Improved chimeric and engineered scaffolds and clusters of multiplex inhibitory RNAs

Abstract

This application relates to the field of RNA interference, and more particularly to RNA interference applied in immunotherapy, such as adoptive cell therapy (ACT). Herein, chimeric clusters of multiple shRNA scaffolds designed to downregulate multiple targets are proposed. Also proposed are polynucleotides, vectors containing shRNAs, and cells expressing such shRNAs alone or in combination with a protein of interest, such as chimeric antigen receptor (CAR) or T cell receptor (TCR). These cells are particularly suitable for use in immunotherapy.

Description

[Technical field]

[0001] This application relates to the field of RNA interference, and more particularly to RNA interference applied in immunotherapy, such as adoptive cell therapy (ACT). Herein, chimeric clusters of multiple shRNA scaffolds designed to downregulate multiple targets are proposed. Also proposed are polynucleotides, vectors containing shRNAs, and cells expressing such shRNAs alone or in combination with a protein of interest, such as chimeric antigen receptor (CAR) or T cell receptor (TCR). These cells are particularly suitable for use in immunotherapy. [Background technology]

[0002] Simultaneous downregulation of multiple targets in an efficient manner in difficult-to-transduce cells is a known problem. Multiplex genome engineering methods are often laborious. When trying to solve the problems faced in multiplexed genome engineering, systems offering the possibility of gene knockdown instead of knockout may be considered, which allows for greater flexibility (e.g., allowing for temporal regulation). Ideally, these systems should be not too laborious (so that individual proteins do not need to be modified for each target, or so that downregulation can be achieved in a single transduction step) and be sufficiently efficient and specific.

[0003] One possible solution is RNA interference (RNAi). Several mechanisms of RNAi gene regulation exist in plants and animals. The first is through the expression of small non-coding RNAs called microRNAs ("miRNAs"). miRNAs can target specific messenger RNAs ("mRNAs") for degradation, thereby promoting gene silencing.

[0004] Given the importance of the microRNA pathway in regulating gene activity, researchers are currently exploring the extent to which small interfering RNA ("siRNA"), an artificially designed molecule, can mediate RNAi. siRNA can trigger the cleavage of target molecules such as mRNA, and like miRNA, siRNA relies on base complementarity to recognize target molecules.

[0005] The class of molecules known as siRNA includes small hairpin RNAs ("shRNAs"). shRNAs are single-stranded molecules that include a sense region and an antisense region that can hybridize with the sense region. shRNAs can form a stem and loop structure, with the sense region and the antisense region forming part or all of the stem. One advantage of using shRNAs is that they can be delivered or transcribed as separate entities that can be incorporated either as a single unit or as part of a multi-component system, neither of which is reasonably possible when siRNAs have two separate strands. However, like other siRNAs, shRNAs still target mRNAs based on base complementarity.

[0006] Many conditions, diseases, and disorders are caused by the interactions of multiple proteins, and as a result, researchers are seeking effective ways to simultaneously deliver multiple siRNAs into cells and organisms. One of the delivery options is the use of vector technology to express shRNAs in cells where they are processed by the endogenous miRNA pathway. Using a separate vector for each shRNA can be cumbersome. As a result, researchers have begun to explore the use of vectors that can express multiple shRNAs. Unfortunately, the published literature describes several challenges when expressing multiple shRNAs from a single vector. Some of the problems researchers have faced include: (a) the risk of vector recombination and loss of shRNA expression, (b) reduced functionality of shRNAs due to position effects in the multiplex cassette (e.g., as a result of secondary structures), (c) the complexity of shRNA cloning, (d) saturation of RNAi processing, (e) cytotoxicity, and (f) undesirable off-target effects.

[0007] Furthermore, although siRNAs have been shown to be effective for short-term gene inhibition in certain transformed mammalian cell lines, their use in primary cell cultures and for stable transcript knockdown has proven much more challenging. Knockdown efficacy is known to vary widely, ranging from more than 10% to less than 90% (e.g., Taxman et al., 2006), and thus requires further optimization. Typically, efficacy decreases when multiple inhibitors are expressed, making this optimization even more important in these situations. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Taxman DJ, Livingstone LR, Zhang J, Conti BJ, locca HA, Williams KL, Lich JD, Ting JP, Reed W. Criteria for effective design, construction, and gene knockdown by shRNA vectors. BMC Biotechnol. 2006 Jan 24;6:7. Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, there remains a need to develop efficient cassettes and vectors for the delivery of multiplexed RNA interference molecules, which is true for cellular applications in general but is less explored in the field of ACT, and there is a high need for efficient systems in these cells.

[0010] Thus, there is a need in the art to provide a system that allows for cell therapy with multiplexed knockdown of targets without the need for multi-step production methods (thus making manufacturing relatively easy and reducing cost) and that offers flexibility (e.g., by making the changes reversible, allowing for attenuation of knockdown (e.g., to avoid toxicity) or by swapping out one target for another). [Means for solving the problem]

[0011] (Summary of the invention) Surprisingly, it has been demonstrated herein that by using scaffolds of the miR-17 miRNA family cluster (i.e., one of the miR-17-92 paralogs), particularly multiplexed scaffolds, particularly as found in the miR-106a-363 ​​cluster, the miR-17-92 cluster, the miR106b-25 cluster, and combinations thereof, particularly chimeric combinations thereof, shRNAs can not only be successfully multiplexed in cells, particularly engineered immune cells, but multiple targets can also be downregulated very efficiently. The chimeric combinations can be chimeric clusters (using scaffolds from these three different clusters to create a new cluster) or chimeric scaffolds (at least the lower stem portion of the scaffold is derived from a different miRNA than the upper stem and / or loop portion), or a combination of both.

[0012] Therefore, the object of the present invention is defined in the following items:

[0013] 1. A nucleic acid molecule comprising at least one RNA interference molecule having a modified scaffold, the modified scaffold comprising a lower stem region and an upper stem / loop region, the lower stem region of the scaffold being of a miR scaffold from the miR-17 family cluster, and at least a portion of the upper stem / loop region of the scaffold being modified to differ from the wild-type / native sequence.

[0014] 2. The nucleic acid molecule according to item 1, wherein the lower stem region of the modified scaffold is selected from a miR-17 scaffold, a miR-18a scaffold, a miR-19a scaffold, a miR-20a scaffold, a miR-19b-l scaffold, a miR-92-1 scaffold, a miR-106a scaffold, a miR-18b scaffold, a miR-20b scaffold, a miR-19b-2 scaffold, a miR-92-2 scaffold, a miR-363 scaffold, a miR-106b scaffold, a miR-25 scaffold, and a miR-93 scaffold.

[0015] 3. The nucleic acid molecule according to item 1 or 2, wherein the modified scaffold is a chimeric scaffold, and wherein at least a portion of the upper stem / loop region is not from the same miR scaffold as the lower stem region, and wherein at least a portion of the upper stem / loop region is selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold.

[0016] 4. The nucleic acid molecule according to any one of items 1 to 3, wherein the at least one RNA interference molecule is at least two multiplexed RNA interference molecules.

[0017] 5. A nucleic acid molecule comprising at least two RNA interference molecules having different scaffolds, wherein the at least two different scaffolds have a lower stem region of a miR scaffold from the miR-17 family cluster; At least one RNA interference molecule comprises a chimeric scaffold, in which at least a portion of the upper stem / loop region is not derived from the same miR scaffold as the lower stem region, and at least a portion of the upper stem / loop region is selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold; and / or A nucleic acid molecule, wherein the at least two RNA interference molecule derived scaffolds are miR-17 family scaffolds derived from different miR-17 family clusters.

[0018] 6. A vector suitable for expression in a modified immune cell, comprising a nucleic acid molecule according to any one of items 1 to 5.

[0019] 7. A first exogenous nucleic acid molecule encoding a protein of interest; A modified cell comprising: a second nucleic acid molecule comprising at least one RNA interference molecule having a modified scaffold, wherein the lower stem region of the scaffold is that of a miR scaffold from the miR-17 family cluster, and at least a portion of the upper stem / loop region of the scaffold is modified to differ from the wild-type / native sequence.

[0020] 8. A first exogenous nucleic acid molecule encoding a protein of interest; a second nucleic acid molecule comprising at least two RNA interference molecules having different scaffolds, the at least two different scaffolds comprising a lower stem region of a miR scaffold from a miR-17 family cluster; At least one RNA interference molecule comprises a chimeric scaffold, in which at least a portion of the upper stem / loop region is not derived from the same miR scaffold as the lower stem region, and at least a portion of the upper stem / loop region is selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold; and / or The at least two scaffolds derived from RNA interference molecules are miR-17 family scaffolds derived from different miR-17 family clusters; Modified cells.

[0021] 9. The modified cell according to item 7 or 8, which is a modified immune cell.

[0022] 10. The modified immune cell of item 9, wherein the immune cell is selected from a T cell, a NK cell, a NKT cell, a macrophage, a stem cell, a progenitor cell, and an iPSC cell.

[0023] 11. The modified cell according to any one of items 7 to 10, wherein the protein of interest is a receptor, in particular a chimeric antigen receptor or a TCR.

[0024] 12. The modified cell according to any one of items 7 to 11, wherein the at least one RNA interference molecule is at least two multiplexed RNA interference molecules under the control of one promoter.

[0025] 13. The modified cell of item 12, wherein the at least two multiplexed RNA interference molecules are at least three multiplexed RNA interference molecules.

[0026] 14. The molecule targeted by the at least one RNA interference molecule is selected from the group consisting of MHC class I genes, MHC class II genes, MHC co-receptor genes (e.g., HLA-F, HLA-G), TCR chains, NKBBiL, LTA, TNF, LTB, LST1, NCR3, AIF1, LY6, heat shock proteins (e.g., HSPA1L, HSPA1A, HSPA1B), complement cascade, and regulatory receptors (e.g., NOTCH4). , TAP, HLA-DM, HLA-DO, RING1, CD52, CD247, HCP5, B2M, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, 2B4, A2AR, BAX, BLIMP1, C160(POLR3A), CBL-B, CCR6, CD7, CD27, CD28, CD38, CD95, CD96, CD123, CD272(BTLA), CD276(aka B7-H3), CIITA, CTLA4, DGK[DGKA, DGKB, DGKD, DGKE, DKGG, DGKH, DGKI, DGKK, DGKQ, DGKZ], DNMT3A, DR4, DR5, EGR2, FABP4, FABP5, FASN, GMCSF, HPK1, IL-10R[IL10RA, IL10RB], IL2, LAG3 (CD223), LFA1, NEAT1, NFkB (including RELA, RELB, NFkB2, NFkBl, REL), NKG2A, NR4A (including NR4A1, NR4A2, NR4A3), PD1, PI3KCD, PPP2RD2, PRAS40, RAPTOR, SHIP1, SOAT1, SOCS1, T-BET, TCF7 (aka 14. The nucleic acid molecule according to any one of items 1 to 5, the vector according to item 6, or the modified cell according to any one of items 7 to 13, which is selected from TCF-1), TET2, TGFBR1, TGFBR2, TGFBR3, TIGIT, TIM3 (aka HAVCR2 or CD366), TOX, TOX2, VISTA (aka VSIR or B7-H5), ZC3H12A (also known as Regnase 1 or MCPIP) and ZFP36L2.

[0027] 15. A nucleic acid molecule according to any one of items 1 to 5, a vector according to item 6, or a modified cell according to any one of items 7 to 14 for use as a medicament.

[0028] 16. A nucleic acid molecule according to any one of items 1 to 5, a vector according to item 6, or a modified cell according to any one of items 7 to 14 for use in the treatment of cancer.

[0029] 17. A method for treating cancer, comprising administering to a subject in need thereof an appropriate dose of a cell according to any one of items 7 to 14, thereby ameliorating at least one symptom.

[0030] 18. The nucleic acid molecule according to any one of items 1 to 5 or the vector according to item 6, wherein the lower stem region and / or up to 10 nucleotides flanking this region from the 3' and / or 5' side have been modified differently from the wild-type / native sequence.

[0031] It is therefore an object of the present invention to provide a vector comprising a nucleic acid sequence comprising at least one RNA interference molecule with a scaffold selected from those present in the miR-106a-363 ​​cluster, in particular a scaffold selected from the miR-106a scaffold, the miR-18b scaffold, the miR-20b scaffold, the miR-19b-2 scaffold, the miR-92-2 scaffold and the miR-363 scaffold. According to a particular embodiment, the vector is suitable for expression in eukaryotic cells, in particular immune cells. The RNA interference molecule typically also comprises a target sequence that is not present in the wild-type / natural scaffold sequence. Typically, this is achieved by replacing the wild-type / naturally occurring target sequence in the microRNA scaffold (typically referred to as the mature sequence) with an optimal target sequence, e.g. a target sequence that matches the mRNA sequence encoding the target protein. Most particularly, the target sequence has a length of 18-23 nucleic acids. The complementary strand of the target sequence is typically referred to as the passenger sequence.

