodiments, two or more inhibitory RNAs have different targets.

[0146] The expression cassettes, vectors and virions according to the invention are particularly suitable for the treatment of chronic inflammatory conditions, in particular ex vivo cell therapy, as they are able to buffer the increase in MHC-I expression in ex vivo therapeutic cells when the cells are exposed to inflammatory stimuli, as shown in Figure 18 and Example 13 below.

[0147] Synthetic microRNA In one embodiment of the present invention, there is provided a synthetic microRNA comprising or consisting of a sequence selected from SEQ ID NOs: 1-17, 64-127, or a functional variant or fragment thereof. In a further embodiment of the present invention, there is provided a synthetic pre-microRNA comprising or consisting of a sequence selected from SEQ ID NOs: 1-17, 64-127, or a functional variant or fragment thereof. The synthetic microRNA or pre-microRNA is a non-naturally occurring or artificial microRNA molecule. According to the present invention, the inhibitory RNA or polynucleotide sequence encoding the inhibitory RNA of SEQ ID NOs: 1-17, 64-127 is a synthetic microRNA that is a non-naturally occurring, artificial sequence. The present invention also provides functional variants or fragments thereof, as described in the "Polynucleotides" section.

[0148] In a further embodiment of the present invention, the synthetic microRNA or synthetic pre-microRNA is an endogenously modified microRNA. An endogenously modified microRNA molecule refers to a molecule in which a natural microRNA molecule originates in a cell and the modification of the microRNA molecule occurs in the cell. Such technologies used to modify endogenous microRNA include, but are not limited to, CRISPR and adenosine deaminase acting on RNA (ADAR).

[0149] In another embodiment of the present invention, there is provided a synthetic microRNA adapted to target messenger RNA transcribed from a gene, preferably the B2M gene. Optionally, the synthetic microRNA comprises a sequence according to any one of SEQ ID NOs: 1-17, 64-127, or a functional variant or fragment thereof, and optionally a sequence according to any one of SEQ ID NOs: 18-34, 128-191, or a functional variant or fragment thereof.

[0150] The synthetic RNAs according to the present invention are particularly suitable for the treatment of chronic inflammatory conditions, in particular ex vivo cell therapy, as they are able to buffer the increase in MHC-I expression in ex vivo therapeutic cells when the cells are exposed to inflammatory stimuli, as shown in Figure 18 and Example 13 below.

[0151] Cells and Cellular Therapy According to the present invention, any type of cell can be used. In one embodiment of the present invention, the cell is a eukaryotic cell, preferably a mammalian cell. The cell can be a stem cell, such as an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), or an adult stem cell.

[0152] Thus, in some embodiments, the cells can be any type of stem cell, preferably an induced pluripotent stem cell (iPSC) or a cell derived via differentiation of a stem cell, e.g., a totipotent or pluripotent stem cell, e.g., an induced pluripotent stem cell. iPSCs can be differentiated into virtually any cell type.

[0153] In certain embodiments, the cells are differentiated cells, including, but not limited to, dendritic cells, lymphocytes, erythrocytes, platelets, hematopoietic cells, pancreatic islet cells, liver cells, muscle cells, keratinocytes, cardiomyocytes, neurons, skeletal muscle cells, ocular cells, mesenchymal cells, fibroblasts, lung cells, gastrointestinal cells, vascular cells, endocrine cells, and adipocytes. In certain other particular embodiments, the present invention provides methods of treating disease states in solid organs.

[0154] In some preferred embodiments, the cells are iPSCs or cells derived from iPSCs.

[0155] In a preferred embodiment of the invention, the cell is a pancreatic cell. In a preferred embodiment of the invention, the cell is a pancreatic beta cell.

[0156] In some preferred embodiments, the cells are bone marrow cells.

[0157] In some preferred embodiments, the cells are hematopoietic stem cells (HSCs).

[0158] 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). Optionally, the cell is a T cell, preferably a cytotoxic T cell, such as a T cell adapted for T cell therapy, e.g., CAR-T cell therapy.

[0159] In some embodiments, the cells are primary T cells, pan T cells, CD3+ T cells, CD3+ and CD4+ T cells, CD8+ T cells, CD4+ helper T cells (optionally Th1, Th2, Th17, Th9, Tfh or Th22 cells), memory T cells, CD4+ Treg cells (optionally FOXP3+ Treg cells or FOXP3- Treg cells).

[0160] In some embodiments, the cell is a macrophage, eg, an M1 macrophage, an M2 macrophage (eg, an M2a, M2b, M2c, and M2d macrophage), a Treg macrophage, or a tumor-associated macrophage (TAM).

[0161] The cells of the invention can be genetically engineered, e.g., transfected with an inhibitory RNA or a polynucleotide sequence encoding an inhibitory RNA, expression cassette, vector, virion, or modified miRNA molecule of the invention. Such transfection can be performed ex vivo, and compositions comprising such transfected cells can then be used in the methods disclosed herein.

[0162] In one embodiment of the present invention, a cell is provided that comprises an expression cassette, vector, virion, synthetic microRNA, or pre-microRNA of the present invention. In another embodiment of the present invention, a cell or cell population obtained from any of the methods of the present invention is provided. In a preferred embodiment, the cell or cell population comprises a modified endogenous miRNA obtained by any of the methods of the present invention.

[0163] In some embodiments, the cells are heterozygous knock-in cell lines for an inhibitory RNA of interest. In some embodiments, the cells are homozygous knock-in cell lines for an inhibitory RNA of interest.

[0164] In another embodiment of the invention, there is provided a cell comprising or expressing a synthetic microRNA according to the invention, wherein the cell has been modified to express a synthetic microRNA such that the synthetic microRNA preferably targets a messenger RNA transcribed from the B2M gene. In a more preferred embodiment, the synthetic microRNA degrades the messenger RNA transcribed from the B2M gene, preferably such that the messenger RNA is partially silenced and B2M protein expression is modified by 1% to 90%, optionally 5% to 90%, optionally 10% to 90% compared to control cells.

[0165] In some preferred embodiments, cells according to aspects of the invention exhibit greater silencing, reduction, or alteration of B2M functional activity when the cells are differentiated into progenitor cells (e.g., iPSC-derived pancreatic cells or iPSC-derived monocytes) compared to when the cells are undifferentiated iPSCs. In some embodiments, the functional activity of B2M in differentiated progenitor cells is silenced, reduced, or altered by at least 10%, 20%, preferably at least 30%, 40%, 50%, more preferably at least 60%, 70%, and most preferably at least 80%, 90%, or 95% compared to when the cells are undifferentiated iPSCs.

[0166] In another embodiment, the cells or compositions are provided for use in a method of treating an autoimmune disease or cancer in a subject, the method comprising administering to the subject a therapeutic amount of the cells or compositions. The autoimmune disease can be any autoimmune disease, preferably type 1 diabetes.

[0167] Autoimmune disorders or cancer are characterized by chronic inflammation in patients, and therefore, any cells or cell compositions transplanted as an intervention will be exposed to inflammatory stimuli. Basal and inducible expression of MHC-I on the cell surface can be affected (upregulated) by inflammatory stimuli. The cells or compositions for use in the method of treating autoimmune diseases or cancer in a subject according to the present invention are particularly suitable for treating chronic inflammatory conditions, particularly ex vivo cell therapy, because they can buffer the increase in MHC-I expression in cells when the cells are exposed to inflammatory stimuli, as shown in Figure 18 and Example 13 below.

[0168] In certain embodiments, the cells administered to the recipient may or may not integrate into an organ requiring such therapy. In certain embodiments, the cells of the present invention differentiate into the desired cell type either before or after transplantation and provide the necessary cellular function without themselves being integrated into tissue at the transplantation site. For example, in certain embodiments for treating diabetes, the cells of the present invention are transplanted into a diabetic patient as pluripotent stem cells or as differentiated pancreatic beta islet cells. In some preferred embodiments for treating diabetes, particularly type 1 diabetes, the cells of the present invention are transplanted into a diabetic patient as differentiated pancreatic beta islet cells (e.g., differentiated from stem cells). The transplanted cells do not need to reconstitute a functioning pancreas, but only need to secrete insulin in response to glucose levels. In certain embodiments, the cells are transplanted into an ectopic location and do not fully integrate into the pancreas. Transplantation of the pluripotent cells of the present invention, the differentiated cells of the present invention, or tissue differentiated and developed ex vivo from the cells of the present invention are all contemplated by the present invention. In certain preferred embodiments, the cells are human cells, and the patient is a human patient.

[0169] The present invention also provides a method of treating an autoimmune disease or cancer in a subject, preferably wherein the autoimmune disease is type I diabetes, the method comprising administering to the subject a therapeutic amount of the cells or composition. Furthermore, the present invention provides use of a cell composition according to the present invention in the manufacture of a medicament for treating an autoimmune disease or cancer in a subject, preferably wherein the autoimmune disease is type I diabetes.

[0170] In a further embodiment of the present invention, a cell is provided containing or expressing a sequence encoding a synthetic inhibitory RNA, preferably a synthetic microRNA, wherein the synthetic inhibitory RNA is adapted to target a gene, preferably the B2M gene. In a preferred embodiment of the present invention, the sequence encoding the synthetic inhibitory RNA, preferably a synthetic microRNA, encodes one or more of SEQ ID NOs: 1-17, 64-127. In an alternative preferred embodiment, the sequence encoding the synthetic microRNA encodes one or more of SEQ ID NOs: 18-34, 128-191. In yet another alternative preferred embodiment, the sequence encoding the synthetic microRNA comprises one or more of SEQ ID NOs: 35-51.

[0171] In one embodiment of the present invention, the sequence encoding the synthetic inhibitory RNA, preferably a microRNA, is genomic or episomal. The term genomic refers to a sequence that has been inserted into the genome, for example, by HDR. Genomic insertion can include a single insertion or multiple insertions (e.g., 2, 3, 4, or more). In this context, "insertion" also includes modifying an endogenous miRNA-encoding sequence to encode the relevant inhibitory RNA, as discussed below. The term episomal refers to a sequence that is introduced into a cell, for example, by vector transduction, and therefore the sequence is not integrated into the genome of the cell; preferably, the sequence is stably maintained in the cell. In a preferred embodiment, the sequence encoding the synthetic inhibitory RNA is genomic.

[0172] In some embodiments of the present invention, a cell comprises a modified genome in which an endogenous miRNA has been modified to redirect it to target B2M. In some embodiments, the genome modification can be homozygous, heterozygous, or hemizygous. In some other embodiments, a cell comprises a modified genome in which an expression cassette has been inserted such that an inhibitory RNA molecule is expressed in the cell. Thus, the present invention contemplates both redirecting a cell's endogenous inhibitory RNA (particularly, miRNA) to target B2M, and inserting a suitable expression construct into the genome to express an inhibitory RNA (particularly, miRNA) that targets B2M. In the case of insertion, the inhibitory RNA (particularly, miRNA) can be a modified form of an inhibitory RNA (particularly, miRNA) found in the cell.