[0032] According to specific embodiments, at least one of the scaffolds of one or more RNA interference molecules is a scaffold selected from miR-106a scaffold, miR-18b scaffold, and miR-20b scaffold.In other words, according to these specific embodiments, a vector is provided that includes a nucleic acid sequence encoding at least one RNA interference molecule having a scaffold selected from those present in the first three scaffolds of miR-106a-363 ​​cluster, i.e., a scaffold selected from miR-106a scaffold, miR-18b scaffold, and miR-20b scaffold. For example, at least one RNA interference molecule can have a miR-106a scaffold, while other RNA interference molecules can have an independently selected scaffold, such as a scaffold independently selected from a miR-106a scaffold, a miR-18b scaffold, a miR-20b scaffold, a miR-19b-2 scaffold, a miR-92-2 scaffold, and a miR-363 scaffold.

[0033] According to certain embodiments, a plurality of RNA interference molecules will be present in the vector. According to these embodiments, the at least one RNA interference molecule is therefore at least two RNA interference molecules, in particular at least two multiplexed RNA interference molecules. According to these embodiments, therefore, a vector is provided comprising a nucleic acid sequence encoding at least two RNA interference molecules with a scaffold selected from those present in the miR-106a-363 ​​cluster, the miR-17-92 cluster, and the miR106b-25 cluster. In particular, at least one of the scaffolds is chimeric (the lower stem region of the scaffold is from a different miRNA scaffold than the upper stem and / or loop region of the scaffold), most particularly, the upper stem and loop region of the scaffold are from a scaffold of the miR-17 family. According to another (but not exclusive) embodiment, the at least two RNA interference molecules have scaffolds selected from miR-17 family scaffolds from at least two different clusters selected from miR-106a-363 ​​cluster, miR-17-92 cluster, and miR106b-25 cluster. In other words, the at least two RNA interference molecules have scaffolds selected from at least two of the following three groups: miR-106a scaffold and miR-20b scaffold; miR-17 scaffold and miR-20a scaffold; miR-106b scaffold and miR-93 scaffold.

[0034] When there are at least two multiplexed RNA interference molecules, these two or more molecules may have the same or different scaffolds. However, it is particularly envisaged that no more than three of the scaffolds are identical, and even more particularly, it is envisaged that no more than two identical scaffolds are used. This is to avoid recombination between identical scaffold sequences (see Example 5). For this reason, it is particularly envisaged to use chimeric clusters and / or clusters with chimeric scaffold sequences, as outlined above.

[0035] According to a specific embodiment, the scaffold present in the vector is exclusively selected from the 15 scaffolds present in the miR-106a-363 ​​cluster, the miR-17-92 cluster, and the miR106b-25 cluster (i.e., selected from the miR-17 scaffold, the miR-18a scaffold, the miR-19a scaffold, the miR-20a scaffold, the miR-19b-1 scaffold, the miR-92-1 scaffold, the miR-106a scaffold, the miR-18b scaffold, the miR-20b scaffold, the miR-19b-2 scaffold, the miR-92-2 scaffold, the miR-363 scaffold, the miR-106b scaffold, the miR-25 scaffold, and the miR-93 scaffold). As outlined above, these can also be chimeric scaffolds, with the lower stem portion selected from one of these 15 scaffolds and the upper stem and loop portion selected from the miR-17 family scaffolds (i.e. selected from miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b scaffolds). However, it is also envisaged to further combine these with different scaffold sequences, in particular different unrelated sequences (to avoid recombination) such as the miR-196a2 sequence.

[0036] According to certain embodiments, the scaffold sequence may be modified to reduce the number of mismatches and / or bulges in the stem region. More particularly, when one of the scaffold sequences used is a miR-18b scaffold, the scaffold may be modified (and modified compared to the wild-type / native sequence) to reduce the number of mismatches and / or bulges in the stem region (see Example 3).

[0037] According to a further aspect, there is provided herein a modified cell comprising a nucleic acid molecule comprising at least two RNA interference molecules having different scaffolds, wherein the at least two different scaffolds have lower stem regions of miR scaffolds from a miR-17 family cluster, and at least one RNA interference molecule has a chimeric scaffold, wherein at least a portion of the upper stem / loop region is not from the same miR scaffold as the lower stem region, and wherein the at least a portion of the upper stem / loop region is selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold, and / or the scaffolds from the at least two RNA interference molecules are miR-17 family scaffolds from different miR-17 family clusters.

[0038] The RNA interference molecules typically also contain a target sequence that is not present in the wild-type / native scaffold sequence. For this purpose, the mature sequence of each miRNA scaffold is replaced with an optimal target sequence. The target sequence typically has a length of 18-23 nucleic acids. It is particularly envisaged that the target sequence is directed against a sequence present in the modified cell, in particular a target sequence. That is, at least one RNA interference molecule has a sequence that targets (by base pair complementarity) a sequence in the modified cell that encodes a protein to be downregulated.

[0039] According to a further embodiment, a first exogenous nucleic acid molecule encoding a protein of interest; and a second nucleic acid molecule comprising at least one RNA interference molecule having a modified scaffold, wherein the lower stem region of the scaffold is of a miR scaffold from the miR-17 family cluster and at least a portion of the upper stem / loop region of the scaffold is modified to differ from the wild-type / native sequence.

[0040] According to a further embodiment, the modified cells comprise: A first exogenous nucleic acid molecule encoding a protein of interest A second nucleic acid molecule comprising at least two RNA interference molecules having different scaffolds, wherein the at least two different scaffolds have lower stem regions of miR scaffolds derived from a miR-17 family cluster, and at least one RNA interference molecule has a chimeric scaffold, wherein at least a portion of the upper stem / loop region is not derived from the same miR scaffold as the lower stem region, and wherein at least a portion of the upper stem / loop region is selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold, and / or the scaffolds from the at least two RNA interference molecules are miR-17 family scaffolds derived from different miR-17 family clusters.

[0041] It should be understood that the first and second exogenous nucleic acid molecules can be provided in a single vector, or alternatively, can be provided as separate nucleic acid molecules.

[0042] According to certain embodiments, the at least one RNA interference molecule comprises a target sequence within the scaffold that is different from the wild-type / native target sequence of the scaffold (i.e., different from the mature strand of the miRNA scaffold). The target sequence is typically 18-23 nucleotides in length. According to certain embodiments, the RNA interference molecule is directed against a target in the modified cell by base pair complementarity of the target sequence.

[0043] When there are at least two multiplexed RNA interference molecules, these two or more molecules may have the same or different scaffolds. However, it is particularly envisaged that no more than three of the scaffolds are identical, and even more particularly, it is envisaged that no more than two identical scaffolds are used. This is to avoid recombination between identical scaffold sequences (see Example 5).

[0044] The modified cell is in particular a eukaryotic cell, more particularly a modified mammalian cell, more particularly a modified human cell. According to a particular embodiment, the cell is a modified immune cell. Exemplary immune cells are selected from T cells, NK cells, NKT cells, macrophages, stem cells, progenitor cells, and iPSC cells.

[0045] According to certain embodiments, the modified cell further comprises a nucleic acid encoding a protein of interest. In particular, this protein of interest is a receptor, in particular a chimeric antigen receptor or a TCR. The chimeric antigen receptor or modified TCR can be directed against any target, typical examples being CD19, CD20, CD22, CD30, BCMA, B7H3, B7H6, NKG2D, HER2, HER3, GPC3, MUC1, MUC16, TAG72, but many others are also suitable. According to certain embodiments, there can be one or more proteins of interest. In such a case, the second (or further) protein can be a receptor, or can be, for example, a cytokine, a chemokine, a hormone, an antibody, a histocompatibility antigen (e.g., HLA-E), a tag, or any other protein that has therapeutic or diagnostic value or allows detection.

[0046] According to certain embodiments, the first and second nucleic acid molecules are present in a single vector, such as a eukaryotic expression plasmid, a minicircle DNA, or a viral vector (e.g., derived from lentivirus, retrovirus, adenovirus, adeno-associated virus, and Sendai virus).

[0047] The at least two multiplexed RNA interference molecules can be at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or even more molecules, depending on the number of target molecules to be downregulated and the practical considerations of co-expressing the multiplexed molecules. According to a particular embodiment, at least three multiplexed RNA interference molecules are used. According to a further particular embodiment, at least one of the at least three RNA interference molecules has a scaffold selected from miR-106a scaffold and miR-20b scaffold. According to another embodiment, at least one of the at least three RNA interference molecules has a scaffold selected from miR-106a scaffold and miR-18b scaffold.

[0048] According to certain embodiments, the scaffold sequence may be modified to reduce the number of mismatches and / or bulges in the stem region. More particularly, when one of the scaffold sequences used is a miR-18b scaffold, the scaffold may be modified (and modified compared to the wild-type / native sequence) to reduce the number of mismatches and / or bulges in the stem region (see Example 3).

[0049] A "multiplex" is a polynucleotide that encodes multiple molecules of the same type, such as multiple siRNAs or shRNAs or miRNAs. Within a multiplex, when the molecules are of the same type (e.g., all shRNAs), they may be identical or may contain different sequences. Between molecules of the same type, there may be an intervening sequence, such as a linker, as described herein. One example of a multiplex of the present invention is a polynucleotide that encodes multiple tandem miRNA-based shRNAs. A multiplex may be single-stranded, double-stranded, or may have both single-stranded and double-stranded regions.

[0050] According to a particular embodiment, said at least two multiplexed RNA interference molecules are under the control of one promoter. Typically, this promoter is not a U6 promoter, since this promoter is associated with toxicity, especially at high expression levels. For the same reason, it is possible to consider excluding the H1 promoter (a weaker promoter than U6) or even PolIII promoters in general (although they may be suitable under certain conditions). According to a particular embodiment, said promoter is selected from PolII promoters and PolIII promoters. According to a particular embodiment, said promoter is a wild-type / natural or synthetic PolII promoter. According to a particular embodiment, the promoter is a PolII promoter selected from the group consisting of cytomegalovirus (CMV) promoter, elongation factor 1 alpha (EF1α) promoter (core or full length), phosphoglycerate kinase (PGK) promoter, composite beta-actin promoter (CAG promoter) with upstream CMV IV enhancer, ubiquitin C (UbC) promoter, spleen limited focus forming virus (SFFV) promoter, Rous sarcoma virus (RSV) promoter, interleukin 2 promoter, murine stem cell virus (MSCV) long terminal repeat (LTR), gibbon ape leukemia virus (GALV) LTR, simian virus 40 (SV40) promoter, and tRNA promoter. These promoters are among the most commonly used polymerase II promoters to drive mRNA expression, and common housekeeping gene promoters can be used as well.

[0051] According to certain embodiments, at least two multiplexed RNA interference molecules can be shRNA molecules or miRNA molecules. Most particularly, they are miRNA molecules. The difference between shRNA molecules and miRNA molecules is that miRNA molecules are processed by Drosha, whereas conventional shRNA molecules are not (which is associated with toxicity, Grimm et al., Nature 441:537-541 (2006)).

[0052] According to a specific embodiment, the different miRNA molecules are under the control of one promoter.

[0053] According to a particular embodiment, at least two of the multiplexed RNA interference molecules are directed to the same target. However, even RNA interference molecules directed to the same target may have different scaffold sequences and / or different target sequences. According to a further specific embodiment, at least two of the multiplexed RNA interference molecules have the same scaffold but different target sequences. According to another specific embodiment, at least two of the multiplexed RNA interference molecules have different scaffolds but the same target sequences. According to a specific embodiment, at least two of the multiplexed RNA interference molecules are identical.

[0054] According to another embodiment, the at least two multiplexed RNA interference molecules are all different. According to a further specific embodiment, the at least two multiplexed RNA interference molecules are all directed to different targets. It should be noted that the RNA interference molecules directed to different targets may have the same scaffold (but have different target sequences).

[0055] Any suitable molecule present in the modified cells can be targeted by the RNA interference molecule. Typical examples of possible targets are: MHC class I genes, MHC class II genes, MHC co-receptor genes (e.g., HLA-F, HLA-G), TCR chains, CD3 chains, NKBBiL, LTA, TNF, LTB, LST1, NCR3, AIF1, LY6, heat shock proteins (e.g., HSPA1L, HSPA1A, HSPA1B), complement cascade, regulatory receptors (e.g., NOTCH4), TAP, HLA-DM, HLA -DO, RING1, CD52, CD247, HCP5, B2M, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, 2B4, A2AR, BAX , BLIMP1, C160(POLR3A), CBL-B, CCR6, CD7, CD27, CD28, CD38, CD95, CD96, CD123, CD272(BTLA), CD276(aka B7-H3), CIITA, CTLA4, DGK[DGKA, DGKB, DGKD, DGKE, DKGG, DGKH, DGKI, DGKK, DGKQ, DGKZ], DNMT3A, DR4, DR5, EGR2, FABP4, FABP5, FASN, GMCSF, HPK1, IL-10R[IL10RA, IL10RB], IL2, LAG3 (CD223), LFA1, NEAT1, NFkB (including RELA, RELB, NFkB2, NFkBl, REL), NKG2A, NR4A (including NR4A1, NR4A2, NR4A3), PD1, PI3KCD, PPP2RD2, PRAS40, RAPTOR, SHIP1, SOAT1, SOCS1, T-BET, TCF7 (aka TCF-1), TET2, TGFBR1, TGFBR2, TGFBR3, TIGIT, TIM3 (aka HAVCR2 or CD366), TOX, TOX2, VISTA (aka VSIR or B7-H5), ZC3H12A (also known as Regnase 1 or MCPIP) and ZFP36L2.

[0056] Particularly suitable constructs have been identified that are miRNA-based. Accordingly, modified cells are provided that include a polynucleotide that includes a microRNA-based shRNA coding region, the microRNA-based shRNA coding region comprising: a sequence encoding one or more artificial miRNA-based shRNA nucleotide sequences, each artificial miRNA-based shRNA nucleotide sequence comprising: miRNA scaffold sequence, the active or mature sequence, and Within each artificial miRNA-based shRNA nucleotide sequence, including a passenger or star sequence, the active sequence is at least 70% complementary to the passenger sequence.

[0057] According to certain embodiments, the active sequence is at least 80% complementary to the passenger sequence, and may be at least 90% or more complementary to the passenger sequence.

[0058] A particular advantage is that the miRNA-based shRNA nucleotide sequences of the present invention can be multiplexed. Thus, modified cells are provided that include a polynucleotide comprising a multiplexed microRNA-based shRNA coding region, said multiplexed microRNA-based shRNA coding region comprising: A sequence encoding two or more artificial miRNA-based shRNA nucleotide sequences, each artificial miRNA-based shRNA nucleotide sequence comprising: miRNA scaffold sequence, the active or mature sequence, and Within each artificial miRNA-based shRNA nucleotide sequence, including a passenger or star sequence, the active sequence is at least 70% complementary to the passenger sequence.

[0059] Both the active sequence and the passenger sequence of each of the artificial miRNA-based shRNA nucleotide sequences are typically 18-40 nucleotides long, more particularly 18-30 nucleotides long, more particularly 18-25 nucleotides long, and most particularly 18-23 nucleotides long. The active sequence can also be 18 or 19 nucleotides long. Typically, the passenger sequence has the same length as the active sequence, although the possible presence of bulges means that they are not necessarily the same length.

[0060] Typically, these microRNA scaffold sequences are separated by linkers. According to certain embodiments, at least a portion of the 5' and / or 3' linker sequences are used with their respective scaffolds.

[0061] The artificial sequence can be, for example, a wild-type / naturally occurring scaffold (e.g., a miR cluster or fragment thereof, e.g., miR-106a-363 ​​cluster) in which the endogenous miR sequence has been replaced with a modified shRNA sequence against a specific target, or it can be a repeat of a single miR scaffold (such as, for example, the miR-20b scaffold) in which the endogenous miR sequence has been replaced with a modified shRNA sequence against a specific target, or it can be a chimeric sequence combining elements (e.g., lower stem, upper stem and loop regions) from two different miRNA scaffolds, artificial miR-like sequences, or a combination thereof.

[0062] The modified cell typically further comprises a nucleic acid molecule encoding a protein of interest, such as a chimeric antigen receptor or TCR, and may be a modified immune cell, as described above.

[0063] The expression of the at least one RNA interference molecule or the co-expression of the multiplexed RNA interference molecules results in the suppression of at least one gene, and typically multiple genes, in the modified cell, which may contribute to a higher therapeutic efficacy.

[0064] The modified cells described herein are also provided for use as a medicament. According to certain embodiments, the modified cells are provided for use in the treatment of cancer.

[0065] This is equivalent to providing a method of treating cancer comprising administering to a subject in need thereof an appropriate dose of the modified cells described herein, thereby ameliorating at least one symptom.

[0066] The modified cells can be autologous immune cells (cells obtained from the patient) or allogeneic immune cells (cells obtained from another subject). [Brief description of the drawings]

[0067] [Figure 1] Schematic representation of the clustered scaffold, showing regions such as the target sequence, the upper stem, the lower stem and the scaffold. [Diagram 2] CAR expression vectors (e.g., CD19, BCMA, B7H3, B7H6, NKG2D, HER2, HER3, GPC3) without (top) or with (bottom) miRNA scaffolds are shown, with designs allowing simultaneous expression of CAR and multiple shRNAs (e.g., 2, 4, 6, 8, ...) from the same vector. LTR: long terminal repeat; promoter (e.g., EF1α, PGK, SFFV, CAG, ...); marker protein (e.g., truncated CD34, CD19); multiplexed shRNA. [Diagram 3]The use of wild type / native mRNA clusters increases transduction efficiency compared to repetitively modified single scaffolds. T cells were transduced with different vectors encoding CD19CAR and three to six multiplexed scaffolds according to the design shown in Figure 2. CD34 was used as a reporter gene and the % of CD34+ T cells measured by FACS 4 days after transduction is shown in the lower panel. The upper panel shows the same but after purification (amount of cells eluted from the purification column divided by the amount of cells loaded onto the purification column). 1-2: scaffolds from the miR-17-92 cluster, four (miR-19a, miR-20a, miR-19bl, miR-92al) and three scaffolds (miR-19a, miR-20a, miR-19bl) respectively; 3-5: scaffolds from the miR-106a-363 ​​cluster, six (all), three (last three) and four (last four) respectively; 6: all three scaffolds from the 106b-25 cluster; 7: all three scaffolds from the miR-23a-27a-24-2 cluster; 8-9: four and three repeats of the miR-196a2 scaffold sequence, respectively; 10: mock vector with only the CD34 tag. The target genes included in the constructs were B2M, CD52 and CD247 in the triplex scaffold and TRAC as an additional gene in the tetraplex scaffold. The hexaplex scaffold targeted each target gene twice, using two different target sequences per target. [Figure 4]Comparison of CD247 (CD3 zeta) knockdown between the 23a-27a-24-2 cluster and the miR-106a-363 ​​cluster, assessed by TCR expression by FACS. 1: mock vector with only the CD34 tag; 2: all three scaffolds derived from the miR-23a-27a-24-2 cluster (CD247 target sequence in the miR-24-2 scaffold); 3-5: scaffolds derived from the miR-106a-363 ​​cluster, six (all), three (last 3) and four (last 4), respectively. The CD247 target sequence is in the miR-363 scaffold; in 3, an additional different sequence is included in the miR-20b scaffold. [Diagram 5] The miRNA106a-363 ​​cluster and the design of the construct used are shown in FIG. [Figure 6] RNA expression in primary T cells from healthy donors transduced with a retroviral vector encoding a second generation CD19-directed CAR, a truncated CD34 selection marker, together with 3x shRNA or 6x shRNA targeting CD247, B2M or CD52 introduced into the 106a-363 ​​miRNA cluster. No shRNA (tCD34) was used as a control. Two days after transduction, cells were enriched using CD34-specific magnetic beads and further amplified in IL-2 (100 lU / mL) for six days. mRNA expression of CD247, B2M, and CD52 was assessed by qRT-PCR using cyclophilin as a housekeeping gene. [Figure 7] Comparison of different shRNA target sequences allowing fine tuning of knockdown levels. 12 different target sequences, all directed against CD247, were evaluated on the miR-20b scaffold. T cells were harvested 12 days after activation (10 days after transduction). TCRab levels were measured by FACS: MFI is presented as a bar graph. All shRNAs achieved at least 50% knockdown, some much more efficient. [Figure 8]Knockdown of CD95 in miR-18b scaffold. Shown are sequences selected from 31 different target sequences, all directed against CD95, and evaluated in miR-18b scaffold. T cells were harvested 16 days after activation (14 days after transduction). CD95 levels were measured by FACS: MFI is presented as a bar graph. The most efficient shRNAs achieved approximately 30% knockdown. [Figure 9] Comparison of miR-106a, miR-18b and miR-20b scaffold structures. The target sequence (here 20 bp long) and passenger strand are shown as rectangles. miR-106a and miR-20b have a mismatch at position 18 of the scaffold (position 14 of the target sequence), while the scaffold of miR-18b is larger, with mismatches at positions 6, 11 and 15 of the target sequence (indicated by arrows 2, 3 and 4, respectively), and two nucleic acid bulges in the passenger strand between positions 1 and 2 of the target sequence (indicated by arrow 1). [Figure 10A] Modifications of the miR-18b scaffold improve knockdown efficiency. Figure 10A shows modifications made to the miR-18b scaffold: removal of the bulge, removal of individual mismatches, and removal of the bulge and the first two mismatches. Figure 10B shows the effect of knockdown of CD95 in these miR-18b scaffolds: constructs with fewer mismatches or bulges compared to the wild type / native sequence achieve higher knockdown efficiency. Knockdown is measured as in Figure 8. [Figure 10B] Modifications of the miR-18b scaffold improve knockdown efficiency. Figure 10A shows modifications made to the miR-18b scaffold: removal of the bulge, removal of individual mismatches, and removal of the bulge and the first two mismatches. Figure 10B shows the effect of knockdown of CD95 in these miR-18b scaffolds: constructs with fewer mismatches or bulges compared to the wild type / native sequence achieve higher knockdown efficiency. Knockdown is measured as in Figure 8. [Figure 11]Evaluation of target sequence length. The effect of target sequence length on knockdown efficiency was evaluated for both target sequences against B2M (left panel) and CD247 (right panel). Constructs are optionally labeled with two lengths (19-20, 21-22, or 22-23) since the wild type / native scaffold sequence is identical to the target sequence at that position. Results shown are for the miR-106a scaffold, similar results were obtained for the miR-20b scaffold (not shown). Cluster: control with irrelevant sequence; as additional controls, target sequences against CD247 and B2M, respectively, were used. [Figure 12A] Figure 12A-C: Evaluation of simultaneous knockdown of various genes using different permutations of scaffolds. A: FACS data showing expression of B2M / HLA (left panel) and CD247 / CD3 zeta (right panel) for the indicated duplex and triplex scaffolds. B: MFI of FACS data of panel A, now including expression of CD95 for triplex scaffolds. C: MFI of FACS data showing expression of B2M, CD247, and CD95 for the indicated constructs. [Figure 12B] Figure 12A-C: Evaluation of simultaneous knockdown of various genes using different permutations of scaffolds. A: FACS data showing expression of B2M / HLA (left panel) and CD247 / CD3 zeta (right panel) for the indicated duplex and triplex scaffolds. B: MFI of FACS data of panel A, now including expression of CD95 for triplex scaffolds. C: MFI of FACS data showing expression of B2M, CD247, and CD95 for the indicated constructs. [Figure 12C]Figure 12A-C: Evaluation of simultaneous knockdown of various genes using different permutations of scaffolds. A: FACS data showing expression of B2M / HLA (left panel) and CD247 / CD3 zeta (right panel) for the indicated duplex and triplex scaffolds. B: MFI of FACS data of panel A, now including expression of CD95 for triplex scaffolds. C: MFI of FACS data showing expression of B2M, CD247, and CD95 for the indicated constructs. [Figure 13] Evaluation of scaffold changes and their impact on KD efficacy. A) Flow cytometry data of four different CAR T cells containing no shRNA (top), duplex shRNA against B2M (in miR-106a scaffold) and CD3 zeta (in miR-20b scaffold) (second from top), a triplex of miR-106a, miR-18b, and miR-20b scaffolds containing a loop mutant in scaffold miR-18b, and a similar triplex in which the upper stem / loop region of scaffold miR-18b was replaced with the upper stem / loop region of miR-17 (bottom). Expression of HLA-I (left panel), CD95 (middle) or TCR expression (right panel) is shown. [Figure 14A] Assessment of target sequence variation when multiplexes show stable microprocessing. A) Flow cytometry expression of shRNA targets in BCMA CAR T cells transduced without shRNA (top), duplexes or triplexes (B2M, CD3 zeta, CD95) against CD3 zeta and B2M, containing various target sequences of B2M. The left panel shows HLA class I expression, the middle panel shows CD95 expression, and the right panel shows TCR expression. B) Mean fluorescence intensity of each target protein normalized to the no-shRNA group. [Figure 14B]Assessment of target sequence variation when multiplexes show stable microprocessing. A) Flow cytometry expression of shRNA targets in BCMA CAR T cells transduced without shRNA (top), duplexes or triplexes (B2M, CD3 zeta, CD95) against CD3 zeta and B2M, containing various target sequences of B2M. The left panel shows HLA class I expression, the middle panel shows CD95 expression, and the right panel shows TCR expression. B) Mean fluorescence intensity of each target protein normalized to the no-shRNA group. [Figure 15A] Functional assessment of different shRNA targets when knocked down by chimeric triplex scaffolds. A) shRNA against B2M protects BCMA CAR T cells against NK killing compared to B2M knockout with Crispr cas9. B) shRNA against B2M inhibits T cell allo-recognition similar to B2M knockout. C) shRNA against CD95 protects against FasL-induced apoptosis. [Figure 15B] Functional assessment of different shRNA targets when knocked down by chimeric triplex scaffolds. A) shRNA against B2M protects BCMA CAR T cells against NK killing compared to B2M knockout with Crispr cas9. B) shRNA against B2M inhibits T cell allo-recognition similar to B2M knockout. C) shRNA against CD95 protects against FasL-induced apoptosis. [Figure 15C] Functional assessment of different shRNA targets when knocked down by chimeric triplex scaffolds. A) shRNA against B2M protects BCMA CAR T cells against NK killing compared to B2M knockout with Crispr cas9. B) shRNA against B2M inhibits T cell allo-recognition similar to B2M knockout. C) shRNA against CD95 protects against FasL-induced apoptosis. [Figure 16A]Target gene knockdown using a chimeric fourplex shRNA cluster containing four shRNA scaffolds derived from three different miR17-92 paralog clusters. A) Diagram of the fourplex shRNA construct. B) Flow cytometry expression profiles of BCMA CAR T cells without shRNA (top histogram), BCMA CAR T with duplex shRNA (target antigens: B2M, CD3 zeta) and fourplex (targets: B2M, CD28, MICA, CD3 zeta). C) Relative expression of MICA measured by qPCR. [Figure 16B] Target gene knockdown using a chimeric fourplex shRNA cluster containing four shRNA scaffolds derived from three different miR17-92 paralog clusters. A) Diagram of the fourplex shRNA construct. B) Flow cytometry expression profiles of BCMA CAR T cells without shRNA (top histogram), BCMA CAR T with duplex shRNA (target antigens: B2M, CD3 zeta) and fourplex (targets: B2M, CD28, MICA, CD3 zeta). C) Relative expression of MICA measured by qPCR. [Figure 16C] Target gene knockdown using a chimeric fourplex shRNA cluster containing four shRNA scaffolds derived from three different miR17-92 paralog clusters. A) Diagram of the fourplex shRNA construct. B) Flow cytometry expression profiles of BCMA CAR T cells without shRNA (top histogram), BCMA CAR T with duplex shRNA (target antigens: B2M, CD3 zeta) and fourplex (targets: B2M, CD28, MICA, CD3 zeta). C) Relative expression of MICA measured by qPCR. [Figure 17A]Target gene knockdown using a chimeric fiveplex shRNA cluster containing five shRNA scaffolds derived from three different miR17-92 paralog clusters. A) Flow cytometry expression profiles of BCMA CAR T cells without shRNA (top histogram), a fiveplex shRNA with non-optimized target sequence duplex (fiveplex 1) and BCMA CAR T containing two fiveplex clusters with the same optimized target sequences in different order (shRNA target antigens: B2M, CD3 zeta, CD28, MICA, CD95). B) Relative expression of MICA measured by qPCR. Fp1, 2, 3: fiveplex 1, 2, 3. No sh: CAR T with no shRNA construct. [Figure 17B] Target gene knockdown using a chimeric fiveplex shRNA cluster containing five shRNA scaffolds derived from three different miR17-92 paralog clusters. A) Flow cytometry expression profiles of BCMA CAR T cells without shRNA (top histogram), a fiveplex shRNA with non-optimized target sequence duplex (fiveplex 1) and BCMA CAR T containing two fiveplex clusters with the same optimized target sequences in different order (shRNA target antigens: B2M, CD3 zeta, CD28, MICA, CD95). B) Relative expression of MICA measured by qPCR. Fp1, 2, 3: fiveplex 1, 2, 3. No sh: CAR T with no shRNA construct. [Figure 18A]Target gene knockdown using fiveplex chimeric constructs. A) Diagram of shRNA fiveplex. B) Flow cytometry expression comparing anti-BCMA CAR T cells without shRNA (lower histogram), anti-BCMA CAR T cells with triplex containing shRNA (targets: B2M, CD95, CD3 zeta) and anti-BCMA CAR T cells with fiveplex containing shRNA (targets: B2M, CD95, CD3 zeta, MICA, CD28; middle and top histograms, respectively). C) Relative percentage of inhibition normalized to no shRNA group using mean fluorescence intensity. [Figure 18B] Target gene knockdown using fiveplex chimeric constructs. A) Diagram of shRNA fiveplex. B) Flow cytometry expression comparing anti-BCMA CAR T cells without shRNA (lower histogram), anti-BCMA CAR T cells with triplex containing shRNA (targets: B2M, CD95, CD3 zeta) and anti-BCMA CAR T cells with fiveplex containing shRNA (targets: B2M, CD95, CD3 zeta, MICA, CD28; middle and top histograms, respectively). C) Relative percentage of inhibition normalized to no shRNA group using mean fluorescence intensity. [Figure 18C] Target gene knockdown using fiveplex chimeric constructs. A) Diagram of shRNA fiveplex. B) Flow cytometry expression comparing anti-BCMA CAR T cells without shRNA (lower histogram), anti-BCMA CAR T cells with triplex containing shRNA (targets: B2M, CD95, CD3 zeta) and anti-BCMA CAR T cells with fiveplex containing shRNA (targets: B2M, CD95, CD3 zeta, MICA, CD28; middle and top histograms, respectively). C) Relative percentage of inhibition normalized to no shRNA group using mean fluorescence intensity. [Figure 19A]Target gene knockdown using sixplex chimeric constructs. A) Flow cytometry expression (targets from left to right: CD38, B2M, CD95, CD3 zeta, CD28, CD27) comparing anti-BCMA CAR T cells without shRNA (lower histogram) and sixplex containing shRNA (upper histogram). B) Relative percentage of inhibition normalized to no shRNA group using mean fluorescence intensity. [Figure 19B] Target gene knockdown using sixplex chimeric constructs. A) Flow cytometry expression (targets from left to right: CD38, B2M, CD95, CD3 zeta, CD28, CD27) comparing anti-BCMA CAR T cells without shRNA (lower histogram) and sixplex containing shRNA (upper histogram). B) Relative percentage of inhibition normalized to no shRNA group using mean fluorescence intensity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] Detailed Description of the Invention definition The present invention will be described with respect to specific embodiments and with reference to certain drawings, but the present invention is not limited thereto, but only by the claims. Any reference numbers in the claims should not be construed as limiting the scope thereof. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn to scale for illustrative purposes. When the term "comprising" is used in the present specification and claims, it does not exclude other elements or steps. When an indefinite or definite article is used when referring to a singular noun, such as "a" or "an" or "the", the plural of that noun is also included unless specifically stated otherwise.