[0173] Thus, in some embodiments of the invention, the sequence encoding the synthetic microRNA is a genomic sequence encoding a modified endogenous microRNA. Optionally, the cell is a human cell, and the modified genomic sequence encodes the following endogenous microRNA: (i) hsa-mir-191, optionally modified to include SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 100; (ii) hsa-mir-302a, optionally modified to include SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:97, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:109; (iii) hsa-mir-302c, optionally modified to include SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:98, SEQ ID NO:107, SEQ ID NO:108, or SEQ ID NO:111; (iv) hsa-mir-93, optionally modified to include SEQ ID NO: 4 or 16; (v) hsa-mir-106a, optionally modified to include SEQ ID NO: 5 or SEQ ID NO: 67; (vi) hsa-mir-106b, optionally modified to include SEQ ID NO: 6; (vii) hsa-mir-200c, optionally modified to include SEQ ID NO: 8; (viii) hsa-mir-20a, optionally modified to include SEQ ID NO: 9; (iv) hsa-mir-21, optionally modified to include SEQ ID NO: 10; (x) hsa-mir-363, optionally modified to include SEQ ID NO: 13; (xi) hsa-mir-518b, optionally modified to include SEQ ID NO: 14 or SEQ ID NO: 76; (xii) hsa-mir-744, optionally modified to include SEQ ID NO: 15; (xiii) hsa-mir-99b, optionally modified to include SEQ ID NO: 17; (xiv) hsa-mir-320a, optionally modified to include SEQ ID NO: 64, SEQ ID NO: 73, or SEQ ID NO: 89; (xv) hsa-mir-520f, optionally modified to include SEQ ID NO: 65, SEQ ID NO: 77, or SEQ ID NO: 94; (xvi) hsa-mir-652, optionally modified to include SEQ ID NO: 66, SEQ ID NO: 78, or SEQ ID NO: 95; (xvii) hsa-mir-1180, optionally modified to include SEQ ID NO: 68 or SEQ ID NO: 79; (xviii) hsa-mir-15b, optionally modified to include SEQ ID NO: 69 or SEQ ID NO: 81; (xix) hsa-mir-182, optionally modified to include SEQ ID NO: 70, SEQ ID NO: 82, or SEQ ID NO: 99; (xx) hsa-mir-23a, optionally modified to include SEQ ID NO: 71 or SEQ ID NO: 87; (xxi) hsa-mir-26b, optionally modified to include SEQ ID NO: 72; (xxii) hsa-mir-335, optionally modified to include SEQ ID NO: 74 or SEQ ID NO: 90; (xxiii) hsa-mir-361, optionally modified to include SEQ ID NO: 75 or SEQ ID NO: 91; (xxiv) hsa-mir-1307, optionally modified to include SEQ ID NO: 80; (xxv) hsa-mir-205, optionally modified to include SEQ ID NO: 83; (xxvi) hsa-mir-22, optionally modified to include SEQ ID NO: 84; (xxvii) hsa-mir-221, optionally modified to include SEQ ID NO: 85; (xxviii) hsa-mir-222, optionally modified to include SEQ ID NO: 86; (xxix) hsa-mir-30e, optionally modified to include SEQ ID NO: 88; (xxx) hsa-mir-423, optionally modified to contain SEQ ID NO: 92; (xxxi) hsa-mir-519c, optionally modified to include SEQ ID NO: 93; (xxxii) hsa-mir-92b, optionally modified to include SEQ ID NO: 96; (xxxiii) hsa-mir-30, optionally modified to include SEQ ID NO: 101 or SEQ ID NO: 102; (xxxiv) hsa-mir-302b, optionally modified to include SEQ ID NO: 105, SEQ ID NO: 106, or SEQ ID NO: 110; (xxxv) hsa-mir-375, optionally modified to include SEQ ID NO: 112, SEQ ID NO: 113, or SEQ ID NO: 114; (xxxvi) hsa-let-7b, optionally modified to include SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 122, or SEQ ID NO: 125; (xxxvii) hsa-let-7g, optionally modified to include SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 123, or SEQ ID NO: 126, and (xxxviii) hsa-mir-98, optionally modified to include SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 121, SEQ ID NO: 124, or SEQ ID NO: 127. The present invention encodes a modified form of one of the following:

[0174] In preferred embodiments of the present invention, the cells exhibit reduced protein expression, preferably B2M expression. Reduced protein expression means that the level of protein expression is less than that seen under normal conditions. Preferably, reduced protein expression means that the protein is not completely eliminated. In preferred embodiments of the present invention, B2M protein expression is not completely eliminated; preferably, B2M is expressed at a level of 1% to 90%, optionally 5% to 90%, and optionally 10% to 90% compared to control cells. In preferred embodiments, B2M is expressed at a level of 30% to 95% compared to control cells. In preferred embodiments, B2M is expressed at a level of 65% to 95% compared to control cells. In preferred embodiments, B2M is expressed at a level of 65% to 89% compared to control cells. In some embodiments, the control cells are the control parent line (unedited). In some embodiments, the control cells are cells in which endogenous miRNAs are deleted or disrupted in one or both alleles.

[0175] In cells of the invention, expression of a protein (preferably B2M) may be altered, degraded, reduced or silenced in accordance with the invention. In one embodiment of any aspect of the invention, protein expression is altered within a range of 1% to 90%, optionally 5% to 90%, optionally 10% to 90%. Optionally, for example, protein expression may be 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, expression of a protein (e.g., B2M) by a desired amount compared to control cells, e.g., adjust expression to a suitable level. In some embodiments, the expression level of B2M or any other cell surface protein in a cell is determined by the amount of B2M expressed on the surface of the cell. The amount of B2M present on the surface of a cell can be determined by a variety of conventional techniques, for example, by flow cytometry.

[0176] In a preferred embodiment of the invention, the cells exhibit reduced B2M protein expression. In a preferred embodiment of the invention, the cells exhibit reduced B2M protein expression, which leads to reduced HLA and / or MHC-I protein expression at the cell surface.

[0177] In another preferred embodiment of the present invention, the cells are detargeted by the innate and adaptive immune systems, so that an immune response against the cells is not mounted.

[0178] In some preferred embodiments, the cells escape the innate immune system. In some preferred embodiments, the cells escape natural killer cell-mediated immune system activation. In some preferred embodiments, the cells are subject to lower natural killer cell-mediated cytotoxicity compared to control cells (e.g., the parental cell line (unedited) or a B2M KO cell line (in one or both alleles) or a cancer cell line). In some preferred embodiments, the cells experience reduced specific lysis by natural killer cells compared to control cells (e.g., the parental cell line (unedited) or a B2M KO cell line (in one or both alleles) or a cancer cell line). In some preferred embodiments, the cells express lower levels of B2M protein (and therefore lower levels of MHC-I protein cell surface expression) compared to control cells (e.g., the parental cell line (unedited) or a cell in which endogenous miRNAs have been deleted or disrupted in one or both alleles). Partial knockdown of B2M (partial silencing of MHC-I) was shown to help cells escape the innate (NK-mediated) immune system (reduced NK-specific lysis).

[0179] In a further embodiment of the present invention, there is provided a composition comprising the cells or cell populations of the present invention, which are optionally induced pluripotent stem cells or cells derived by differentiation of induced pluripotent stem cells (e.g., pancreatic beta cells). Such compositions may be sterile compositions suitable for parenteral administration.

[0180] Also provided are inhibitory RNAs or polynucleotides encoding inhibitory RNAs, expression cassettes, vectors, virions, synthetic microRNAs or pre-microRNAs, cells, and optionally induced pluripotent stem cells or cells or compositions derived therefrom for use in therapy.

[0181] In another embodiment of the present invention, there is provided an inhibitory RNA or a polynucleotide encoding an inhibitory RNA, expression cassette, vector, virion, synthetic microRNA or pre-microRNA, cell, optionally induced pluripotent stem cell or composition for use in a method of treating an autoimmune disease or cancer in a subject, wherein the autoimmune disease is preferably type 1 diabetes.

[0182] In a further embodiment of the present invention, an inhibitory RNA or a polynucleotide encoding an inhibitory RNA, an expression cassette, a vector, a virion, a synthetic microRNA, or a pre-microRNA is provided for use in a method for degrading messenger RNA transcribed from the B2M gene in a cell, wherein preferably the messenger RNA is partially silenced and B2M protein expression is altered by 1% to 90%, optionally 5% to 90%, and optionally 10% to 90% of expression compared to control cells. Optionally, the cells are then transferred into a mammal, preferably a human, to treat an autoimmune disease or cancer. The autoimmune disease may be any autoimmune disease, preferably type 1 diabetes.

[0183] The cells of the present invention can be differentiated into a variety of cell types of all three lineages, including, but not limited to, hematopoietic cells, mesenchymal cells, pancreatic endoderm cells, cardiac cells, and keratinocyte cells. Generally, each cell type can be analyzed for reactivity with human T cells and NK cells, appropriate differentiation markers, and xenotransplantation in immunodeficient mice to determine its potential for in vivo development. A brief discussion of each differentiated cell type follows.

[0184] In certain embodiments, the cells of the present invention can be differentiated into hematopoietic stem cells or blood cell types derived from hematopoietic stem cells to treat various hematopoietic disorders currently treated by bone marrow transplantation. Patients receiving blood transfusions may become refractory to platelet transfusions due to HLA mismatches. Patients with cytopenia (neutropenia, thrombocytopenia) can be treated by delivering platelet or neutrophil cells derived from the present invention to treat bleeding or infection.

[0185] Furthermore, dendritic cells derived from the stem cells of the present invention are antigen-presenting cells that can be used as cellular vaccines when appropriately engineered. Dendritic cells derived from the donor are recognized as foreign by the direct pathway, while recipient T cells recognize intact donor MHC antigens on the donor cells. In certain embodiments, dendritic cells of the present invention are engineered to express a single-chain fusion HLA class I protein and a unique peptide antigen and used to vaccinate against a specific pathogen or tumor antigen.

[0186] To obtain hematopoietic cells, pluripotent cells are first allowed to form embryoid bodies, and then nonadherent cells are cultured in the presence of hematopoietic cytokines to develop specific cell lineages. Hematopoietic cell differentiation can be analyzed by flow cytometry and colony assays. Different cell populations are sorted based on their surface markers and used to monitor HLA gene expression and reactivity with human NK cells and T cells, as measured by Elispot, mixed lymphocyte reaction, and cytotoxicity assays. The effectiveness of suppressing NK cell-mediated killing can be examined at different stages of differentiation and engraftment. See Bix et al., 1991, Nature 349, pp. 329-331. Hematopoietic stem cells can also be assayed using xenotransplantation models, for example, in immunodeficient mice (SCID-repopulating cells or SRC).

[0073] The cells of the present invention can be differentiated into hematopoietic cells before or after the cells are administered to a patient. In certain preferred embodiments, the cells are human cells, and the patient is human. In vitro hematopoietic differentiation can be performed according to established protocols, see, for example, Slukvin et al., 2006, J Immunol 776:2924-32 and Chang et al., 2006, Blood 108:1515-23.

[0187] In certain other embodiments, the pluripotent cells of the present invention can be differentiated into mesenchymal stem cells. MSCs have the potential to form several differentiated cell types, including bone marrow stromal cells, adipocytes, osteoblasts, and chondrocytes. Therefore, inducing pluripotent stem cells to form MSCs (iMSCs) is useful in treating skeletal and joint conditions. iMSCs can further differentiate into osteoblasts in vivo to form bone. Deyle et al., 2012, Mol Ther. 20(1):204-13. Cellular responses of T cells and NK cells to ESCs, iMSCs, and their more differentiated derivatives, such as osteoblasts, can be examined.

[0188] In certain embodiments, mesenchymal stem cells can be differentiated into non-limiting examples of cell types, such as bone marrow stromal cells, adipocytes, osteoblasts, osteocytes, and chondrocytes. The cells of the present invention are differentiated into mesenchymal stem cells before or after the cells are administered to a patient. In certain preferred embodiments, the cells are human cells and the patient is human. In vitro mesenchymal differentiation can be performed according to established protocols. See, e.g., Deyle et al.

[0189] In yet another specific embodiment, pluripotent cells can be differentiated into insulin-producing pancreatic islet cells. The cells of the present invention can be used to treat insulin-dependent diabetes. Advantageously, the transplanted cells do not need to reconstitute a functioning pancreas. Rather, they only need to secrete insulin in response to glucose levels. Thus, treatment can be successful using different cell doses, using cells that have not fully differentiated into adult cell types, or even when the cells are transplanted into an ectopic location. Certain autoantigens, such as those derived from GAD65 or insulin, can cause autoimmune destruction of beta cells in diabetes (Di Lorenzo et al., 2007, Clin Exp Immunol 148, pp. 1-16). The cells of the present invention can be differentiated into pancreatic cells as previously described, using exposure of the cells to various cytokines and drugs that promote the sequential formation of mesendoderm, definitive endoderm, and pancreatic progenitor cells (Kroon et al., 2008, Nat Biotechnol 26, pp. 443-452). These cells can be further cultured in grafts in immunodeficient mice. The cells of the present invention can be analyzed at various developmental stages for their reactivity with T cells and NK cells. The cells of the present invention can be differentiated into pancreatic islet cells before or after administration to a patient. In certain preferred embodiments, the cells are human cells and the patient is human.