[0069] Moreover, the terms first, second, third, etc., in the specification and claims are used to distinguish between similar elements and not necessarily to describe an order or chronology. The terms so used are interchangeable under appropriate circumstances, with it being understood that the embodiments of the invention described herein are capable of operation in arrangements other than those described or illustrated herein.

[0070] The following terms or definitions are presented solely to aid in the understanding of the invention.

[0071] Practitioners are directed to, inter alia, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (up to Supplement 114), John Wiley & Sons, New York (2016) for definitions and terms of skill in the art. The definitions provided herein should not be construed to be narrower than would be understood by a person of ordinary skill in the art.

[0072] As used herein, an "modified cell" is a cell that has been modified by human intervention (as opposed to a naturally occurring mutation).

[0073] The term "nucleic acid molecule", also referred to interchangeably as "nucleotide" or "nucleic acid" or "polynucleotide", as used herein, refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Nucleic acid molecules include, but are not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that may be single-stranded, or more typically, double-stranded or a mixture of single-stranded and double-stranded regions. Furthermore, "polynucleotide" refers to triple-stranded regions containing RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA that contains one or more modified bases and DNA or RNA whose backbones have been modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA. Thus, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as the DNA and RNA chemical forms characteristic of viruses and cells. "Polynucleotide" also encompasses relatively short nucleic acid strands often referred to as oligonucleotides.

[0074] A "vector" is a replicon, such as a plasmid, phage, cosmid, or virus, into which another nucleic acid segment can be operatively inserted to bring about the replication or expression of the segment. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell capable of stable growth in vitro for several generations. In some examples provided herein, cells are transformed by transfecting the cells with DNA.

[0075] As used herein, "to differ or differs" with respect to a sequence, in particular "different from a wild-type sequence", means that the sequence has been altered compared to the wild-type / natural sequence, either by substitution, deletion and / or insertion of nucleic acids. In particular, "to differ or differs" with respect to differences in the upper stem / loop region may mean that at least one mismatch or bulge has been removed or introduced compared to the wild-type sequence. If no mismatch or bulge has been removed or introduced, a sequence is considered to differ from the wild-type sequence if it has less than 98% sequence identity, less than 95% sequence identity, in particular less than 90% sequence identity over its relevant length. In the context of an upper stem / loop sequence, the relevant length is the length of the upper stem / loop region. Note that in the context of a chimeric sequence in which the upper stem / loop sequence has been replaced, the appropriate wild-type sequence to compare is the sequence of the original scaffold (i.e. the scaffold corresponding to the lower stem region). A sequence that is "differentially modified" means that changes have been purposefully introduced, typically to achieve a more desirable result (e.g., improved downregulation of a target sequence or improved microprocessing of a scaffold).

[0076] The terms "express" and "produce" are used synonymously herein and refer to the biosynthesis of a gene product. These terms include transcription of a gene into RNA. These terms also include translation of RNA into one or more polypeptides, as well as post-transcriptional and post-translational modifications of all natural origins.

[0077] The term "exogenous" as used herein, particularly in the context of cells or immune cells, refers to any substance that is present and active in an individual living cell, but originates outside the cell (as opposed to an endogenous factor). The phrase "exogenous nucleic acid molecule" thus refers to a nucleic acid molecule that has been introduced into an (immune) cell, typically by transduction or transfection. The term "endogenous" as used herein refers to any factor or substance that is present and active in an individual living cell, and originates inside the cell (and thus is typically also produced in non-transduced or non-transfected cells).

[0078] As used herein, "isolated" means that a biological component (e.g., a nucleic acid, peptide, or protein) has been substantially separated, produced separately, or purified from other biological components of the organism in which it naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Thus, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. "Isolated" nucleic acids, peptides, and proteins may be part of a composition, but may still be isolated if such a composition is not part of the natural environment of the nucleic acid, peptide, or protein. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0079] "Multiplexing" as used herein in the context of molecular biology refers to the simultaneous targeting of two or more (i.e., multiple) related or unrelated targets. As used herein, the term "RNA interference molecule" refers to an RNA (or RNA-like) molecule that inhibits gene expression or translation by neutralizing the targeted mRNA molecule. RNA interference molecules neutralize the targeted mRNA molecule by base pair complementarity. Within the RNA interference molecule is a target sequence (typically 18-23 nucleic acids) that can hybridize to the targeted nucleic acid molecule. Examples include siRNA (including shRNA) or miRNA molecules. Thus, as used herein, a "multiplexed RNA interference molecule" is two or more molecules that exist simultaneously for the simultaneous downregulation of one or more targets. Typically, each of the multiplexed molecules is directed against a specific target, although the two molecules can be directed against the same target (and even can be identical).

[0080] As used herein, a "promoter" is a regulatory region of nucleic acid usually located adjacent to a gene region and provides a control point for regulated gene transcription.

[0081] A "multiplex" is a polynucleotide that encodes multiple molecules of the same type, such as multiple siRNAs or shRNAs or miRNAs. Within a multiplex, when the molecules are of the same type (e.g., all shRNAs), they may be identical or may contain different sequences. Between molecules of the same type, there may be an intervening sequence, such as a linker, as described herein. One example of a multiplex of the present invention is a polynucleotide that encodes multiple miRNA-based shRNAs. A multiplex may be single-stranded, double-stranded, or may have both single-stranded and double-stranded regions.

[0082] As used herein, a "chimeric antigen receptor" or "CAR" refers to a chimeric receptor (i.e., composed of moieties from different sources) that has at least a binding moiety with specificity for an antigen (which may be derived, for example, from an antibody, receptor, or its cognate ligand) and a signaling moiety capable of transmitting a signal in an immune cell (e.g., the CD3 zeta chain, the Fc epsilon Rl gamma domain, the CD3 epsilon domain, the recently described DAP10 / DAP12 signaling domain, or other signaling or co-signaling moieties such as domains from CD28, 4-1BB, OX40, ICOS, DAP10, DAP12, CD27, and CD2 as costimulatory domains can also be used). A "chimeric NK receptor" is a CAR in which the binding moiety is derived or isolated from a NK receptor.

[0083] As used herein, "TCR" refers to a T cell receptor. In the context of adoptive cell transfer, it typically refers to a modified TCR, i.e., a TCR that has been modified to recognize a specific antigen, most typically a tumor antigen. As used herein, "endogenous TCR" refers to a TCR that is endogenously present on an unmodified cell (typically a T cell). The TCR is a disulfide-linked membrane-anchored heterodimeric protein, usually composed of highly variable alpha (α) and beta (β) chains expressed as part of a complex with the invariant CD3 chain molecule. The TCR receptor complex is an octomeric complex of the variable TCR receptor α and β chains with the CD3 co-receptor (including the CD3γ chain, the CD3δ chain, and two CD3ε chains) and two CD3ζ chains (aka the CD247 molecule). As used herein, the term "functional TCR" refers to a TCR that is capable of transmitting a signal upon binding to its cognate ligand. Typically, allogeneic therapy involves modifications to reduce or impair the TCR function, for example by knocking out or knocking down at least one of the TCR chains. The endogenous TCR in the modified cells is considered functional if it retains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or even at least 90% of the signaling capacity (or T cell activation) compared to a cell with an endogenous TCR that has not been modified. Assays for assessing signaling capacity or T cell activation are known to those skilled in the art, and include, in particular, ELISAs measuring interferon gamma. According to another embodiment, the endogenous TCR is considered functional if it has not been modified to disrupt TCR function.