[0190] In certain other specific embodiments, the cells can be differentiated into cardiomyocytes. The common clinical problems of myocardial infarction and congestive heart failure can be treated by transplanting healthy cardiomyocytes, which engraft and re-establish a functioning myocardium. The cardiomyocytes of the present invention allow these treatments to proceed using pre-packaged cells, avoiding the immunosuppression currently required for allogeneic heart transplants. Physiologically relevant tests, such as electrical conduction and contraction studies, can be performed on cardiomyocytes derived from the cells of the present invention. The cells of the present invention can be differentiated into cardiomyocytes before or after the cells are administered to a patient. In certain embodiments, the cells of the present invention are differentiated into cardiomyocytes to treat diseases, including, but not limited to, myocardial infarction and congestive heart failure. In vitro cardiomyocyte differentiation can be performed according to established protocols. See, for example, Lafiamme et al., 2007, Nat Biotechnol 25, 1015-1024.

[0191] In yet other specific embodiments, the pluripotent cells can be differentiated into keratinocytes. Severe burns and genetic skin conditions require treatment using skin transplants, which are currently performed using various cell sources, such as pig skin grafts and cultured autologous human keratinocytes. The keratinocytes of the present invention could potentially provide a major clinical advancement by enabling burns to be treated as an emergency using prepackaged cells and by treating genetic diseases such as epidermolysis bullosa with "normal" cells that do not require correction of the underlying genetic mutation. In many cases, the cells only need to engraft long enough for adjacent host cells to repopulate the affected area. For in vivo differentiation, the cells of the present invention can be embedded in a polyvinyl alcohol sponge (PVA)-collagen gel implant for transplantation into a recipient. The cells of the present invention can be differentiated into keratinocytes either before or after transplantation. In certain preferred embodiments, the cells are human cells and the patient is human.

[0192] Other cell types or therapeutic applications will be apparent to those skilled in the art.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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).

[0203] method In one embodiment of the present invention, a method is provided for modifying the expression of a protein, optionally B2M, in a cell. Further provided is a method for modifying the translation of an RNA transcript derived from a protein-coding gene, optionally B2M, in a cell. Optionally, the method comprises introducing into the cell an inhibitory RNA or a polynucleotide encoding the inhibitory RNA, an expression cassette, a vector, a virion, or a synthetic microRNA or pre-microRNA of the invention. Introduction into the cell can be by any suitable method, for example, transfection.

[0204] In another embodiment of the invention, there is provided a method for altering the translation of messenger RNA transcripts derived from protein-coding genes and the subsequent expression of proteins in a cell, comprising introducing into the cell an inhibitory RNA or a polynucleotide encoding an inhibitory RNA, an expression cassette, a vector, a virion, or a synthetic microRNA or pre-microRNA of the invention.

[0205] In yet another embodiment of the present invention, there is provided a method of modifying (preferably reducing) the translation of a messenger RNA transcript derived from a protein-coding gene (preferably the B2M gene) and the subsequent expression of the protein (preferably B2M) in a cell, comprising introducing into the cell a DNA editing agent that confers silencing specificity of a microRNA for a target RNA of interest, wherein the sequence of the microRNA comprises any one of SEQ ID NOs: 1-17, 64-127 (or a variant or fragment thereof as discussed above), whereby the microRNA is modified such that translation of the messenger RNA transcript into a protein molecule is modified.

[0206] In other aspects of the invention, methods employ gene editing techniques that utilize 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).

[0207] 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.

[0208] Thus, there is provided a method of modifying the translation of an RNA transcript derived from a protein-coding gene, optionally 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 any one of SEQ ID NOs: 1-17, 64-127, whereby the non-coding RNA molecule is modified so as to modify translation of the RNA transcript into a protein molecule.

[0209] According to certain embodiments of the invention, the cell is a eukaryotic cell, preferably a mammalian cell. According to other certain embodiments of the invention, the cell is an induced pluripotent stem cell or a cell derived from the differentiation of an induced pluripotent stem cell. Preferably, the cell is a pancreatic beta cell. Alternatively, it may be preferred that the cell is an immune cell.

[0210] In one embodiment, the cell is a human cell and the method includes the steps 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-182, hsa-mir-206b, hsa-mir-208c, 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-182, hsa-mir-206c, hsa-mir-208d, hsa-mir-208e, hsa-mir-208f ... The method includes editing the sequence of an endogenous microRNA selected from the group consisting of a-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, hsa-let-7g, and hsa-mir-98.

[0211] In one embodiment of the invention, the protein is B2M, or a messenger RNA transcript derived from a protein-coding gene encodes the B2M protein. In another embodiment of the invention, expression of the protein in the cell is altered by 1% to 90%, optionally 5% to 90%, optionally 10% to 90%, compared to a control cell.

[0212] Protein expression (preferably B2M) may be altered, degraded, reduced, or silenced in accordance with the present invention. In one embodiment of any aspect of the present invention, protein expression is altered within 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% relative 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 expressed on the surface of the cell. The amount of B2M present on the surface of the cell can be determined by various conventional techniques, e.g., flow cytometry.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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).

[0220] 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).

[0221] 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.

[0222] 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.

[0223] 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.).

[0224] 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) nucleotide sequences complementary to regions of the target nucleic acid that flank 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.

[0225] 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 showed 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).

[0226] It has further been demonstrated that synthetic chimeric guide RNAs (sgRNAs) composed of fusions between crRNA and tracrRNA can direct Cas9 to cleave DNA targets complementary to the crRNA in vitro. Transient expression of Cas9 together with synthetic sgRNAs has been demonstrated to be able to produce targeted double-strand breaks (DSBs) in a variety of different species (Cho et al., 2013; Cong et al., 2013; DiCarlo et al., 2013; Hwang et al., 2013a, b; Jinek et al., 2013; Mali et al., 2013).

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] 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). He has historically been classified according to 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-metallo) 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.

[0235] Modified versions of CRISPR, such as dead CRISPR (dCRISPR-endonuclease), can also be used to inhibit CRISPR transcription (CRISPRi) or activate CRISPR transcription (CRISPRa).

[0236] 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.

[0237] 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.

[0238] 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.

[0239] 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 usually 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.

[0240] Aspects of the present invention are demonstrated by the following non-limiting examples. [Example]

[0241] 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).

[0242] 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 the 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, and programmable gene silencing.

[0243] 1. Stability. While siRNA oligos are inherently transient, shRNAs are variably expressed by transgenes that are inactivated by epigenetic mechanisms. In contrast, endogenous gene loci are never unexpectedly silenced, and thus GeiGS® silencing RNAs (sRNAs) are stably expressed.

[0244] 2. Tunability. GEiGS® allows for the redirection of miRNA scaffolds with different levels of expression, thus allowing the abundance of GEiGS® sRNA to be tailored according to cell engineering requirements. This is notoriously difficult to achieve with current RNAi approaches and is not possible with CRISPR KO, which is binary in nature.

[0245] 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 have reached a specific stage in differentiation.

[0246] 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.

[0247] 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.

[0248] The experimentally validated GEiGS® solution 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.

[0249] Example 1 Computational Design of GEiGS Solutions The B2M solution was generated using a computational pipeline with the following inputs: B2M mRNA sequence (GenBank accession: NM_004048.4), human genome reference sequence (GRCh38), miRNA annotation (miRbase v22), and cell type-specific miRNA quantification.

[0250] miRNA expression in human iPSCs and ESCs was quantified using publicly available next-generation sequencing (NGS) datasets: ENCODE accession ENCSR958UOC, ENCSR430YFL.

[0251] 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.

[0252] 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.

[0253] 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 computationally designed GEiGS solutions 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 Drosha enzyme.

[0254] The solutions tested were:

[0255] [Table 1A]

[0256] [Table 1B]

[0257] The sequence of the expanded solution was as follows [SEQ ID NO]: >hsa-mir-302a_11_B2M-exon2-2

[0258] [ka]

[0259] >hsa-mir-302a_35_B2M-exon4

[0260] [ka]

[0261] >hsa-mir-302c_12_B2M-exon2-2

[0262] [ka]

[0263] >hsa-mir-302c_36_B2M-exon4

[0264] [ka]

[0265] >hsa-mir-20a_29_B2M-exon4

[0266] [ka]

[0267] hsa-mir-200c_27_B2M-exon4

[0268]

change

[0269] hsa-mir-21_30_B2M-exon4

[0270]

change

[0271] hsa-mir-363_41_B2M-exon4

[0272]

change

[0273] hsa-mir-93_19_B2M-exon2-2

[0274]

change

[0275] hsa-mir-93_49_B2M-exon4

[0276]

change

[0277] hsa-mir-106a_20_B2M-exon4

[0278]

change

[0279] hsa-mir-106b_21_B2M-exon4

[0280]

change

[0281] hsa-mir-191_8_B2M-exon2-2

[0282]

change

[0283] hsa-mir-191_26_B2M-exon4

[0284]

change

[0285] hsa-mir-744_47_B2M-exon4

[0286]

change

[0287] hsa-mir-518b_43_B2M-exon4

[0288]

change

[0289] hsa-mir-99b_50_B2M-exon4

[0290]

change

[0291] >hsa-mir-302a_23_None (negative control)

[0292] [ka]

[0293] >hsa-mir-200c_13_None (negative control)

[0294] [ka]

[0295] In addition to the negative control constructs listed in Table 1 above, additional miR30-based constructs were purchased from Vector Builder (Neg Ctrl 0).

[0296] 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.

[0297] The cloned solutions were individually transfected into the human iPSC line WTC-11 (Allen Institute of Cell Science) by nucleofection (Lonza). These are undifferentiated iPSCs. 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.

[0298] 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.

[0299] 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.

[0300] 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:

[0301] Gate on all cells expressing DsRed (when compared to untransfected controls) and then extract the mean B2M fluorescence intensity in this subpopulation.

[0302] 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.

[0303] 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.

[0304] 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.

[0305] Example 3 Gene editing of endogenous miRNA scaffolds in desired cell types Solutions 8, 20, 29, and 30 (see Table 1) 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:

[0306] [Table 2A]

[0307] [Table 2B]

[0308] The Sol-30 gRNA and HDR template sequences are shown in Table 3.

[0309] 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.

[0310] 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.

[0311] 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).

[0312] Example 4 Gene editing of endogenous miRNA scaffolds in primary human T lymphocytes Solution 30 (see Table 3) is performed as a complete GEiGS, i.e., by editing the endogenous locus of each miRNA scaffold. Several stimulus-induced miRNAs can 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). Gene editing is 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). The following gRNA and HDR template sequences are used:

[0313] [Table 3]

[0314] 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 primary human T cells using electroporation. The method used in Roth et al. (2018) is recommended for use with fresh cells, bulk T cells or subpopulations sorted by fluorescence-activated cell sorter (FACS), and cells from whole blood or leukapheresis, but can also be used with cells recovered from cryopreservation.

[0315] Immediately after isolation, 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 -1 The cells are stimulated for 2 days with a cytokine cocktail of IL-15, followed by electroporation according to standard methods (see, for example, Roth et al.).

[0316] After electroporation, T cells were incubated at 500U ml -1 Culture the T cells in medium with 100 μg of IL-2. Maintain the T cells at an approximate density of 1 million cells per ml of medium throughout the culture period. Every 2–3 days after electroporation, add additional medium with additional fresh IL-2 to a final concentration of 500 U ml -1 The cells are cultured in a medium containing 1000 ml of PBS and transferred to a larger culture vessel as needed to maintain a density of 1 million cells per ml.

[0317] Five days after transfection, GEiGS-modified cells are analyzed by flow cytometry to assess the level of B2M expression. Cells are also analyzed by PCR, Sanger sequencing, and NGS amplicon sequencing to assess the efficiency of gene editing. At this stage, B2M-low cells can be FACS-sorted to enrich for the fraction of GEiGS-modified cells.

[0318] Example 4.1 Gene editing of endogenous miRNA scaffolds in primary human T lymphocytes Solution 30 (see Example 2 and Table 3) 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).

[0319] 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).

[0320] 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.

[0321] The gRNA in Solution 30 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. Results show a reduction in surface B2M expression (Figure 4). Within the CD3+, mixed T cell population, approximately 24% of cells exhibited B2M silencing (Figure 11a), 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 showed a similar reduction in B2M surface expression (Figure 11b).

[0322] 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 (CTLs) 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 CTLs and NK cells are conveniently isolated from commercial suppliers of peripheral blood mononuclear cells (PBMCs).

[0323] 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.

[0324] 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.

[0325] Thus, we demonstrate that efficient, dose-dependent reduction of HLA surface expression on human cells results in enhanced resistance to alloreactive T lymphocyte-mediated cytotoxicity while avoiding non-MHC-restricted killing.