[0084] The term "immune cells" as used herein refers to cells that are part of the immune system (which may be either the adaptive or innate immune system). Immune cells as used herein are typically immune cells produced for adoptive cell transfer (either autologous or allogeneic). Many different types of immune cells are used for adoptive therapy and are therefore envisioned for use in the methods described herein. Examples of immune cells include, but are not limited to, T cells, NK cells, NKT cells, lymphocytes, dendritic cells, bone marrow cells, macrophages, stem cells, progenitor cells or iPSCs. The latter three are not immune cells per se but can be used in adoptive cell transfer for immunotherapy (see, for example, Jiang et al., Cell Mol Immunol 2014; Themeli et al., Cell Stem Cell 2015). Typically, production begins with stem cells or iPSCs (or may even begin with a dedifferentiation step of immune cells to iPSCs), but production entails a step of differentiation into immune cells prior to administration. Stem cells, progenitor cells and iPSCs (i.e., stem cells, progenitor cells and iPSCs or their differentiated progeny transduced with a CAR described herein) used in the production of immune cells for adoptive transfer are considered immune cells herein. According to certain embodiments, the stem cells contemplated in the method do not include the destruction of a human embryo.

[0085] Particularly contemplated immune cells include white blood cells (leukocytes), including lymphocytes, monocytes, macrophages, and dendritic cells. Particularly contemplated lymphocytes include T cells, NK cells, and B cells, with T cells being especially contemplated. It is noted that in the context of adoptive transfer, immune cells are typically primary cells (i.e., cells that are directly isolated from human or animal tissue and have not been cultured or have only been cultured for a short period of time) and not cell lines (i.e., cells that have been continuously subcultured for an extended period of time and have acquired homogeneous genotypic and phenotypic characteristics). According to a specific embodiment, the immune cells are primary cells (i.e., cells that are directly isolated from human or animal tissue and have not been cultured or have only been cultured for a short period of time) and not cell lines (i.e., cells that have been continuously cultured for an extended period of time and have acquired homogeneous genotypic and phenotypic characteristics). According to another specific embodiment, the immune cells are not cells from a cell line.

[0086] As used herein, "microRNA scaffold", "miRNA scaffold" or even "scaffold" refers to a primary microRNA sequence with well-characterized characteristics including specific microRNA processing requirements into which an RNA sequence can be inserted (typically to replace an existing miRNA sequence with an siRNA against a specific target). A microRNA scaffold is minimally composed of a double-stranded upper stem region (typically of 18-23 nucleotides) connected on both sides to flexible loop sequences, said upper stem region typically being processed by Dicer. Typically, said microRNA scaffold further comprises a lower stem region, and optionally further comprises 5' and 3' flanking sequences or basic segments. A guide or target sequence is inserted into said upper stem region and is a single-stranded sequence of 18-23 nucleotides. As said target sequence recognizes its target by complementary base pairing, this sequence is typically identical to a sequence present in the target or its regulatory region. As used herein, a "target" or a "target protein" refers to a molecule (typically a protein, but which may be a nucleic acid molecule) that must be downregulated (i.e., the expression of which must be reduced in a cell). Note that since miRNAs function at the nucleic acid level, even for proteins, the miRNA target sequence is identical to the sequence that codes for the protein (e.g., the mRNA sequence) or the sequence that regulates the expression of the protein (e.g., the 3'UTR region, etc.).

[0087] Examples of miRNA scaffolds include scaffolds present in wild-type / naturally occurring miRNA clusters such as miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92-2 or miR-363, or engineered scaffolds such as SMART Vector™ micro-RNA adaptive scaffolds (Horizon Discovery, Lafayette, CO, USA). As used herein, "miR-106a" corresponds to human Gene ID 406899, "miR-18b" corresponds to human Gene ID 574033, "miR-20b" corresponds to human Gene ID 574032, "miR-19b-2" corresponds to human Gene ID 406981, "miR-92-2," also known as "miR-92a-2," corresponds to human Gene ID 407049, and "miR-363" corresponds to human Gene ID 574031.

[0088] As used herein, a "microRNA cluster" or "miRNA cluster" refers to a collection of microRNA scaffolds that function together. These may be wild-type / naturally occurring clusters or combinations of miRNA scaffolds that are not found together in nature. Wild-type / naturally occurring microRNA clusters are well described and include, for example, the miR-106a-363 ​​cluster, miR-17-92, miR-106b-25, and miR-23a-27a-24-2 cluster. A miRNA cluster can be considered as a combined scaffold. Thus, as used herein, a cluster or a "combined miRNA scaffold" refers to a combination of multiple miRNA scaffolds that function under the control of one promoter. The multiple miRNA scaffolds may be the same or different and have target sequences for the same or different target proteins and, in the case of the same target, have the same or different target sequences for the target. Such combined scaffolds, when under the control of one promoter, are also referred to as "multiplex scaffolds", "multiplexed scaffolds" or "multiplexed miRNA scaffolds". In some cases, once the number of scaffolds is determined, this can be used instead of the prefix "multiplex". For example, a "duplex scaffold" means that there are two scaffolds, a "triplex scaffold" has three scaffolds, a "tetraplex" or "quadruplex" has four, a "pentaplex" has five, a "hexaplex" has six, etc. Thus, a miRNA cluster with six different miRNA scaffolds (e.g., wild-type / naturally occurring miR-106a-363 ​​cluster) can be considered as a hexaplex miRNA scaffold or a cluster of six miRNAs.

[0089] As used herein, a "miR-17 family cluster" refers to (among other things) one of three paralogous miRNA clusters that contain scaffolds from the miR-17 family: the miR-106a-363 ​​cluster (composed (in order) of the miR-106a scaffold, the miR-18b scaffold, the miR-20b scaffold, the miR-19b-2 scaffold, the miR-92a2 scaffold, and the miR-363 scaffold); The miR-17 to 92 cluster (composed of the miR-17 scaffold, the miR-18a scaffold, the miR-19a scaffold, the miR-20a scaffold, the miR-19b-1 scaffold and the miR-92-1 (and also the miR-92al) scaffold), and the miR-106b to 25 cluster (composed of the miR-106b scaffold, the miR-93 scaffold and the miR-25 scaffold). The "miRNA-17 family" or "miR-17 family" is classified according to the seed sequence and includes miR-17, miR-20a, miR-106a, mir-20b, miR-106b and miR-93. Similarly, other families classified according to seed sequences are the "miR-18 family" (miR-18a, miR-18b), the "miR-19 family" (miR-19a, miR-19b-l, and miR-19b-2), and the "miR-92 family" (miR-92al, miR-92a2, miR-363, and miR-25).

[0090] Thus, a "scaffold from the miR-17 family cluster" is any scaffold selected from a miR-17 scaffold, a miR-18a scaffold, a miR-18b scaffold, a miR-19a scaffold, a miR-19b-l scaffold, a miR-19b-2 scaffold, a miR-20a scaffold, a miR-20b scaffold, a miR-25 scaffold, a miR-92-1 scaffold, a miR-92a2 scaffold, a miR-93 scaffold, a miR-106a scaffold, a miR-106b scaffold, and a miR-363 scaffold.

[0091] In contrast, a "miR-17 family scaffold" as used herein is selected from the more restricted group of six scaffolds from the miR-17 family: miR-17 scaffold, miR-20a scaffold, mir-20b scaffold, miR-93 scaffold, miR-106a scaffold, and miR-106b scaffold.

[0092] FIG. 1 shows a schematic example of a multiplexed scaffold sequence as used herein, showing the upper and lower stem regions, the target sequence, and the individual scaffolds.

[0093] The term "subject" refers to all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles, including humans and non-human animals. In the most notable embodiment of the described methods, the subject is a human.

[0094] The term "treat" or "treatment" refers to success or indicators of success in attenuating or ameliorating an injury, disease state or condition, including objective or subjective parameters such as alleviation, remission, reduction of symptoms, or making the condition more tolerable to the patient, slowing degeneration or decline, reducing the debilitating end point of degeneration, improving the physical or mental well-being of the subject, or prolonging survival. The treatment can be evaluated by objective or subjective parameters, including the results of a physical examination, neurological examination, or psychiatric evaluation.

[0095] As used herein, the phrase "adoptive cell therapy", "adoptive cell transfer", or "ACT" refers to the transplantation of cells, most typically immune cells, into a subject (e.g., a patient). These cells can be from the patient (in the case of autologous therapy) or from another individual (in the case of allogeneic therapy). The goal of the therapy is to improve immune function and characteristics, and in cancer immunotherapy, to enhance the immune response against the cancer. T cells are most commonly used for ACT, but other immune cell types such as NK cells, lymphocytes (e.g., tumor infiltrating lymphocytes (TIL)), dendritic cells, and myeloid cells are also used in applications.

[0096] An "effective amount" or "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of a therapeutic agent, such as the transformed immune cells described herein, may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the therapeutic agent (such as the cells) to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or adverse effects of the therapeutic agent are outweighed by the therapeutically beneficial effects.

[0097] The expression "graft-versus-host disease" or "GvHD" refers to a condition that can occur after allogeneic transplantation. In GvHD, donated bone marrow, peripheral blood (stem) cells or other immune cells view the recipient's body as foreign and the donated cells attack the body. Since donor immunocompetent immune cells such as T cells are the main drivers of GvHD, one strategy to prevent GvHD is by reducing (TCR-based) signaling in these immunocompetent cells, for example by directly or indirectly inhibiting the function of the TCR complex.

[0098] To assess whether targeting multiple genes in the context of adoptive cell transfer (ACT) is feasible without the need for genome editing (and its associated costs and complex manufacturing processes), we decided to test multiplexed RNA interference molecules.

[0099] The underlying approach is based on transcription of RNA from a specific vector that is processed by endogenous RNA processing machinery to generate active shRNAs that can target the mRNA of choice by base recognition, resulting in the destruction of the specific mRNA by the RISC complex. Specific destruction of the targeted mRNA results in a decrease in the expression of the associated protein. Transient knockdown of gene expression can be achieved by transfecting RNA oligonucleotides into the target cells of choice, whereas expression of the desired shRNA from the integrated vector allows stable knockdown of gene expression.

[0100] Successful expression of shRNAs is highly dependent on coupling with a polymerase III (PolIII) promoter (e.g., H1, U6), which generates RNA species lacking 5' cap and 3' polyadenylation, allowing processing of the shRNA duplex. Once transcribed, the shRNA undergoes processing, is exported from the nucleus, and is further processed and loaded into the RNA-induced silencing complex (RISC) for optimal mRNA targeted degradation (Moore et al., 2010). Although effective, the efficiency of transcription driven by PolIII promoters may result in cytotoxicity due to saturation of endogenous microRNA pathways due to excessively high expression of shRNAs from PolIII promoters (Fowler et al., 2016). Furthermore, expression of both therapeutic genes and shRNAs from a single vector has typically been achieved by employing a polymerase II (PolII) promoter driving a therapeutic gene and a PolIII promoter driving a shRNA of interest. Although this is functional, it comes at the expense of vector space and therefore reduces options for including therapeutic genes ( Chumakov et al., 2010 ; Moore et al., 2010 ).

[0101] Embedding the shRNA within a microRNA (mir) framework allows the shRNA to be processed under the control of a PolII promoter (Giering et al., 2008). Importantly, the expression levels of embedded shRNAs tend to be low, thereby avoiding the toxicity observed when using other systems such as the U6 promoter (Fowler et al., 2015). Indeed, mice administered shRNAs driven by a liver-specific PolII promoter showed stable gene knockdown without tolerability issues for more than a year (Giering et al., 2008). However, this was only for one shRNA performed in liver cells, with only a 15% reduction in protein levels (Giering et al., 2008), so it remains to be seen whether higher efficiency can be achieved for multiple targets, especially in immune cells (which are difficult to engineer).

[0102] Surprisingly, starting from the observation that elements of the miR106a-363 ​​cluster, a paralog of the miR-17-92 cluster, are surprisingly efficient for downregulation of targets, particularly for multiplexed downregulation of targets in T cells, it is shown herein that multiplexed downregulation can be further improved by creating chimeric clusters based on scaffolds of the miR-17 family cluster. This can be done in particular by utilizing chimeric scaffolds that incorporate the upper stem and loop regions of the miR-17 family on the lower stem regions of different scaffolds from the miR-17 family cluster, and / or by combining scaffolds from different miR-17 paralogs; in particular, scaffolds from the miR-17 family from different paralog clusters. Expression of multiple microRNA-based shRNAs against different targets (e.g., based on individual scaffolds present in the miR106a-363 ​​cluster) was feasible in T cells without recombination, without toxicity, and while achieving efficient downregulation of multiple targets at the same time.

[0103] It is therefore an object of the present invention to provide specific nucleic acid molecules, vectors and modified cells comprising the miRNA scaffolds. The nucleic acid molecules, vectors and cells are typically provided for use as medicines, such as for use in the treatment of cancer. This is equivalent to providing a method of treating cancer, which involves administering the nucleic acid molecules, vectors or cells described herein to a subject in need thereof, thereby ameliorating at least one symptom of the cancer.