[0326] Example 6 Demonstration of escape from NK cell-mediated immune responses in culture for clonal GEiGS engineered cell lines Natural killer (NK) cells recognize and kill cells that lack surface expression of MHC-I ("loss of self"), and an NK cell assay was set up to determine how their killing activity against GEiGS-modified iPSCs was modulated by B2M silencing.

[0327] Previously isolated CD56+ NK cells were thawed and stimulated to expand with IL-2 / IL-12, IL-2 alone, or no cytokine. On the day of the assay, NK cells were counted and stained using the CellTrace™ CFSE Cell Proliferation Kit. Target cells included K562 cells as a positive control for cell killing. The tumor cell line K562 is known to be a potent stimulator of activated NK cell-mediated killing and therefore serves as a control for assessing the extent of activated NK cell-mediated killing (Zamai L, AR Mariani, G Zauli, L. Rodella, R. Rezzani, FA Manzoli and M Vitale. Kinetics of in vitro natural killer activity against K562 cells as detected by flow cytometry. 1998. Cytometry 32(4):280-285. DOI: 10.1002 / (sici)1097-0320(19980801)32:4<280::aid-cyto4>3.0.co;2-m).

[0328] NK cells were activated with the cytokines IL-2 or IL-12 or a mixture of IL-2 and IL-12, and then the cells were mixed at a defined ratio (usually 5:1 or 1:1). Control (unmodified) iPSCs, B2M - / - iPSCs and GEiGS-modified iPSCs were stained with Zombie NIR. Zombie staining allows for differentiation between cells that died before the start of the killing assay, as opposed to cells that died during the course of the assay. Target cells were typically seeded in 96-well plates at a density of 40,000 cells / well.

[0329] NK cells were then added to the wells at varying ratios to target cells. Typical ratios evaluated were 5 (NK):1 (target), 2.5:1, or 1:1. An extra well of target cells was included, from which maximal, spontaneous release could be calculated at the end of the assay. Plates were then incubated at 37°C, 5% CO2 for a defined period (4-48 hours).

[0330] After incubation, maximum release was determined by lysing the samples with 20% Triton-X. All wells were then stained with propidium iodide (PI). Once staining was complete, the plates were run on a flow cytometer. CFSE staining allowed us to identify NK cells, Zombie NIR-stained cells that died before the killing assay, and PI-stained cells that died during the killing assay. Spontaneous release values ​​were subtracted from PI values, and the remaining amount represented the percentage of target cells killed by NK cells.

[0331] Specific lysis of the K562 cell line was observed in the presence or absence of cytokine stimulation as a positive control (Figure 12). Control iPSCs showed reduced levels of specific lysis (approximately 50% of the maximal level of cell killing observed for K562 cells), but as expected, undifferentiated B2M iPSCs showed reduced levels of specific lysis (approximately 50% of the maximal level of cell killing observed for K562 cells). - / - iPSCs showed elevated levels of specific lysis (Figure 12). This is because the complete absence of B2M leads to the complete absence of MHC-I (a "loss of self" phenotype), allowing these cells to be effectively detected and lysed by NK cells. Undifferentiated, GEiGS-modified iPSC lines (iPSCs S29 / 29 ) do not quantitatively express the same levels of B2M as the control iPSC lines from which they were derived (i.e., they express lower levels of B2M and therefore lower levels of MHC-I cell surface expression), and therefore, across all mixed culture conditions tested, B2M - / - The results showed consistently reduced specific lysis by NK cells compared to cell lines.

[0332] This suggests that partial knockdown of B2M results in partial reduction of MHC-I cell surface expression, which in turn reduces the expression of GEiGS-modified iPSC lines (iPSCs) by activated NK cells in tissue culture. S29 / 29 ) demonstrate that B2M allows cells to escape the innate (NK-mediated) immune system, as indicated by reduced specific lysis of cells. Thus, partial knockdown of B2M results in enhanced cell survival after exposure to cells of the innate (NK-mediated) immune system.

[0333] Example 7 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

[0334] [Table 4]

[0335] 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:

[0336] [Table 5]

[0337] >hsa-mir-191_8_B2M-exon2-2

[0338] [ka]

[0339] >hsa-mir-302c_12_B2M-exon2-2

[0340] [ka]

[0341] >hsa-mir-106a_20_B2M-exon4

[0342] [ka]

[0343] >hsa-mir-20a_29_B2M-exon4

[0344] [ka]

[0345] >hsa-mir-21_30_B2M-exon4

[0346] [ka]

[0347] >hsa-mir-518b_43_B2M-exon4

[0348] [ka]

[0349] Example 8 Demonstration of B2M silencing in pancreatic progenitors identified in culture from clonal GEiGS-modified iPSCs Solution 30 (see Tables 3 and 4) was implemented as a full GEiGS to demonstrate that, as in the case of iPSC-derived pancreatic cells, B2M silencing can be programmed to be deployed only once therapeutic cells reach a specific stage of differentiation. Because this miRNA scaffold is known to be developmentally regulated and expressed in a cell-state-specific manner in pancreatic cells (see also Jin W et al., 2019, Figure 13), the endogenous locus of each miRNA (hsa-mir-21) is modified (as described in Example 3).

[0350] 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 (see, e.g., Figures 14, 15A, and 15B) show that a significant proportion of specific progenitor cells were positive for both pancreatic cell markers, and the majority of PDX-1-positive cells also stained for B2M.

[0351] 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.

[0352] Example 9 Differentiation of the GEiGS Solution 30 knock-in iPSC line into definitive endoderm and pancreatic lineage progenitor cells Homozygous Solution 30 knock-in iPSC lines (miR-21 S30 / +1 ) were differentiated into definitive endoderm and pancreatic lineage cells. The methodology for identifying pancreatic cells from pluripotent stem cells was adapted from Balboa et al., Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells. Nat Biotechnol. 2022 Jul;40(7):1042-1055. Doi: 10.1038 / s41587-022-01219-z. Epub 2022 Mar 3. PMID: 35241836; PMCID: PMC9287162.

[0353] There are many different published protocols describing the identification of pancreatic lineage cells from iPSCs, and those skilled in the art will be aware of other methods for identifying pancreatic cells from pluripotent stem cells. See, for example, the references provided below. 1: Damour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol. 2005 Dec;23(12):1534-41. Doi: 10.1038 / nbt1163. Epub 2005 Oct 28. PMID: 16258519. 2: Schulz TC, Young HY, Agulnick AD, Babin MJ, Baetge EE, Bang AG, Bhoumik A, Cepa I, Cesario RM, Haakmeester C, Kadoya K, Kelly JR, Kerr J, Martinson LA, McLean AB, Moorman MA, Payne JK, Richardson M, Ross KG, Sherrer ES, Song X, Wilson AZ, Brandon EP , Green CE , Kroon EJ , Kelly G , DAmour KA , Robins AJ . A scalable system for production of functional pancreatic progenitors from human embryonic stem cells. PloS One. 2012;7(5):e37004. Doi: 10.1371 / journal.pone.0037004. Epub 2012 May 18. PMID: 22623968; PMCID: PMC3356395. 3: Kumar M, Jordan N, Melton D, Grapin-Botton A. Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate. Dev Biol. 2003 Jan 1;259(1):109~22. Doi: 10.1016 / s0012-1606(03)00183-0. PMID: 12812792. 4: Melton D. The promise of stem cell-derived islet replacement therapy. Diabetologia. May 2021;64(5):1030~1036. Doi: 10.1007 / s00125-020-05367-2. Epub 2021 Jan 16. PMID: 33454830; PMCID: PMC8012315. 5: Peterson QP, Veres A, Chen L, Slama MQ, Kenty JHR, Hassoun S, Brown MR, Dou H, Duffy CD, Zhou Q, Matveyenko AV, Tyrberg B, Soerhede-Winzell M, Rorsman P, Melton DA. A method for the generation of human stem cell-derived alpha cells. Nat Commun. May 7, 2020;11(1):2241. Doi: 10.1038 / s41467-020-16049-3. PMID: 32382023; PMCID: PMC7205884. 6: Pagliuca FW, Millman JR, Gurtler M, Segel M, Van Dervort A, Ryu JH, Peterson QP, Greiner D, Melton DA. Generation of functional human pancreatic β cells in vitro. Cell. 2014 Oct 9;159(2):428-39. Doi: 10.1016 / j.cell.2014.09.040. PMID: 25303535; PMCID: PMC4617632.

[0354] In vitro differentiation of pancreatic cells from iPSCs reproducibly proceeds through a four-step process: definitive endoderm, primitive gut tube, posterior foregut endoderm, and pancreatic progenitor cells. Specific progenitor cells generated after 14 days in continuous culture were isolated and assessed by flow cytometry for staining levels for pancreatic progenitor markers (PDX-1, NKX6.1) and co-staining for B2M (MHC-I).

[0355] It has been demonstrated that Solution 30 miRNA (hsa-mir-21) is expressed at low levels in undifferentiated iPSCs and is upregulated in differentiated cells, particularly pancreatic cells, compared to undifferentiated iPSCs (see Example 8, Figure 13). Therefore, Solution 30 is predicted to be expressed at higher levels in differentiated cells, which would lead to decreased B2M expression compared to control (undifferentiated) iPSC lines.

[0356] Heterozygous Solution 30 knock-in clones were generated and differentiated into definitive endoderm and subsequently into pancreatic progenitor cells. The iPSC clonal line (S30-DP1-F12) is a heterozygous compound genotyped with Solution 30 knocked in one allele and a single-base insertion in the other allele that does not disrupt the transcription of the pre-miRNA or its scaffolding secondary structure. The iPSC line (miR-21) S30 / +1 ) were differentiated for 4 days using established methodologies to identify definitive endoderm (detailed in Balboa D et al., 2022), and the percentage of cells expressing an early marker (CXCR4) was assessed by flow cytometry.

[0357] Heterozygous knock-in line (iPSC miR-21 S30 / +1 ) are the control (unmodified, 50%) and B2M - / - Both iPSCs and iPSCs showed a high percentage of CXCR4-positive cells (approximately 97%) compared to iPSCs (95%) (Figure 14). To confirm that differentiated cells expressed reduced amounts of B2M, the percentage of CXCR4-positive cells was assessed for their B2M expression levels. S30 / +1 The lines showed approximately 80% reduction in B2M expression compared to the control (unmodified) iPSC cell line (FIG. 15a). As expected, B2M− / − iPSCs do not express B2M protein.

[0358] The iPSC lines were further differentiated in culture for 7-10 days to a stage where the definitive endoderm cells express the pancreatic lineage marker, PDX-1. The pancreatic and duodenal homeobox-1 (PDX-1) marker gene is required for pancreatic development and beta cell maturation.

[0359] The results show that a significant proportion of specific progenitor cells at this late stage of development are positive for PDX-1, and the majority of PDX-1-positive cells also stain for B2M (Fig. 15b). S30 / +1 The clonal lines showed a high percentage of PDX-1-expressing cells, which had significantly reduced B2M expression compared to control (unmodified) iPSCs. sRNA from Solution 30 was expressed monoallelically.

[0360] These results demonstrate that B2M expression can be reduced in a differentiation-dependent manner using the GEiGS-based solutions of the present invention. Furthermore, heterozygous knock-in of such solutions is sufficient to achieve significant and sustained knockdown. SEQ ID NOs: 1 or 18 (Solution 8), 5 or 22 (Solution 20), and 9 or 26 (Solution 29) are all expected to produce effects similar to those observed with Solution 30.

[0361] Example 10 Demonstration of B2M silencing in monocytes identified in culture from clonal GEiGS-modified iPSCs Solution 30, implemented as a full GEiGS (see Tables 3 and 4), 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 hsa-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).

[0362] 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, 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.

[0363] Monocytes were generated over a 3-4 week period from an isogenic control parental line (parental control WT / WT line) and a homozygous (KI / KI) clone for Solution 30 (S30 / S30 genotype) 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.

[0364] 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 16a). More than 90% of the cells were double-positive for the known monocyte markers CD14 and CD45. iPSC lines (GEiGS Solution 30 and 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 16b).

[0365] Example 11 Additional GEiGS Solutions A further B2M GEiGS solution was designed as shown in Example 1 and detailed in Table 6. As before, the pipeline input was the following: B2M mRNA sequence (GenBank accession: NM_004048.4), human genome reference sequence (GRCh38), miRNA annotation (miRbase v22), and cell type-specific miRNA quantification.

[0366] miRNA expression in human iPSCs and ESCs was quantified using publicly available next-generation sequencing (NGS) datasets: ENCODE accession ENCSR958UOC, ENCSR430YFL.

[0367] The GEiGS® solution design consisted of an endogenous pre-miRNA (hairpin), also known as the endogenous miRNA scaffold, engineered to encode a novel silencing RNAi trigger in place of its native guide strand. The novel GEiGS® silencing RNA matched B2M mRNA by sequence complementarity and was predicted to lead to silencing of B2M via the RNA interference pathway.

[0368] The extension solutions consist of a computationally designed GEiGS solution and 50–150 or 40 bases of genomic sequence flanking each endogenous pre-miRNA on which the GEiGS solution was based, to avoid omitting important sequences that act as signals for the Drosha enzyme.

[0369] The B2M GEiGS solutions detailed in Table 6 are tested for silencing activity by the plasmid-based ectopic expression assay detailed in Example 2. As before, a wide range of B2M silencing is observed, ranging from 5% to 99%. The most effective solutions exhibited potent silencing activity.

[0370] [Table 6A]

[0371] [Table 6B]

[0372] [Table 6C]

[0373] [Table 6D]

[0374] [Table 6E]

[0375] [Table 6F]

[0376] Example 12 Assessing the effect of homology arm length on the efficiency of homology-directed repair We varied the concentration and type of HDR (homology-directed repair) template with various lengths of homology arms to assess whether this affected the efficiency of editing miRNA loci for insertion of GEiGS templates by homology-directed repair. Single-stranded DNA (ssDNA) may increase precision editing efficiency compared to double-stranded DNA (dsDNA) templates by promoting duplex formation and thus HDR, and by reducing the risk of repair by end-joining, which would lead to undesired editing outcomes [Skarnes WC, Pellegrino E, McDonough JA. Improving homology-directed repair efficiency in human stem cells. Methods. 2019 Jul 15;164-165:18-28. doi: 10.1016 / j.ymeth.2019.06.016. Epub 2019 Jun 16. PMID: 31216442]. Furthermore, shorter homology may allow greater amounts and concentrations of donor to be transfected into cells, thus facilitating efficient HDR editing.

[0377] iPSCs were transfected with 4 μM (micromolar) Cas9-guide RNA complexes and varying concentrations (μM) of HDR template. Various forms of HDR template were tested: short ssDNA (137 bp total length, purchased from Integrated DNA Technologies with AltR HDR modification) with right and left homology arms with 40 bp long modified ends, compared with long ssDNA (632 bp total length, purchased from Integrated DNA Technologies) with right and left homology arms with 350 bp long, or short HDR blocks (dsDNA with modified ends, purchased from Integrated DNA Technologies) with right and left homology arms with 150 bp or 350 bp long (137 bp or 632 bp total length). The efficiency of HDR was assessed by performing next-generation sequencing on the amplified miRNA locus of interest (miR-21 locus) and analyzed using allele quantification software (CRISPresso2) [Clement K, Rees H, Canver MC, Gehrke JM, Farouni R, Hsu JY, Cole MA, Liu DR, Joung JK, Bauer DE, Pinello L. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol. 2019 Mar;37(3):224-226. doi: 10.1038 / s41587-019-0032-3. PubMed PMID: 30809026] (Figure 17). Figure 17 shows that 40 base pair long flanking genomic sequences (homology arms) show a dose-dependent superior effect on HDR efficiency (HDR%) compared to 350 bp long or 150-350 bp long flanking genomic sequences, which are slightly better than the donor-less template alone.

[0378] At similar concentrations (0.357 or 2 μM), short ssDNA resulted in higher editing efficiencies (HDR%) compared to long ssDNA or short dsDNA HDR blocks. Maximum editing efficiency was observed after increasing the concentration of short ssDNA to 8 μM. Testing higher concentrations of long ssDNA or short HDR blocks may not be possible due to physical constraints in the synthesis of these templates.

[0379] Example 13 The ability of GEiGS Solution-29 to buffer against induced expression of B2M following exposure of cells to inflammatory cytokines GEiGS offers the potential for stable and tunable knockdown of B2M, thus providing partial silencing of MHC-I expression rather than complete knockout (KO), making it suitable for generating hypoimmunogenic cells. MHC-I KO is useful for effective evasion of the recipient's adaptive immunity, but exposes therapeutic cells to rejection by cells of the innate immune system.

[0380] The cells or compositions according to the present invention may be used in a method for treating an autoimmune disease or cancer in a subject, the method comprising administering a therapeutic amount of the cells or compositions to the subject. The autoimmune disease may be any autoimmune disease, preferably type I diabetes. To treat diabetes, the cells of the present invention may be transplanted into a diabetic patient as pancreatic beta islet cells differentiated from stem cells.

[0381] Autoimmune disorders or cancer are characterized by chronic inflammation in patients. Therefore, in type 1 diabetes and cancer, cells or cell compositions transplanted as interventions are exposed to inflammatory stimuli. Many different factors can affect the basal and inducible expression of MHC-I on the cell surface. These include factors found in diseased, infected, or inflamed tissues, such as cytokines, growth factors [interferon (IFN), tumor necrosis factor (TNF), and type II interferon (IFN-γ)]. IFN has the ability to activate the expression of major and minor HLA genes, components of the MHC-I complex.

[0382] B2M KO would prevent MHC-I upregulation in therapeutic cells in response to inflammatory cytokines. For GEiGS to be an effective alternative to B2M KO for maintaining immune cells in the spleen even when exposed to inflammation, its silencing effect needs to be demonstrated to be stable, tunable, and sufficiently dynamic to maintain gene silencing.

[0383] Various media conditions were tested to assess their effect on B2M expression in iPSCs. Interferon-gamma (IFN-γ or IFN-g) was included at a concentration of 20 ng / ml, a supraphysiological concentration used to induce IFN-responsive genes in experimental tissue cultures. Exposure of control (unmodified) iPSCs to IFN-γ for 72 hours (WT + IFN-γ) led to a more than six-fold increase in the relative expression level of B2M compared to unstimulated iPSCs alone (WT), as analyzed by flow cytometry using an anti-B2M antibody.

[0384] Unexpectedly, the homozygous (KI / KI) Solution 29 clonal line, which biallelically expresses B2M sRNA, showed elevated cell surface B2M expression in the presence of IFN-γ (S29+INFg), and this increase was significantly reduced (7-fold) compared to that of the parental line (WT+INFg) exposed to IFN-γ for 72 hours, suggesting that GEiGS has a profound buffering effect in preventing MHC-I upregulation in cells in response to inflammatory cytokines (Figure 18). The relative fold change in B2M cell surface expression (MHC-I cell surface expression) between S29 and S29+INF-γ (approximately 10-fold) is approximately identical to the relative fold change between WT and WT+INF-γ (approximately 14-fold). In this experiment, the MHC-I complex is a heterodimer composed of hypervariable HLA proteins and beta-2 microglobulin (B2M), so the cell surface levels of B2M indicate the cell surface levels of MHC-I.

[0385] This demonstrates that the GEiGS solution of the present invention, which targets B2M, can also reduce B2M (and therefore MHC-I) levels in chronic inflammatory conditions. This indicates that such a solution may be of therapeutic benefit to patients, even those suffering from chronic inflammatory diseases. Furthermore, this example illustrates that the effect of inhibitory RNA is not limited by the copy number of the target transcript in the cell.

[0386] SEQ ID NO: 1 or 18 (Solution 8), SEQ ID NO: 5 or 22 (Solution 20), SEQ ID NO: 9 or 26 (Solution 29) are all predicted to produce effects similar to those observed with Solution 30.

[0387] Example 14 Demonstration of the ability of GEiGS to silence one or multiple target genes in cells when distinct solutions from a single construct are ectopically expressed. The constructs were used to ectopically express a B2M-targeting solution and eGFP (Solution 179) or two separate B2M-silencing solutions as concatemers (Solution 29 and Solution 43, or two copies of Solution 29) under a constitutive promoter via transfection into iPSCs as described in Example 2. Plasmid maps for expression of one or more solutions are shown in Figure 19a.

[0388] Two separate iPSC lines were used: a characterized clonal cell line in which one copy of the sox-2 gene had been modified to incorporate mEGFP without disrupting the protein coding sequence (Allen Institute, AICS-0074-026:WTC-mEGFP-sox2-cl26), and the parental control iPSC line used to generate this tagged line (WTC, Coriell Medical Institute, Cell Line Biorepository, sample GM25256). Cells were harvested 3 days post-transfection and analyzed by flow cytometry as previously described (Example 2).

[0389] B2M and eGFP GEiGS Solution 101 consists of a miR-30 scaffold engineered to incorporate an eGFP trigger sequence (miR-30_GFP_Leb_130) and target GFP alone, with 130 bp of extended homology arms designated as adjacent scaffold sequences (Fellman et al.). The miR-30 GFP sequence is extracted from the publication Lebbink RJ, Lowe M, Chan T, Khine H, Wang X, McManus MT. Polymerase II promoter strength determines efficacy of microRNA-adapted shRNAs. PLoS One. 2011;6(10):e26213. doi: 10.1371 / journal.pone.0026213. Epub 2011 Oct 21. PMID: 22031824; PMCID: PMC3198731. Solution 179 consists of a polycistronic sequence (two separate modified miRNA scaffolds (miRNA-20a and miRNA-30)) consisting of Solution 29 (targeting B2M) and miR-30_eGFP_Leb_130 (targeting GFP).

[0390] Compared to negative control vector transfection, Solution 101 demonstrated efficient GFP silencing without affecting the level of B2M expression in undifferentiated iPSCs (Figure 19b). Solution 179, consisting of two distinct engineered miRNA scaffolds (miRNA-20a and miRNA-30), was effective in silencing both B2M and eGFP with apparent equal efficiency (Figure 19b).

[0391] This demonstrates that multiple GEiGS solutions expressed from the same construct can be effective in silencing multiple targets.

[0392] Two separate solutions targeting B2M GEiGS Solution 177 was created, consisting of separate solutions with diverse sequences targeting the B2M transcript (trigger): Solution 29 (based on modified miRNA-20a) combined with Solution 43 (based on modified miR-518b). Solution 178 was created, consisting of two copies of Solution 29 (targeting B2M).

[0393] Compared to negative control vector transfections, Solution 177 (two different solutions targeting B2M) appeared more effective at silencing B2M than Solution 43 alone, demonstrating a slight additive effect and that the two separate solutions did not interfere with each other's ability to silence B2M (Figure 20). Solution 178, which contains two copies of Solution 29 and the same trigger sequence, had no additive effect on silencing B2M compared to Solution 29 alone (Figure 20).

[0394] Example 15 Analysis of potential off-target effects caused by GEiGS demonstrates that GEiGS is highly specific and shows no off-target effects. A systematic unbiased whole-transcriptome analysis of the Solution 29 (S29) GEiGS strain and matched controls was performed to assess whether GEiGS-mediated silencing causes off-target effects.

[0395] We generated isogenic GEiGS-modified and control iPSC cell lines from the same parental iPSC line and measured gene expression using RNA-seq. Differential gene expression (DGE) analysis using the parental line as a reference was performed to detect transcriptional changes in an unbiased manner across the genome. This approach detects both direct changes in gene expression caused by GEiGS (specific target silencing, potential silencing of unintended targets, and potential upregulation of cognate targets of redirected miRNAs) and indirect changes caused by knock-on effects from direct changes in gene expression. RNA-seq and DEG analysis were performed using standard techniques.

[0396] The GEiGS lines analyzed were homozygous for Solution 29 knock-in (S29 KI / KI), a B2M-targeted GEiGS solution based on MIR20A as a scaffold (see Example 3 for details on the implementation of Solution 29 GEiGS). The following control lines were also generated: (i) B2M homozygous KO (B2M KO / KO) generated by CRISPR KO to detect transcriptional changes specifically caused by B2M silencing, and (ii) hsa-mir-20a homozygous KO (hsa-mir-20a KO / KO) to detect changes in gene expression caused by loss of activity of miRNA genes hijacked and used as scaffolds in Solution 29.