[0104] According to a first aspect, there is provided a nucleic acid molecule comprising at least one RNA interference molecule having a modified scaffold, wherein the lower stem region of the scaffold is that of a miR scaffold from the miR-17 family cluster and at least a portion of the upper stem / loop region of the scaffold is modified to differ from the wild-type / native sequence.

[0105] The miR-17 family cluster comprises 15 scaffolds, and therefore the lower stem region of the modified scaffold will be selected from a miR-17 scaffold, a miR-18a scaffold, a miR-19a scaffold, a miR-20a scaffold, a miR-19b-l scaffold, a miR-92-1 scaffold, a miR-106a scaffold, a miR-18b scaffold, a miR-20b scaffold, a miR-19b-2 scaffold, a miR-92-2 scaffold, a miR-363 scaffold, a miR-106b scaffold, a miR-25 scaffold, and a miR-93 scaffold. Where at least part of the upper stem / loop region is altered differently from the wild-type / native sequence, this may mean that only the loop region is altered to this effect, only the upper stem region is altered to this effect, either the upper stem region and part of either or both the loop are altered to this effect, or all of the upper stem and loop regions are altered.

[0106] According to a particular embodiment, the modified scaffolds disclosed herein are chimeric scaffolds.Typically, they are chimeric scaffolds derived from two scaffolds from the miR-17 family cluster.In particular, at least one of the two scaffolds is also a miR-17 family scaffold, i.e., selected from the miR-17 scaffold, the miR-20a scaffold, the miR-106a scaffold, the miR-20b scaffold, the miR-106b scaffold, and the miR-93 scaffold. Most particularly, said chimeric scaffold is selected from the miR-17 family cluster scaffolds (i.e. miR-17 scaffold, miR-18a scaffold, miR-19a scaffold, miR-20a scaffold, miR-19b-1 scaffold, miR-92-1 scaffold, miR-106a scaffold, miR-18b scaffold, miR-20b scaffold, miR-19b-2 scaffold, miR-92-2 scaffold, miR-363 scaffold, etc. The scaffold comprises a lower stem region selected from a miR-17 family scaffold (i.e., selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold) and at least a portion of the upper stem / loop region, in particular all of the upper stem / loop region selected from a miR-17 family scaffold (i.e., selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold).

[0107] Thus, in a further embodiment, there is provided a nucleic acid molecule comprising at least one RNA interference molecule having a modified scaffold, wherein the lower stem region of the scaffold is selected from the group consisting of miR-17 scaffold, miR-18a scaffold, miR-19a scaffold, miR-20a scaffold, miR-19b-1 scaffold, miR-92-1 scaffold, miR-106a scaffold, miR-18b scaffold, miR-20b scaffold, miR-19b-2 scaffold, miR-92-2 scaffold, miR-363 scaffold, and the like. a miR scaffold selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold, and an upper stem and loop region of said scaffold selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold, and wherein the lower stem region of said scaffold is different from the upper stem and loop region of said scaffold.

[0108] In addition to the scaffold, at least one RNA interference molecule will typically also comprise a target sequence not present in the wild-type / native scaffold sequence, in particular the target sequence has a length of 18-23 nucleic acids, more particularly 18-21 nucleic acids, in particular 18-20 nucleic acids.

[0109] According to further particular embodiments, the nucleic acid molecule comprising at least one RNA interference molecule comprises at least two multiplexed RNA interference molecules, at least one of which has a scaffold. When there are at least two multiplexed RNA interference molecules, these two or more molecules may have the same or different scaffolds. In principle, the additional RNA interference molecule may have any type of suitable scaffold, either wild type / natural or synthetic, but it is particularly envisaged that said additional RNA interference molecule (or molecules) have a scaffold selected from the miR-17 family cluster, i.e., miR-17 scaffold, miR-18a scaffold, miR-19a scaffold, miR-20a scaffold, miR-19b-l scaffold, miR-92-1 scaffold, miR-106a scaffold, miR-18b scaffold, miR-20b scaffold, miR-19b-2 scaffold, miR-92-2 scaffold, miR-363 scaffold, miR-106b scaffold, miR-25 scaffold and miR-93 scaffold. As shown in the examples, especially in examples 5-8, such combinations of scaffolds can result in successful multiplexing. Importantly, not all of the scaffolds need to have an upper stem / loop region that is altered to be different from the wild-type / native sequence, however, according to certain embodiments, when at least two multiplexed RNA interference molecules are present, all of the scaffolds have a lower stem region derived from a miR-17 family cluster.

[0110] As noted, the scaffolds may be the same or different, however, it is particularly envisaged that no more than three of the scaffolds are identical, and even more particularly, it is envisaged that no more than two identical scaffolds are used, in order to avoid recombination between identical scaffold sequences or other factors that would decrease the miRNA processing (see Example 5).

[0111] Thus, according to a further aspect, there is provided a nucleic acid molecule comprising at least two RNA interference molecules having different scaffolds, said at least two different scaffolds comprising a lower stem region of a miR scaffold from the miR-17 family cluster, At least one RNA interference molecule has a chimeric scaffold, in which at least a portion of the upper stem / loop region is not derived from the same miR scaffold as the lower stem region, and at least a portion of the upper stem / loop region is selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold; and / or The scaffolds derived from the at least two RNA interference molecules are miR-17 family scaffolds selected from at least two different groups consisting of at least two different miR-17 family clusters, i.e., miR-17 and miR-20a (miR-17-92 cluster), miR-106a and miR-20b (from 106a-363 ​​cluster), and miR-106b and miR-93 (from miR-106b-25 cluster).

[0112] Again, it is specifically envisaged that at least a portion of the upper stem / loop region is the entire upper stem / loop region.

[0113] According to a specific embodiment, the scaffolds present in the nucleic acid molecule are exclusively selected from the miR-17 family cluster (optionally further modified), however, it is also envisaged to further combine them with different scaffold sequences, in particular with different unrelated sequences (to avoid recombination), such as the miR-196a2 sequence and / or the miR-23a-27a-24-2 cluster.

[0114] According to further specific embodiments, the nucleic acid molecule comprising at least one RNA interference molecule comprises at least two multiplexed RNA interference molecules under the control of one promoter. According to further specific embodiments, the at least two multiplexed RNA interference molecules are at least three multiplexed RNA interference molecules. According to further embodiments, the at least two multiplexed RNA interference molecules are at least four multiplexed RNA interference molecules, the at least two multiplexed RNA interference molecules are at least five multiplexed RNA interference molecules, and the at least two multiplexed RNA interference molecules are at least six multiplexed RNA interference molecules.

[0115] According to certain embodiments, the scaffold sequence may be modified to reduce the number of mismatches and / or bulges in the stem region. As used herein, "mismatch" refers to a base pair that is not a complimentary Watson-Crick base pair. As used herein, "bulge" refers to an unpaired stretch of nucleotides (typically 1-5, in particular 1-3) located within one strand of a nucleic acid duplex. More specifically, when one of the scaffold sequences used is a miR-18b scaffold, the scaffold may be modified (and modified compared to the wild-type / native sequence) to reduce the number of mismatches and / or bulges in the stem region (see Example 3). This can be done by restoring base pair complementarity (in the case of a mismatch), typically by matching the passenger strand with the target strand, or, in the case of a bulge, by removing the extra unpaired nucleotides.

[0116] According to certain embodiments, said at least two multiplexed RNA interference molecules can be shRNA molecules or miRNA molecules. Most particularly, they are miRNA molecules. The difference between shRNA molecules and miRNA molecules is that miRNA molecules are processed by Drosha, whereas conventional shRNA molecules are not (which is associated with toxicity, Grimm et al., Nature 441:537-541 (2006)).

[0117] According to certain embodiments, the miRNA molecules can be provided as individual miRNA scaffolds under the control of one promoter. Each selected scaffold typically corresponds to one miRNA (Figure 1), and the scaffolds can be repeated or combined with other scaffolds to obtain the expression of multiple RNA interference molecules (Figures 1-2). However, when repeated or combined with additional scaffolds, it is typically assumed that all of the multiplexed RNA interference molecules are under the control of one promoter (i.e., the promoter is not repeated when repeating individual scaffolds or adding additional scaffolds).

[0118] Particularly suitable scaffold sequences for miRNA multiplexing are those found in bona fide polycistronic miRNA clusters or parts thereof, where the endogenous miRNA target sequence is replaced with the shRNA target sequence of interest. Particularly suitable miR scaffold clusters for this purpose are the miR-106a-363, miR-17-92, miR-106b-25, and miR-23a-27a-24-2 clusters, and particularly contemplated are the miR-106a-363 ​​cluster and fragments thereof (i.e., one or more individual scaffolds). Of note, in order to conserve vector payload, it is also particularly contemplated to use parts of such wild-type / native clusters rather than the entire sequence (this is particularly useful since not all miRNAs are equidistant and not all linker sequences may be necessary). Indeed, it is shown herein (Example 5) that scaffolds can be used outside the context of the cluster and can be combined in various ways. Other considerations can also be taken into account, such as, for example, which miRNA is most efficiently processed in the cell. For example, the miR-17-92 cluster is composed of (in order) the miR-17, miR-18a, miR-19a, miR-20a, miR-19b-l and miR-92-1 (and also miR-92a) scaffolds, and particularly useful fragments of the cluster are the scaffold sequences from miR-19a through miR-92-1 (i.e., four of the six miRNAs) and their linkers, or the scaffold sequences from miR-19a through miR-19b-l (three of the six miRNAs). Similarly, the 106a-363 ​​cluster is composed of (in order) the miR-106a scaffold, the miR-18b scaffold, the miR-20b scaffold, the miR-19b-2 scaffold, the miR-92-2 (also miR-92a2) scaffold and the miR-363 scaffold (see Figure 5).Particularly useful fragments of the clusters are the scaffold sequences from miR-106a to miR-20b (i.e., three of the six miRNAs) (see Example 5), from miR-20b to miR-363 (i.e., four of the six miRNAs) or from miR-19b-2 to miR-363 (i.e., three of the six miRNAs) (see Figure 6). Both wild-type / natural linker sequences can be used, as well as fragments thereof or artificial linkers (again, to reduce the payload of the vector).

[0119] As miRNA scaffolds from the miR-106a-363 ​​cluster are particularly contemplated, particularly contemplated linkers are the 5' and 3' sequences of each scaffold (see FIG. 1). Linker sequences can be, for example, 150bp, 140bp, 130bp, 120bp, 110bp, 100bp, 90bp, 80bp, 70bp, 60bp, 50bp, 40bp, 30bp, 20bp, 10bp or less on either side of the scaffold. If two non-adjacent scaffolds are used in the cluster (e.g., as in Example 5), the linker will of course not be identical to that found in the cluster. Nevertheless, hybrid linkers can be made, for example, by using the 30, 60 or 90bp present in the 3' of one scaffold in the cluster and then fusing it with a linker consisting of 30, 60, 90bp 5' of the selected scaffold.

[0120] The miRNA scaffolds are in particular used as is, i.e. without any modification to the scaffold sequence. In particular, the lower stem sequence is kept identical to that found in each miRNA scaffold. Preferably, the loop sequences in the upper stem are also not altered, although experiments have shown that these are primarily flexible structures and can be adapted in length and sequence as long as the upper stem structure is not affected. Although not preferred, the skilled artisan will understand that scaffolds with such modified loops are within the scope of the present application. Within the upper stem of the scaffold, a target sequence is found. The wild type / natural target sequence of the miR-106a-363 ​​cluster is 22-23 bp long. As shown in Example 4, the dimensions of the target sequence can be shortened without adverse effects. The target sequence can be 18-23 bp long, with sequences of 18-21 bp being particularly envisaged, and sequences of 18-20 bp being further envisaged. If shorter sequences are required, there is no problem in using target sequences of 18 or 19 bp.

[0121] As is evident for targeting, the target sequence is part of the scaffold, which obviously requires adaptation of the target. The miRNA scaffold has some mismatches in its structure, so the question is whether these mismatches should be retained or not. As shown in Example 3 (and Figure 9), the mismatches found at position 14 of the target sequences of miR-106a and miR-20b can be retained without negatively affecting the downregulation of the target, which means that the passenger strand is not completely complementary to the guide strand. As also shown in Example 3 (and Figure 10), when multiple mismatches are present (e.g., in the miR-18b scaffold), the passenger strand can be made more complementary to the guide strand to achieve more efficient knockdown (if necessary). It should be noted that this modification is not necessary to achieve significant knockdown levels, but removing mismatches at positions 6, 11, and 15 of the target sequence (corresponding to bp 20 and 70, 25 and 65, and 29 and 61 of the scaffold (see FIG. 9)) systematically improves knockdown. The same is true for the bulge (nucleotides 75 and 76 of the miR-18b scaffold). Increasing the complementarity of the target and passenger strands by removing mismatches or bulges in the passenger strand may also improve downregulation of other scaffolds, but this is not yet necessary, as testing different target sequences always results in satisfactory knockdown levels.