[0397] Each experimental sample contained at least two biological replicates (independent clones with the same genotype) in two technical replicates (independent cultures of the same clone). DGE analysis was performed using the R package edgeR with a false positive rate (FDR) of 0.05 as the statistical significance threshold and no effect size cutoff.

[0398] The results are shown in Figures 21a-c. When comparing B2M KO / KO with the parental cell line, we found that CRISPR KO of B2M led to a highly specific and significant reduction in mRNA levels transcribed from the B2M gene (91% downregulation); B2M was the only differentially expressed gene (Figure 21a). No other changes in gene expression were detected, indicating that loss of MHC-I expression in steady-state cultures of iPSCs has no consequences for cell physiology (Figure 21a).

[0399] In the hsa-mir-20a KO, loss of the edited miRNA did not result in any expression changes, providing experimental evidence of functional redundancy in the miRNA network utilized by GEiGS (Figure 21b).

[0400] In GEiGS-modified cells in which Solution 29 was introduced into the endogenous hsa-mir-20a locus, there was again a highly specific and significant reduction in the level of mRNA transcribed from the B2M gene compared to the parental cell line; B2M was again the only differentially expressed gene (Figure 21c). This demonstrates that GEiGS is highly specific, silencing only the intended target and not other unintended targets.

[0401] Notably, off-target effects previously associated with silencing of unintended targets when using siRNA or shRNA were not detected, further supporting the application of GEiGS for highly specific silencing of target genes while avoiding off-target effects such as silencing of unintended targets and potential loss-of-function effects after redirection of miRNA genes used as scaffolds.

[0402] Example 16 Demonstration that B2M silencing in myeloid cells results in loss of cell surface MHC-I (as evidenced by loss of HLA-A / B / C expression using antibody labeling) 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.

[0403] 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).

[0404] 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.

[0405] After several days of differentiation in culture, cells were isolated for analysis of target MHC-I protein expression (B2M or HLA-ABC). Relative B2M and HLA-ABC expression in GEiGS-modified iPSCs, monocytes, and macrophages (miR-21 redirected to target B2M, i.e., Solution 30) compared with control cells in which endogenous miR-21 was knocked out (KO / KO). GEiGS and control lines were differentiated in vitro first into monocytes and then into macrophages. B2M and HLA-ABC were assayed by flow cytometry and showed effective silencing in myeloid cells (84–88% silencing), but not at the iPSC stage (Figure 22).

[0406] The relative B2M protein expression levels between Solution 30 undifferentiated iPSCs and their undifferentiated homozygous KO control iPSC lines were nearly identical, indicating that disrupting the endogenous miR-21 locus did not result in nonspecific B2M silencing effects at the iPSC stage. When iPSCs were subsequently differentiated from the wild-type control (unedited) to myeloid progenitor cells, monocytes, and macrophages, the homozygous miR-21 KO and homozygous Solution 30 (KI / KI) cell lines were able to differentiate effectively with identical efficiency, but the homozygous Solution 30 (KI / KI) cell line showed specific reduction of B2M protein expression only at the myeloid progenitor, monocyte, and macrophage stages (Figure 22a). This demonstrates that modifying the miR-21 locus to incorporate Solution 30 does not disrupt the ability of stem cells to differentiate into myeloid lineage cells, and that B2M cell surface expression is specifically reduced only in myeloid lineage-stage cells.

[0407] Antibodies against cell surface-expressed classical HLA-A / B / C showed that, as predicted for B2M silencing by Solution 30, there was a reduction in cell surface expression of HLA-A / B / C, as B2M is required for correct processing and cell surface distribution of MHC-I complex proteins.

[0408] This example shows that silencing B2M results in reduced cell surface expression of HLA-A / B / C and therefore of the MHC-I complex.

[0409] Example 17 B2M Solution Screen Based on known B2M silencing specificity in other human cell types (e.g., iPSCs), we performed a screen using validated solutions targeting B2M with different predicted levels of silencing in activated T cells.

[0410] The objective was to demonstrate the utility of lentivirus (LV)-mediated solution delivery, as opposed to plasmid-based delivery, into T cells, a cell type notoriously difficult to ectopically express solutions in. A dual reporter system, in which the 3'UTR sequence of the B2M transcript was placed contiguous with the coding sequence for copGFP, was also used to validate this system. Screening for copGFP loss in cells expressing a validated solution (trigger) against the B2M sequence, rather than relying on detection of residual B2M protein expression on the T cell surface using flow cytometry, was a useful test of this alternative analytical approach for solution validation and supported the more rapid identification of effective solutions, e.g., intracellular transcription factor targets. Effective solutions possess B2M silencing specificity as a result of perfect complementarity binding to the cognate B2M target gene sequence.

[0411] Constructs for lentiviral (LV) delivery studies and validation of a novel reporter system for B2M silencing based on copGFP loss in T cells are described (Figure 23a / b). Constructs with high (Solution 29), moderate (Solution 12), low (Solution 50), and no B2M silencing activity (scrambled) are shown in Figure 23a. Activity predictions were based on the previous efficacy seen or predicted in Examples 2, 6, and 15 and Figures 10, 12, and 21 for Solution 29, in Examples 8 and 13 for Solution 12, and in Example 2 and Figure 8 for Solution 50.

[0412] To validate LV-based solution delivery, this reporter system was transduced into the Jurkat cell line. Jurkat cells were transduced with a pooled mixture of individual LV constructs, Solution 12, Solution 29, and Solution 50, or scrambled (negative control), to generate B2M Jurkat reporter pools of cells. Separate multiplicities of infection (MOIs) were assessed for each pool of LV constructs (1, 5, and 10). dsRED expression from each solution construct was used to monitor and gate for the emergence of solution-expressing cell pools. Cell surface detection of the low-affinity NGF-receptor protein was used to gate for cell pools expressing the copGFP sequence containing the B2M 3'UTR binding sequence from each solution.

[0413] The gating strategy for identifying cells expressing both dsRED (solution sequence) and copGFP is outlined in Figure 23b. Levels of copGFP expression were monitored 3, 6, and 9 days after transduction, and verification of copGFP disappearance in B2M Jurkat reporter cells containing effective solution was confirmed using residual B2M cell surface expression by flow cytometry.

[0414] As shown in Figure 24a, GFP loss as a result of transcript degradation by solution binding to the B2M 3'UTR in the constructs showed the expected pattern of silencing specificity as Solution 29 > Solution 12 > Solution 50. There was a close correlation between the quantitative amount of copGFP loss and the detectable amount of remaining B2M protein staining, and the silencing effect demonstrated by these distinct analytical approaches was both dose (MOI) and time post-transduction dependent (days).

[0415] When analyzed by flow cytometry and gated for solution-expressing cells (high dsRED) and copGFP expression, the B2M reporter Jurkat pool displayed a pattern of B2M silencing solution effect that closely mimicked that predicted for each individual solution when expressed alone, confirming that LV pool transduction of T cells may be a valid alternative to a plasmid-based system for further solution design testing and validation (Figure 24b).

[0416] This demonstrates that lentiviral solution delivery can lead to successful knockdown of a target gene of interest, as detectable by copGFP disappearance, as well as that different levels of target gene expression can be achieved using specially designed solutions.

[0417] The reader's attention is directed to all articles and documents published herewith, filed contemporaneously with or previously filed in connection with this application, and the contents of all such articles and documents are incorporated herein by reference.

[0418] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually inconsistent.

[0419] Each feature disclosed in this specification (including any accompanying claims, abstract, and drawings), unless expressly stated otherwise, may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0420] The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel, or any novel combination of, features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel, or any novel combination of method or process steps so disclosed.

[0421] (References) TIFF2026504098000040.tif210160

Claims

1. 1. An inhibitory RNA, or a polynucleotide sequence encoding an inhibitory RNA, preferably a microRNA, wherein the inhibitory RNA is: (i) any one of SEQ ID NOs: 1-17, 64-127; (ii) a variant of any one of SEQ ID NOs: 1-17, 64-127; (iii) a fragment of any one of SEQ ID NOs: 1 to 17, 64 to 127, and (iv) a DNA sequence complementary to any one of (i), (ii), or (iii); An inhibitory RNA or polynucleotide sequence comprising or consisting of a sequence selected from:

2. below: (i) any one of SEQ ID NOs: 18-34, 128-191; (ii) a variant of any one of SEQ ID NOs: 18-34, 128-191; (iii) a fragment of any one of SEQ ID NOs: 18-34, 128-191, and (iv) a DNA sequence complementary to any one of (i), (ii), or (iii); 2. The inhibitory RNA or polynucleotide sequence of claim 1, comprising or consisting of a sequence selected from:

3. below: (i) any one of SEQ ID NOs: 35-51; (ii) a variant of any one of SEQ ID NOs: 35 to 51, and (iii) a fragment of any one of SEQ ID NOs: 35 to 51 3. A polynucleotide sequence encoding the inhibitory RNA of claim 1 or 2, comprising or consisting of a sequence selected from:

4. 4. The inhibitory RNA or polynucleotide sequence encoding the inhibitory RNA according to any one of claims 1 to 3, which is RNA.

5. 4. The inhibitory RNA or polynucleotide sequence encoding the inhibitory RNA according to any one of claims 1 to 3, which is DNA.

6. 6. An inhibitory RNA or a polynucleotide sequence encoding an inhibitory RNA according to any one of claims 1 to 5, which is a synthetic nucleic acid sequence.

7. 7. An inhibitory RNA or a polynucleotide sequence encoding an inhibitory RNA according to any one of claims 1 to 6, comprising or encoding a microRNA scaffold.

8. The inhibitory RNA or polynucleotide sequence encoding the inhibitory RNA of claim 7, wherein the microRNA scaffold is a human pre-microRNA scaffold.

9. Human pre-microRNA scaffolds include: (i) hsa-mir-191, (ii) hsa-mir-302a, (iii) hsa-mir-302c, (iv) hsa-mir-93, (v) hsa-mir-106a, (v i) hsa-mir-106b, (vii) hsa-mir-200c, (viii) hsa-mir-20a, (iv) hsa-mir-21, (x) hsa-mir-363, (x i) hsa-mir-518b, (xii) hsa-mir-744, (xiii) hsa-mir-99b, (xiv) hsa-mir-320a, (xv) hsa-mir-520 f, (xvi)hsa-mir-652, (xvi)hsa-mir-1180, (xvii)hsa-mir-15b, (xviii)hsa-mir-182, (xix)hsa-m ir-23a, (xx)hsa-mir-26b, (xxi)hsa-mir-335, (xxii)hsa-mir-361, (xxiii)hsa-mir-1307, (xxiv )hsa-mir-205, (xxv)hsa-mir-22, (xxvi)hsa-mir-221, (xxvii)hsa-mir-222, (xxviii)hsa-mir-3 0e, (xxix)hsa-mir-423, (xxx)hsa-mir-519c, (xxxi)hsa-mir-92b, (xxxii)hsa-mir-30, (xxxiii) hsa-mir-302b, (xxxiv)hsa-mir-375, (xxxv)hsa-let-7b, (xxxv)hsa-mir-98 and (xxxvi)hsa-let-7g The inhibitory RNA or polynucleotide sequence encoding the inhibitory RNA according to claim 8, selected from any one of the following:

10. Any one of the polynucleotides of SEQ ID NOs: 1-17, 64-127 is inserted into a human pre-microRNA sequence, preferably (i) SEQ ID NO: 1 or 7 is inserted into hsa-mir-191 (SEQ ID NO: 18 or 24, respectively); (ii) SEQ ID NO: 2 or 11 is inserted into hsa-mir-302a (SEQ ID NO: 19 or 28, respectively); (iii) SEQ ID NO: 3 or 12 is inserted into hsa-mir-302c (SEQ ID NO: 20 or 29, respectively); (iv) SEQ ID NO: 4 or 16 is inserted into hsa-mir-93 (SEQ ID NO: 21 or 33, respectively); (v) SEQ ID NO: 5 is inserted into hsa-mir-106a (SEQ ID NO: 22); (vi) SEQ ID NO: 6 is inserted into hsa-mir-106b (SEQ ID NO: 23); (vii) SEQ ID NO: 8 is inserted into hsa-mir-200c (SEQ ID NO: 25); (viii) SEQ ID NO: 9 is inserted into hsa-mir-20a (SEQ ID NO: 26); (iv) SEQ ID NO: 10 is inserted into hsa-mir-21 (SEQ ID NO: 27); (x) SEQ ID NO: 13 is inserted into hsa-mir-363 (SEQ ID NO: 30); (xi) SEQ ID NO: 14 is inserted into hsa-mir-518b (SEQ ID NO: 31); (xii) SEQ ID NO: 15 is inserted into hsa-mir-744 (SEQ ID NO: 32); (xiii) SEQ ID NO: 17 is inserted into hsa-mir-99b (SEQ ID NO: 34); (xiv) SEQ ID NO: 64, SEQ ID NO: 73, or SEQ ID NO: 89 is inserted into hsa-mir-320a (SEQ ID NO: 128, SEQ ID NO: 137, or SEQ ID NO: 153, respectively); (xv) SEQ ID NO: 65, SEQ ID NO: 77, or SEQ ID NO: 94 is inserted into hsa-mir-520f (SEQ ID NO: 129, SEQ ID NO: 141, or SEQ ID NO: 158, respectively); (xvi) SEQ ID NO: 66, SEQ ID NO: 78, or SEQ ID NO: 95 is inserted into hsa-mir-652 (SEQ ID NO: 130, SEQ ID NO: 142, or SEQ ID NO: 159, respectively); (xvii) SEQ ID NO: 67 is inserted into hsa-mir-106a (SEQ ID NO: 131); (xviii) SEQ ID NO: 68 or SEQ ID NO: 79 is inserted into hsa-mir-1180 (SEQ ID NO: 132 or SEQ ID NO: 143, respectively); (xix) SEQ ID NO: 69 or SEQ ID NO: 81 is inserted into hsa-mir-15b (SEQ ID NO: 133 or SEQ ID NO: 145, respectively); (xx) SEQ ID NO: 70, SEQ ID NO: 82, or SEQ ID NO: 99 is inserted into hsa-mir-182 (SEQ ID NO: 134, SEQ ID NO: 146, or SEQ ID NO: 163, respectively); (xxi) SEQ ID NO: 71 or SEQ ID NO: 87 is inserted into hsa-mir-23a (SEQ ID NO: 135 or SEQ ID NO: 151, respectively); (xxii) SEQ ID NO: 72 is inserted into hsa-mir-26b (SEQ ID NO: 136); (xxiii) SEQ ID NO: 74 or SEQ ID NO: 90 is inserted into hsa-mir-335 (SEQ ID NO: 138 or SEQ ID NO: 154, respectively); (xxiv) SEQ ID NO: 75 or SEQ ID NO: 91 is inserted into hsa-mir-361 (SEQ ID NO: 139 or SEQ ID NO: 155, respectively); (xxv) SEQ ID NO: 76 is inserted into hsa-mir-518b (SEQ ID NO: 140); (xxvi) SEQ ID NO: 80 is inserted into hsa-mir-1307 (SEQ ID NO: 144); (xxvii) SEQ ID NO: 83 is inserted into hsa-mir-205 (SEQ ID NO: 147); (xxviii) SEQ ID NO: 84 is inserted into hsa-mir-22 (SEQ ID NO: 148); (xxix) SEQ ID NO: 85 is inserted into hsa-mir-221 (SEQ ID NO: 149); (xxx) SEQ ID NO: 86 is inserted into hsa-mir-222 (SEQ ID NO: 150); (xxxi) SEQ ID NO: 88 is inserted into hsa-mir-30e (SEQ ID NO: 152); (xxxii) SEQ ID NO: 92 is inserted into hsa-mir-423 (SEQ ID NO: 156); (xxxiii) SEQ ID NO: 93 is inserted into hsa-mir-519c (SEQ ID NO: 157); (xxxiv) SEQ ID NO: 96 is inserted into hsa-mir-92b (SEQ ID NO: 160); (xxxv) SEQ ID NO: 97, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 109 is inserted into hsa-mir-302a (SEQ ID NO: 161, SEQ ID NO: 167, SEQ ID NO: 168, or SEQ ID NO: 173, respectively); (xxxvi) SEQ ID NO: 98, SEQ ID NO: 107, SEQ ID NO: 108, or SEQ ID NO: 111 is inserted into hsa-mir-302c (SEQ ID NO: 162, SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 175, respectively); (xxxvii) SEQ ID NO: 100 is inserted into hsa-mir-191 (SEQ ID NO: 164); (xxxviii) SEQ ID NO: 101 or SEQ ID NO: 102 is inserted into hsa-mir-30 (SEQ ID NO: 165 or SEQ ID NO: 166, respectively); (xxxix) SEQ ID NO: 105, SEQ ID NO: 106, or SEQ ID NO: 110 is inserted into hsa-mir-302b (SEQ ID NO: 169, SEQ ID NO: 170, or SEQ ID NO: 174, respectively); (xxxx) SEQ ID NO:112, SEQ ID NO:113, or SEQ ID NO:114 is inserted into hsa-mir-375 (SEQ ID NO:176, SEQ ID NO:177, or SEQ ID NO:178, respectively); (xxxxi) SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 122, or SEQ ID NO: 125 is inserted into hsa-let-7b (SEQ ID NO: 179, SEQ ID NO: 183, SEQ ID NO: 186, or SEQ ID NO: 189, respectively); (xxxxii) SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 123 or SEQ ID NO: 126 is inserted into hsa-let-7g (SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 187 or SEQ ID NO: 190, respectively), and / or (xxxxiii) SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 121, SEQ ID NO: 124, or SEQ ID NO: 127 is inserted into hsa-mir-98 (SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 185, SEQ ID NO: 188, or SEQ ID NO: 191, respectively); 10. An inhibitory RNA or a polynucleotide sequence encoding an inhibitory RNA according to any one of claims 1 to 9.

11. Optionally, the polynucleotide sequence comprises genomic sequences flanking a human pre-microRNA, the polynucleotide sequence being: (i) SEQ ID NO: 35 (including SEQ ID NO: 2 or SEQ ID NO: 19), (ii) SEQ ID NO: 36 (including SEQ ID NO: 11 or SEQ ID NO: 28); (iii) SEQ ID NO: 37 (including SEQ ID NO: 3 or SEQ ID NO: 20); (iv) SEQ ID NO: 38 (including SEQ ID NO: 12 or SEQ ID NO: 29), (v) SEQ ID NO: 39 (including SEQ ID NO: 9 or SEQ ID NO: 26), (vi) SEQ ID NO: 40 (including SEQ ID NO: 8 or SEQ ID NO: 25), (vii) SEQ ID NO: 41 (including SEQ ID NO: 10 or SEQ ID NO: 27), (viii) SEQ ID NO: 42 (including SEQ ID NO: 13 or SEQ ID NO: 30); (iv) SEQ ID NO: 43 (including SEQ ID NO: 4 or SEQ ID NO: 21), (x) SEQ ID NO: 44 (including SEQ ID NO: 16 or SEQ ID NO: 33), (xi) SEQ ID NO: 45 (including SEQ ID NO: 5 or SEQ ID NO: 22); (xii) SEQ ID NO: 46 (including SEQ ID NO: 6 or SEQ ID NO: 23), (xiii) SEQ ID NO: 47 (including SEQ ID NO: 1 or SEQ ID NO: 18), (xvi) SEQ ID NO: 48 (including SEQ ID NO: 7 or SEQ ID NO: 24), (xv) SEQ ID NO: 49 (including SEQ ID NO: 15 or SEQ ID NO: 32; (xvi) SEQ ID NO: 50 (including SEQ ID NO: 14 or SEQ ID NO: 31, and / or (xvii) SEQ ID NO: 51 (including SEQ ID NO: 17 or SEQ ID NO: 34) 11. A polynucleotide sequence encoding the inhibitory RNA of any one of claims 1 to 10, comprising:

12. The inhibitory RNA targets the B2M RNA, optionally an exon sequence contained in the messenger RNA from the B2M gene, optionally exon 2-1, 2-2 or exon 4, and preferably the inhibitory RNA is (i) SEQ ID NO: 2 or SEQ ID NO: 19, which targets B2M gene exon 2-2; (ii) SEQ ID NO: 11 or SEQ ID NO: 28, which targets B2M gene exon 4; (iii) SEQ ID NO: 3 or SEQ ID NO: 20, which targets B2M gene exon 2-2; (iv) SEQ ID NO: 12 or SEQ ID NO: 29, which targets B2M gene exon 4; (v) SEQ ID NO: 9 or SEQ ID NO: 26, which targets B2M gene exon 4; (vi) SEQ ID NO: 8 or SEQ ID NO: 25, which targets B2M gene exon 4; (vii) SEQ ID NO: 10 or SEQ ID NO: 27, which targets B2M gene exon 4; (viii) SEQ ID NO: 13 or SEQ ID NO: 30, which targets B2M gene exon 4; (iv) SEQ ID NO: 4 or SEQ ID NO: 21, which targets B2M gene exon 2-2; (x) SEQ ID NO: 16 or SEQ ID NO: 33, targeting B2M gene exon 4; (xi) SEQ ID NO: 5 or SEQ ID NO: 22, which targets B2M gene exon 4; (xii) SEQ ID NO: 6 or SEQ ID NO: 23, which targets B2M gene exon 4; (xiii) SEQ ID NO: 1 or SEQ ID NO: 18, which targets B2M gene exon 2-2; (xvi) SEQ ID NO: 7 or SEQ ID NO: 24, which targets B2M gene exon 4; (xv) SEQ ID NO: 15 or SEQ ID NO: 32, which targets B2M gene exon 4; (xvi) SEQ ID NO: 14 or SEQ ID NO: 31, which targets B2M gene exon 4; (xvii) SEQ ID NO: 17 or SEQ ID NO: 34, which targets B2M gene exon 4; (xviii) SEQ ID NOs: 64 to 66 or 128 to 130, which target B2M exon 2-1; (xix) SEQ ID NOs: 67-78 or 129-142, which target B2M exon 2-2, and / or (xx) SEQ ID NOs: 79-96 or 143-160 targeting B2M exon 4 11. The inhibitory RNA or polynucleotide sequence encoding the inhibitory RNA of any one of claims 1, 2, 4 or 6 to 10, comprising:

13. An expression cassette comprising a polynucleotide sequence encoding an inhibitory RNA described in any one of claims 1 to 12 operably linked to one or more regulatory elements, preferably a promoter, optionally a constitutive or inducible promoter, suitable for enabling transcription of the inhibitory RNA in a cell.

14. 14. The expression cassette of claim 13, wherein the promoter is EF1 alpha.

15. A vector comprising a polynucleotide sequence encoding an inhibitory RNA according to any one of claims 1 to 12 or an expression cassette according to claim 13 or 14.

16. 16. The vector of claim 15, comprising DNA encoding a regulatory element, preferably a promoter, optionally a constitutive or inducible promoter, suitable for allowing transcription of the inhibitory RNA in the cell.

17. 17. The vector of claim 16, wherein the cell is a mammalian cell, preferably a human cell.

18. 18. The vector of any one of claims 15 to 17, which is a viral vector.

19. 19. The vector of claim 18, which is a lentivirus, adenovirus, adeno-associated virus (AAV), retrovirus, alphavirus, herpesvirus, arenavirus, measles virus, poxvirus, or paramyxovirus vector.

20. 18. The vector of any one of claims 15 to 17, which is a plasmid.

21. 21. The vector of claim 20, which is plasmid VB210602-1567ytv.

22. 22. A virion comprising the vector of any one of claims 15 to 21.

23. A synthetic microRNA comprising or consisting of a sequence selected from any one of SEQ ID NOs: 1 to 17, 64 to 127, or a functional variant or fragment thereof.

24. A synthetic pre-microRNA comprising or consisting of a sequence selected from any one of SEQ ID NOs: 18-34, 128-191, or a functional variant or fragment thereof.

25. 25. The synthetic microRNA or synthetic pre-microRNA of claim 23 or 24, which is a modified endogenous microRNA or pre-microRNA molecule.

26. 1. A synthetic microRNA adapted to target messenger RNA transcribed from the B2M gene, the synthetic microRNA comprising a sequence according to any one of SEQ ID NOs: 1-17, 64-127 or a functional variant or fragment thereof, and optionally a sequence according to any one of SEQ ID NOs: 18-34, 128-191 or a functional variant or fragment thereof.

27. 27. A cell comprising or expressing the synthetic microRNA of any one of claims 23 to 26, wherein the cell is modified to express the synthetic microRNA such that the synthetic microRNA targets a messenger RNA transcribed from the B2M gene.

28. 28. The cell of claim 27, wherein the synthetic microRNA degrades messenger RNA transcribed from the B2M gene, preferably such that the messenger RNA is partially silenced and B2M protein expression is altered in the range of 10% to 90% expression compared to control cells.

29. A cell comprising or expressing a sequence encoding a synthetic inhibitory RNA, preferably a synthetic microRNA, wherein the synthetic inhibitory RNA is adapted to target B2M.

30. 30. The cell of claim 29, wherein the sequence encoding the synthetic inhibitory RNA, preferably a synthetic microRNA, encodes one or more of SEQ ID NOs: 1 to 17, 64 to 127.

31. 30. The cell of claim 29, wherein the sequence encoding the synthetic microRNA encodes one or more of SEQ ID NOs: 18-34, 128-191.

32. 30. The cell of claim 29, wherein the sequence encoding the synthetic microRNA comprises one or more of SEQ ID NOs: 35-51.

33. 33. The cell according to any one of claims 29 to 32, wherein the sequence encoding the synthetic inhibitory RNA, preferably a microRNA, is genomic or episomal, preferably genomic.

34. The synthetic microRNA-encoding sequence is a genomic sequence encoding a modified endogenous microRNA, and optionally the cell is a human cell, and the modified genomic sequence encodes the following endogenous microRNA: (i) hsa-mir-191, optionally modified to include SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 100; (ii) hsa-mir-302a, optionally modified to include SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:97, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:109; (iii) hsa-mir-302c, optionally modified to include SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:98, SEQ ID NO:107, SEQ ID NO:108, or SEQ ID NO:111; (iv) hsa-mir-93, optionally modified to include SEQ ID NO: 4 or 16; (v) hsa-mir-106a, optionally modified to include SEQ ID NO: 5 or SEQ ID NO: 67; (vi) hsa-mir-106b, optionally modified to include SEQ ID NO: 6; (vii) hsa-mir-200c, optionally modified to include SEQ ID NO: 8; (viii) hsa-mir-20a, optionally modified to include SEQ ID NO: 9; (iv) hsa-mir-21, optionally modified to include SEQ ID NO: 10; (x) hsa-mir-363, optionally modified to include SEQ ID NO: 13; (xi) hsa-mir-518b, optionally modified to include SEQ ID NO: 14 or SEQ ID NO: 76; (xii) hsa-mir-744, optionally modified to include SEQ ID NO: 15; (xiii) hsa-mir-99b, optionally modified to include SEQ ID NO: 17; (xiv) hsa-mir-320a, optionally modified to include SEQ ID NO: 64, SEQ ID NO: 73, or SEQ ID NO: 89; (xv) hsa-mir-520f, optionally modified to include SEQ ID NO: 65, SEQ ID NO: 77, or SEQ ID NO: 94; (xvi) hsa-mir-652, optionally modified to include SEQ ID NO: 66, SEQ ID NO: 78, or SEQ ID NO: 95; (xvii) hsa-mir-1180, optionally modified to include SEQ ID NO: 68 or SEQ ID NO: 79; (xviii) hsa-mir-15b, optionally modified to include SEQ ID NO: 69 or SEQ ID NO: 81; (xix) hsa-mir-182, optionally modified to include SEQ ID NO: 70, SEQ ID NO: 82, or SEQ ID NO: 99; (xx) hsa-mir-23a, optionally modified to include SEQ ID NO: 71 or SEQ ID NO: 87; (xxi) hsa-mir-26b, optionally modified to include SEQ ID NO: 72; (xxii) hsa-mir-335, optionally modified to include SEQ ID NO: 74 or SEQ ID NO: 90; (xxiii) hsa-mir-361, optionally modified to include SEQ ID NO: 75 or SEQ ID NO: 91; (xxiv) hsa-mir-1307, optionally modified to include SEQ ID NO: 80; (xxv) hsa-mir-205, optionally modified to include SEQ ID NO: 83; (xxvi) hsa-mir-22, optionally modified to include SEQ ID NO: 84; (xxvii) hsa-mir-221, optionally modified to include SEQ ID NO: 85; (xxviii) hsa-mir-222, optionally modified to include SEQ ID NO: 86; (xxix) hsa-mir-30e, optionally modified to include SEQ ID NO: 88; (xxx) hsa-mir-423, optionally modified to contain SEQ ID NO: 92; (xxxi) hsa-mir-519c, optionally modified to include SEQ ID NO: 93; (xxxii) hsa-mir-92b, optionally modified to include SEQ ID NO: 96; (xxxiii) hsa-mir-30, optionally modified to include SEQ ID NO: 101 or SEQ ID NO: 102; (xxxiv) hsa-mir-302b, optionally modified to include SEQ ID NO: 105, SEQ ID NO: 106, or SEQ ID NO: 110; (xxxv) hsa-mir-375, optionally modified to include SEQ ID NO: 112, SEQ ID NO: 113, or SEQ ID NO: 114; (xxxvi) hsa-let-7b, optionally modified to include SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 122, or SEQ ID NO: 125; (xxxvii) hsa-let-7g, optionally modified to include SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 123, or SEQ ID NO: 126, and (xxxviii) hsa-mir-98, optionally modified to include SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 121, SEQ ID NO: 124, or SEQ ID NO:

127.

34. The cell of any one of claims 29 to 33, wherein the cell encodes a modified form of one of the following:

35. 35. The cell of any one of claims 29 to 34, which exhibits reduced B2M expression, preferably not completely abolished, and optionally wherein B2M is expressed at a level of 1% to 90%, 5% to 90%, or 10% to 90% compared to control cells.

36. 36. The cell of any one of claims 29 to 35, which is detargeted by the innate and adaptive immune systems.

37. 37. The cell according to any one of claims 29 to 36, which is a eukaryotic cell, preferably a mammalian cell, more preferably a human cell.

38. 38. The cell according to any one of claims 29 to 37, which is an induced pluripotent stem cell or a cell derived by differentiation of an induced pluripotent stem cell.

39. 39. The cell of any one of claims 29 to 38, which is a pancreatic beta cell or an immune cell.

40. 26. A cell comprising an expression cassette according to claim 13 or 14, a vector according to any one of claims 15 to 21, a virion according to claim 22 or a synthetic microRNA or pre-microRNA according to any one of claims 23 to 25.

41. 41. A composition comprising the cells of any one of claims 29 to 40.

42. 42. An inhibitory RNA or a polynucleotide encoding an inhibitory RNA according to any one of claims 1 to 12, an expression cassette according to claims 13 or 14, a vector according to any one of claims 15 to 21, a virion according to claim 22 or a synthetic microRNA or pre-microRNA according to any one of claims 23 to 26, a cell according to any one of claims 27 to 40 or a composition according to claim 41 for use in therapy.

43. 42. An inhibitory RNA or a polynucleotide encoding an inhibitory RNA according to any one of claims 1 to 12, an expression cassette according to claim 13 or 14, a vector according to any one of claims 15 to 21, a virion according to claim 22 or a synthetic microRNA or pre-microRNA according to any one of claims 23 to 26, a cell according to any one of claims 27 to 40 or a composition according to claim 41 for use in a method of treating an autoimmune disease or cancer in a subject.

44. 44. The inhibitory RNA or polynucleotide encoding an inhibitory RNA, expression cassette, vector, virion or synthetic microRNA or pre-microRNA according to claim 43, wherein the autoimmune disease is preferably type I diabetes.

45. 27. The inhibitory RNA or polynucleotide encoding the inhibitory RNA of any one of claims 1 to 12, the expression cassette of claim 13 or 14, the vector of any one of claims 15 to 21, the virion of claim 22 or the synthetic microRNA or pre-microRNA of any one of claims 23 to 26 for use in a method for degrading messenger RNA transcribed from the B2M gene in a cell, wherein preferably the messenger RNA is partially silenced and B2M protein expression is altered in the range of 10% to 90% expression compared to control cells.

46. 46. ​​The inhibitory RNA or polynucleotide encoding an inhibitory RNA, expression cassette, vector, virion or synthetic microRNA or pre-microRNA for use according to claim 45, wherein the cells are then transferred into a mammal, preferably a human, to treat an autoimmune disease or cancer, preferably wherein the autoimmune disease is type 1 diabetes.

47. 42. A cell according to any one of claims 27 to 40 or a composition according to claim 41 for use in a method of treating an autoimmune disease or cancer in a subject, the method comprising administering to the subject a therapeutic amount of the cell or composition.

48. 48. The cell or composition for use according to claim 47, wherein the autoimmune disease is type I diabetes.

49. A method of treating an autoimmune disease or cancer in a subject, preferably wherein the autoimmune disease is type I diabetes, the method comprising administering to the subject a therapeutic amount of a cell or composition.

50. 42. Use of a cell according to any one of claims 27 to 40 or a composition according to claim 41 in the manufacture of a medicament for treating an autoimmune disease or cancer in a subject, preferably wherein the autoimmune disease is type I diabetes.

51. A method for modifying protein expression in a cell, the method comprising the step of introducing into the cell an inhibitory RNA or a polynucleotide encoding the inhibitory RNA described in any one of claims 1 to 12, an expression cassette described in claim 13 or 14, a vector described in any one of claims 15 to 21, a virion described in claim 22, or a synthetic microRNA or pre-microRNA described in any one of claims 23 to 26.

52. 26. A method for modifying the translation of messenger RNA transcripts derived from protein-coding genes in a cell and the subsequent expression of proteins, comprising introducing into the cell an inhibitory RNA or a polynucleotide encoding the inhibitory RNA described in any one of claims 1 to 12, an expression cassette described in claims 13 or 14, a vector described in any one of claims 15 to 21, a virion described in claim 22, or a synthetic microRNA or pre-microRNA described in any one of claims 23 to 26.

53. 1. A method for modifying the translation of messenger RNA transcripts derived from protein-coding genes in a cell and the subsequent expression of proteins, comprising introducing into the cell a DNA editing agent that confers silencing specificity of a microRNA for a target RNA of interest, wherein the sequence of the microRNA comprises any one of SEQ ID NOs: 1-17, 64-127, whereby the microRNA is modified such that translation of the messenger RNA transcript into a protein molecule is modified.

54. 54. The method of any one of claims 51 to 53, wherein the cell is a eukaryotic cell, preferably a mammalian cell, more preferably a human cell.

55. The cells are human cells, and the method comprises the steps of: detecting 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-182, or hsa-mir-23a.

55. The method of claim 54, comprising editing the sequence of an endogenous microRNA selected from the group consisting of 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, has-let-7g, and hsa-mir-98.

56. 56. The method of any one of claims 51 to 55, wherein the cell is an induced pluripotent stem cell or a cell derived by differentiation of an induced pluripotent stem cell.

57. 57. The method of any one of claims 51 to 56, wherein the cell is a pancreatic beta cell or an immune cell.

58. 58. The method of any one of claims 51 to 57, wherein the protein is B2M or a messenger RNA transcript derived from a protein-coding gene encodes the B2M protein.

59. 59. The method of any one of claims 51 to 58, wherein the expression of the protein in the cell is modified in the range of 10% to 90% compared to a control cell.

60. 60. The method of any one of claims 53 to 59, wherein the DNA editing substance comprises a DNA editing system, preferably wherein 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.

61. 61. The method of any one of claims 53 to 60, wherein the DNA editing substance does not contain an endonuclease.

62. 62. The method of any one of claims 53 to 61, wherein the DNA editing substance comprises an endonuclease.

63. 63. The method of Claim 62, wherein the endonuclease comprises Cas9.

64. 64. The method of any one of claims 61 to 63, wherein the endonuclease comprises a catalytically inactive endonuclease.

65. 65. The method of any one of claims 53 to 64, wherein the DNA editing agent is linked to a reporter for monitoring expression in the cell.

66. 66. A cell or cell population obtained from the method of any one of claims 51 to 65.

67. 67. A cell or cell population comprising a modified endogenous miRNA obtained by the method of any one of claims 51 to 66.

68. 68. A composition comprising a cell or cell population according to any one of claims 65 to 67.

69. 69. The composition of claim 68 for use in cell therapy.

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