[0122] Each RNA interference molecule can target different molecules, can target the same molecule, or a combination thereof (i.e., multiple RNA molecules are directed to one target, while only one RNA interference molecule is directed to different targets). When the RNA interference molecules are directed to the same target, they can target the same region, or they can target different regions. In other words, when the RNA interference molecules are directed to the same target, they can be the same or different. Examples of such combinations of RNA interference molecules are shown in the Examples section.

[0123] Thus, according to a particular embodiment, at least two of the multiplexed RNA interference molecules are directed to the same target. According to a further particular embodiment, these at least two RNA interference molecules use the same miRNA scaffold. They can be directed to the same target by using the same target sequence (according to these specific embodiments, at least two of the multiplexed RNA interference molecules are the same) or by using different target sequences (according to these specific embodiments, at least two of the multiplexed RNA interference molecules have the same scaffold but different target sequences). According to another embodiment, the at least two multiplexed RNA interference molecules directed to the same target have different miRNA scaffold sequences. In that case, they can have the same target sequence or they can have different target sequences directed to the same target.

[0124] According to another embodiment, the at least two multiplexed RNA interference molecules are all different. According to a further specific embodiment, the at least two multiplexed RNA interference molecules are all directed against different targets.

[0125] Any suitable molecule present in the modified cell can be targeted by the present RNA interference molecules. Exemplary contemplated targets are: MHC class I genes, MHC class II genes, MHC coreceptor genes (e.g., HLA-F, HLA-G), TCR chains, NKBBiL, LTA, TNF, LTB, LST1, NCR3, AIF1, LY6, heat shock proteins (e.g., HSPA1L, HSPA1A, HSPA1B), complement cascade, regulatory receptors (e.g., NOTCH4), TAP, HLA-DM, HLA-DO, RING1, CD52, CD247, HCP5, B2M, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, 2B4, A2AR, BAX, BLIMP1, C160 (POLR3A), CBL-B, CCR6, CD7, CD27, CD28, CD38, CD95, CD96, CD123, CD272 (BTLA), CD276 (aka B7-H3), CIITA, CTLA4, DGK[DGKA, DGKB, DGKD, DGKE, DKGG, DGKH, DGKI, DGKK, DGKQ, DGKZ], DNMT3A, DR4, DR5, EGR2, FABP4, FABP5, FASN, GMCSF, HPK1, IL-10R[IL10RA, IL10RB], IL2, LAG3 (CD223), LFA1, NEAT1, NFkB (including RELA, RELB, NFkB2, NFkBl, REL), NKG2A, NR4A (including NR4A1, NR4A2, NR4A3), PD1, PI3KCD, PPP2RD2, PRAS40, RAPTOR, SHIP1, SOAT1, SOCS1, T-BET, TCF7 (aka TCF-1), TET2, TGFBR1, TGFBR2, TGFBR3, TIGIT, TIM3 (aka HAVCR2 or CD366), TOX, TOX2, VISTA (aka VSIR or B7-H5), ZC3H12A (also known as Regnase 1 or MCPIP) and ZFP36L2.

[0126] According to a further aspect of the invention, said nucleic acid molecule is not used as such, but is provided in a suitable vector, i.e. a vector allowing expression in a cell. According to a particular embodiment, said vector is suitable for expression in a eukaryotic cell, in particular an immune cell.

[0127] Thus, a vector suitable for expression in modified immune cells is provided, comprising the nucleic acid molecule described herein.All the features disclosed for said nucleic acid molecule also apply mutatis mutandis to said vector.In other words, a vector is provided, comprising at least one RNA interference molecule with modified scaffold, wherein the lower stem region of said scaffold is that of a miR scaffold from miR-17 family cluster, and at least a part of the upper stem / loop region of said scaffold is modified to be different from the natural sequence. According to further embodiments, the lower stem region of the modified scaffold is selected from a miR-17 scaffold, a miR-18a scaffold, a miR-19a scaffold, a miR-20a scaffold, a miR-19b-l scaffold, a miR-92-1 scaffold, a miR-106a scaffold, a miR-18b scaffold, a miR-20b scaffold, a miR-19b-2 scaffold, a miR-92-2 scaffold, a miR-363 scaffold, a miR-106b scaffold, a miR-25 scaffold, and a miR-93 scaffold. According to further embodiments, the modified scaffold is a chimeric scaffold, wherein at least a portion of the upper stem / loop region is not from the same miR scaffold as the lower stem region, and wherein at least a portion of the upper stem / loop region is selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold.

[0128] According to a particular embodiment, the at least one RNA interference molecule present in said vector is at least two RNA interference molecules, in particular at least two multiplexed RNA interference molecules.

[0129] According to a further particular embodiment, a vector is provided comprising a nucleic acid molecule comprising at least two RNA interference molecules having different scaffolds, said at least two different scaffolds comprising a lower stem region of a miR scaffold from the miR-17 family cluster, At least one RNA interference molecule has a chimeric scaffold, at least a portion of the upper stem / loop region is not from the same miR scaffold as the lower stem region, and at least a portion of the upper stem / loop region is selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold; and / or The scaffolds derived from the at least two RNA interference molecules are miR-17 family scaffolds derived from different miR-17 family clusters. The at least two multiplexed RNA interference molecules can be at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or even more molecules, depending on the number of target molecules to be downregulated and the practical considerations of co-expressing the multiplexed molecules. The miR-17 family cluster has 15 scaffolds, which can be replicated without losing knockdown activity (Example 5) and individual scaffolds from different clusters can be combined (Example 7), so up to 12 scaffolds can in principle be multiplexed, although in practice fewer numbers are often used.

[0130] A "multiplex" is a polynucleotide that encodes multiple molecules of the same type, such as multiple siRNAs or shRNAs or miRNAs. Within a multiplex, when the molecules are of the same type (e.g., all shRNAs), they may be identical or may contain different sequences. Between molecules of the same type, there may be an intervening sequence, such as a linker, as described herein. One example of a multiplex of the present invention is a polynucleotide that encodes multiple tandem miRNA-based shRNAs. A multiplex may be single-stranded, double-stranded, or may have both single-stranded and double-stranded regions.

[0131] According to a particular embodiment, at least two multiplexed RNA interference molecules are under the control of one promoter. Typically, when multiple RNA interference molecules are expressed, this is done by integrating multiple copies of shRNA-expression cassettes. These typically have identical promoter sequences, resulting in frequent recombination events that remove repeated sequence fragments. As a solution, several different promoters are typically used in the expression cassettes (e.g., Chumakov et al., 2010). However, according to the present embodiment, recombination is avoided by using only one promoter. Although expression is typically low, this is advantageous in terms of toxicity, since too many siRNAs can be toxic to the cell (e.g., by interfering with the endogenous siRNA pathway). Using only one promoter has the additional advantage that all shRNAs are co-regulated and expressed at similar levels. Surprisingly, as shown in the examples, multiple shRNAs can be transcribed from one promoter without a significant drop in efficacy.

[0132] Typically, the promoter used to express the RNA interference molecule is not a U6 promoter, since this promoter is associated with toxicity, especially at high expression levels. For the same reason, it is possible to consider excluding the H1 promoter (a weaker promoter than U6) or even the PolIII promoter in general (although it may be suitable under certain conditions). Thus, according to a specific embodiment, the promoter used to express the RNA interference molecule is not an RNA PolIII promoter. RNA PolIII promoters lack temporal and spatial control and do not allow controlled expression of miRNA inhibitors. In contrast, many RNA PolII promoters allow tissue-specific expression, and both inducible and repressible RNA PolII promoters exist. Although tissue-specific expression is often not required in the context of the present invention (because the cells are selected before modification), it is still advantageous to have a specific promoter for, for example, immune cells, since it has been shown that the differences in RNAi efficacy with various promoters were particularly pronounced in immune cells (Lebbink et al., 2011). According to a specific embodiment, the promoter is selected from a PolII promoter and a PolIII promoter. According to a particular embodiment, the promoter is a natural or synthetic PolII promoter. Suitable promoters include, but are not limited to, cytomegalovirus (CMV) promoter, elongation factor 1 alpha (EF1α) promoter (core or full length), phosphoglycerate kinase (PGK) promoter, composite beta-actin promoter (CAG promoter) with upstream CMV IV enhancer, ubiquitin C (UbC) promoter, spleen limited focus forming virus (SFFV) promoter, Rous sarcoma virus (RSV) promoter, interleukin 2 promoter, murine stem cell virus (MSCV) long terminal repeat (LTR), gibbon ape leukemia virus (GALV) LTR, simian virus 40 (SV40) promoter, and tRNA promoter. These promoters are among the most commonly used polymerase II promoters to drive mRNA expression.

[0133] The vectors disclosed herein are particularly suitable for use in cells used for ACT. Thus, one of the objects of the present invention is to provide modified cells comprising a nucleic acid molecule encoding at least one RNA interference molecule as described herein. The RNA interference molecule also typically comprises a target sequence not present in the natural scaffold sequence. The target sequence typically has a length of 18-23 nucleic acids. It is particularly envisaged that the target sequence is directed to a sequence present in the modified cell, in particular a target sequence. That is, the at least one RNA interference molecule comprises a sequence that targets (by base pair complementarity) a sequence in the modified cell that encodes a protein to be downregulated, or a regulatory region of the target protein.Examples of such targets include, but are not limited to, MHC class I genes, MHC class II genes, MHC co-receptor genes (e.g., HLA-F, HLA-G), TCR chains, NKBBiL, LTA, TNF, LTB, LST1, NCR3, AIF1, LY6, heat shock proteins (e.g., HSPA1L, HSPA1A, HSPA1B), complement cascade, regulatory receptors (e.g., NOTCHI4), TA P, HLA-DM, HLA-DO, RING1, CD52, CD247, HCP5, B2M, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, 2B4, A2 AR, BAX, BL1MP1, C160(POLR3A), CBL-B, CCR6, CD7, CD27, CD28, CD38, CD95, CD96, CD123, CD272(BTLA), CD276(aka B7-H3), CIITA, CTLA4, DGK[DGKA, DGKB, DGKD, DGKE, DKGG, DGKH, DGKI, DGKK, DGKQ, DGKZ], DNMT3A, DR4, DR5, EGR2, FABP4, FABP5, FASN, GMCSF, HPK1, IL-10R[IL10RA, IL10RB], IL2, LAG3 (CD223), LFA1, NEAT1, NFkB (including RELA, RELB, NFkB2, NFkBl, REL), NKG2A, NR4A (including NR4A1, NR4A2, NR4A3), PD1, PI3KCD, PPP2RD2, PRAS40, RAPTOR, SHIP1, SOAT1, SOCS1, T-BET, TCF7 (aka TCF-1), TET2, TGFBR1, TGFBR2, TGFBR3, TIGIT, TIM3 (aka HAVCR2 or CD366), TOX, TOX2, VISTA (aka VSIR or B7-H5), ZC3H12A (also known as Regnase 1 or MCPIP), and ZFP36L2.

[0134] In addition to the RNA interference molecule, the vector often contains further elements, typically also a nucleic acid encoding a protein of interest, such as a CAR. According to further particular embodiments, the at least two multiplexed RNA interference molecules and the protein of interest are both under the control of one promoter. This also reduces vector burden (as there is no separate promoter used to express the protein of interest) and provides the advantage of co-regulated expression. This may be advantageous, for example, when the protein of interest is a cancer-targeting CAR and the RNA interference molecule is intended to have an additive or synergistic effect in tumor eradication. Examples of useful RNA targets include (but are not limited to) CD247, TRAC (both of which downregulate the TCR complex, making cells more suitable for allogeneic therapy), B2M (to expand tissue compatibility), CD52 (to make cells resistant to CD52-directed chemotherapy), CD95 (to make cells insensitive to CD95-induced cell death), checkpoint molecules (e.g., PD-1, PD-L1, CTLA4), and many others.

[0135] As mentioned above, the nucleic acid molecules and vectors described herein are particularly useful for modifying cells for ACT. Thus, modified immune cells are provided that comprise the nucleic acid molecules or vectors described herein. All the features disclosed for the nucleic acid molecules and vectors apply mutatis mutandis to said modified cells.

[0136] Cells that contain at least one RNA interference molecule or at least two RNA interference molecules can have advantages, especially therapeutic advantages. RNA interference molecules can in fact be directed against targets whose (over)expression is undesirable. However, typically, the modified cells provided herein further contain at least one protein of interest.

[0137] therefore, a first exogenous nucleic acid molecule encoding a protein of interest; A second nucleic acid molecule comprising at least one RNA interference molecule having a modified scaffold, wherein the lower stem region of the scaffold is of a miR scaffold from the miR-17 family cluster, and at least a portion of the upper stem / loop region of the scaffold is modified to differ from the native sequence; A modified cell is provided, comprising:

[0138] According to further embodiments, the lower stem region of the modified scaffold is selected from a miR-17 scaffold, a miR-18a scaffold, a miR-19a scaffold, a miR-20a scaffold, a miR-19b-l scaffold, a miR-92-1 scaffold, a miR-106a scaffold, a miR-18b scaffold, a miR-20b scaffold, a miR-19b-2 scaffold, a miR-92-2 scaffold, a miR-363 scaffold, a miR-106b scaffold, a miR-25 scaffold, and a miR-93 scaffold. According to further embodiments, the modified scaffold is a chimeric scaffold, wherein at least a portion of the upper stem / loop region is not from the same miR scaffold as the lower stem region, and wherein at least a portion of the upper stem / loop region is selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold.

[0139] moreover, a first exogenous nucleic acid molecule encoding a protein of interest; a second nucleic acid molecule comprising at least two RNA interference molecules having different scaffolds, the at least two different scaffolds comprising a second nucleic acid molecule having a lower stem region of a miR scaffold from a miR-17 family cluster; At least one RNA interference molecule has a chimeric scaffold, at least a portion of the upper stem / loop region is not from the same miR scaffold as the lower stem region, and at least a portion of the upper stem / loop region is selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold; and / or The scaffolds derived from the at least two RNA interference molecules are miR-17 family scaffolds derived from different miR-17 family clusters. A modified cell comprising:

[0140] According to a particular embodiment, the modified cells are modified immune cells, in particular, the immune cells are selected from T cells, NK cells, NKT cells, macrophages, stem cells, progenitor cells, and iPSC cells.

[0141] When there are at least two multiplexed RNA interference molecules, these two or more molecules may have the same or different scaffolds. However, it is particularly envisaged that no more than three of the scaffolds are the same, and it is particularly envisaged that no more than two identical scaffolds are used. This is to avoid recombination between identical scaffold sequences or overloading the miRNA processing capacity of cells (see Example 5). For the same reason, when there are multiple target sequences for the same target, it is particularly envisaged that either different target sequences are used in different scaffolds or the same target sequence is used. It is particularly envisaged that the same target sequence may be in the same scaffold, but it is not more than twice present.

[0142] Any further additional target protein can provide, for example, an additive, complementary or even synergistic effect or can be used for different purposes. For example, said target protein can be a CAR for tumors, and the RNA interference molecule can interfere with tumor function, for example, by targeting immune checkpoints, directly downregulating tumor targets, or targeting the tumor microenvironment. Alternatively or additionally, one or more of said RNA interference molecules can prolong the persistence of therapeutic cells or otherwise modify physiological responses (e.g., interfere with GvHD or host-versus-graft reaction).

[0143] The proteins of interest can in principle be any protein depending on the situation. Typically, however, they are proteins with a therapeutic function. They may include, for example, secreted therapeutic proteins such as interleukins, cytokines or hormones. However, according to certain embodiments, the proteins of interest are not secreted. Instead of therapeutic proteins, the proteins of interest may perform different functions, such as diagnostics or detection. Thus, the proteins of interest may be tags or reporter genes. Typically, the proteins of interest are receptors. According to further particular embodiments, the receptors are chimeric antigen receptors or TCRs. The chimeric antigen receptor can be for any target expressed on the surface of a target cell, exemplary examples include, but are not limited to, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD56, CD70, CD123, CD133, CD138, CD171, CD174, CD248, CD274, CD276, CD279, CD319, CD326, CD340, B These include CMA, B7H3, B7H6, CEACAM5, EGFRvlll, EPHA2, mesothelin, NKG2D, HER2, HER3, GPC3, Flt3, DLL3, IL1RAP, KDR, MET, mucin1, IL13Ra2, FOLH1, FAP, CA9, FOLR1, ROR1, GD2, PSCA, GPNMB, CSPG4, ULBP1, ULBP2, but many more exist and are suitable. Most CARs are scFv-based (i.e., the binding moiety is an scFv against a specific target, and the CAR is typically named after the target), while some CARs are receptor-based (i.e., the binding moiety is a part of a receptor, and the CAR is typically named after the receptor). An example of the latter is NKG2D-CAR.

[0144] The modified TCR can be directed against any target of a cell, including an intracellular target. In addition to the targets present on the cell surface, typical targets of TCRs include, but are not limited to, NY-ESO-1, PRAME, AFP, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, gp100, MART-1, tyrosinase, WT1, p53, HPV-E6, HPV-E7, HBV, TRAIL, thyroglobulin, KRAS, HERV-E, HA-1, CMV, and CEA.

[0145] According to these particular embodiments in which an additional protein of interest is present, the first and second nucleic acid molecules in the modified cell are typically present in one vector, such as a eukaryotic expression plasmid, a minicircle DNA, or a viral vector (e.g., derived from lentivirus, retrovirus, adenovirus, adeno-associated virus, and Sendai virus). According to further specific embodiments, the viral vector is selected from lentivirus vectors and retrovirus vectors. Especially in the latter case, the vector load (i.e., the total size of the construct) is important, and the use of compact multiplex cassettes is particularly advantageous.

[0146] Of note, the cells described herein may contain multiple proteins of interest: for example, a receptor protein and a reporter protein (see FIG. 2), or alternatively, a receptor protein, an interleukin and a tag protein.

[0147] The modified cells are in particular eukaryotic cells, more particularly modified mammalian cells, and even more particularly modified human cells. According to particular embodiments, the cells are modified immune cells. Exemplary immune cells are selected from T cells, NK cells, NKT cells, macrophages, stem cells, progenitor cells, and iPSC cells.

[0148] The cells disclosed herein typically contain multiplexed RNA interference molecules, which can be directed against one or more targets that need to be downregulated (either intracellular targets, or extracellular targets if the shRNA is secreted).

[0149] In other words, the invention disclosed herein has identified a particularly suitable construct that is miRNA-based. Accordingly, modified cells are provided that include a polynucleotide that includes a microRNA-based shRNA coding region, the microRNA-based shRNA coding region being a sequence encoding one or more artificial miRNA-based shRNA nucleotide sequences, each artificial miRNA-based shRNA nucleotide sequence comprising: a miRNA scaffold sequence; an active or mature sequence; and a passenger or star sequence, wherein within each artificial miRNA-based shRNA nucleotide sequence, the active sequence is at least 70% complementary to the passenger sequence.

[0150] According to certain embodiments, the active sequence is at least 80% complementary to the passenger sequence, and may be at least 90% or more complementary to the passenger sequence.

[0151] A particular advantage is that the miRNA-based shRNA nucleotide sequences of the present invention can be multiplexed. Thus, modified cells are provided that include a polynucleotide comprising a multiplexed microRNA-based shRNA coding region, the multiplexed microRNA-based shRNA coding region comprising: A sequence encoding two or more artificial miRNA-based shRNA nucleotide sequences, each artificial miRNA-based shRNA nucleotide sequence comprising: miRNA scaffold sequence, the active or mature sequence, and Within each artificial miRNA-based shRNA nucleotide sequence, including a passenger or star sequence, the active sequence is at least 70% complementary to the passenger sequence.

[0152] The miRNA-based shRNA nucleotide sequence is in particular selected from the miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92-2 and miR-363 sequences. Both the active and passenger sequences of each artificial miRNA-based shRNA nucleotide sequence are typically 18-40 nucleotides long, more particularly 18-30 nucleotides long, more particularly 18-25 nucleotides long and most particularly 18-23 nucleotides long. The active sequence may also be 18 or 19 nucleotides long. Typically, the passenger sequence has the same length as the active sequence, although the possible presence of a bulge means that they are not necessarily the same length.

[0153] Typically, these microRNA scaffold sequences are separated by a linker. In microRNA clusters, the linker can be up to 500, 400, 300, 200, 150, 100 nucleotides long. When multiplexing scaffold sequences, it may be an objective to use a natural linker sequence (found 5' and 3' of the miRNA scaffold sequence) of sufficient length to ensure that any potential regulatory sequences are included. For example, 50, 100, or 150 nucleotides adjacent to the scaffold sequence can be used. Another objective may be to reduce the payload of the vector and reduce the linker length, which can be, for example, 30-60 nucleotides long, although shorter chains also work. Indeed, it has been surprisingly found that the length of the linker does not play an essential role and can be very short (less than 10 nucleotides) or even absent without interfering with shRNA function. According to certain embodiments, at least a portion of the 5' and / or 3' linker sequence is used with its respective scaffold, typically at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 120 nucleotides, at least 150 nucleotides, or at least 200 nucleotides of the 5' and / or 3' linker sequence.

[0154] The miRNA-based shRNA nucleotide sequence is considered to be an artificial sequence, because the endogenous miR sequence is replaced with a modified shRNA sequence against a specific target, even though the scaffold sequence may be of natural origin. The artificial sequence may be, for example, a scaffold of natural origin (e.g., a miR cluster or a fragment thereof, e.g., miR-106a-363 ​​cluster) in which the endogenous miR sequence is replaced with a modified shRNA sequence against a specific target, a repeat of a single miR scaffold (e.g., miR-20b scaffold, etc.) in which the endogenous miR sequence is replaced with a modified shRNA sequence against a specific target, an artificial miR-like sequence, or a combination thereof.

[0155] The modified cell typically further comprises a nucleic acid molecule encoding a protein of interest, such as a chimeric antigen receptor or TCR, and may be a modified immune cell, as described above.

[0156] The expression of the at least one RNA interference molecule or the co-expression of the multiplexed RNA interference molecules results in the suppression of at least one gene, and typically multiple genes, in the modified cell, which may contribute to a higher therapeutic efficacy.

[0157] The modified cells described herein are also provided for use as a medicament. According to certain embodiments, the modified cells are provided for use in the treatment of cancer. Examples of types of cancer that can be treated include, but are not limited to, adenocarcinoma, adrenal cortical carcinoma, anal cancer, astrocytoma, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, Ewing's sarcoma, eye cancer, fallopian tube cancer, gastric cancer, glioblastoma, head and neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, myelodysplastic syndrome, multiple myeloma, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, peritoneal cancer, pharyngeal cancer, prostate cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, skin cancer, small intestine cancer, gastric cancer, testicular cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, and Wilms' tumor.

[0158] According to certain embodiments, the cells may be provided for the treatment of liquid or blood cancers, such as leukemias (including, a.o. acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL)), lymphomas (a.o. Hodgkin's lymphoma and non-Hodgkin's lymphomas, such as B-cell lymphomas (including, for example, DLBCL), T-cell lymphomas, Burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, and small lymphocytic lymphoma), multiple myeloma, or myelodysplastic syndromes (MDS).

[0159] This is equivalent to saying that a method of treating cancer is provided, comprising administering to a subject in need thereof an appropriate dose of a modified cell as described herein (i.e., a modified cell comprising exogenous nucleic acid molecules encoding at least two multiplexed RNA interference molecules, and optionally comprising an additional nucleic acid molecule encoding a protein of interest), thereby ameliorating at least one symptom associated with said cancer. Cancers contemplated for treatment include, but are not limited to, adenocarcinoma, adrenal cortical carcinoma, anal cancer, astrocytoma, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, Ewing's sarcoma, eye cancer, fallopian tube cancer, gastric cancer, glioblastoma, head and neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, myelodysplastic syndrome, multiple myeloma, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, peritoneal cancer, pharyngeal cancer, prostate cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, and Wilms' tumor. According to further particular embodiments, there is provided a method of treating hematological cancer comprising administering to a subject in need thereof an appropriate dose of modified cells described herein, thereby ameliorating at least one symptom of said cancer.

[0160] According to another embodiment, the cells can be provided for use in the treatment of autoimmune diseases. Examples of types of autoimmune diseases that can be treated include, but are not limited to, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), multiple sclerosis (MS), type 1 diabetes, amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease), spinal muscular atrophy (SMA), Crohn's disease, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, psoriatic arthritis, Addison's disease, ankylosing spondylitis, Behcet's disease, celiac disease, Coxsackie myocarditis, endometriosis, fibromyalgia, Graves' disease, Hashimoto's thyroiditis, Kawasaki disease, Meniere's disease, myasthenia gravis, sarcoidosis, scleroderma, Sjogren's syndrome, thrombocytopenic purpura (TTP), ulcerative colitis, vasculitis, and vitiligo.

[0161] This is equivalent to providing a method of treating an autoimmune disease, comprising administering to a subject in need thereof an appropriate dose of modified cells as described herein, thereby ameliorating at least one symptom associated with said autoimmune disease. Examples of autoimmune diseases that can be treated are described above.

[0162] According to yet another embodiment, the cells can be provided for use in the treatment of infectious diseases. "Infectious disease" is used herein to refer to any type of disease caused by the presence of a foreign organism (pathogen) in or on a subject or organism with the disease. Infections are usually thought to be caused by microorganisms or parasitic microorganisms such as viruses, prions, bacteria, and viroids, although larger organisms such as macroparasites and fungi can also infect. Said organisms capable of causing infection are referred to herein as "pathogens" (if they cause disease) and "parasites" (if they benefit at the expense of the host organism, thereby reducing the biological fitness of the host organism, even if no obvious disease is present), and include, but are not limited to, viruses, bacteria, fungi, protozoa (e.g., Plasmodium, Phytophthora) and protozoa (e.g., Plasmodium, Entamoeba, Giardia, Toxoplasma, Cryptosporidium, Trichomonas, Leishmania, Trypanosoma) (parasitic microorganisms) and macroparasites, e.g., worms (e.g., nematodes such as roundworms, filariae, hookworms, pinworms and whipworms, or flat animals such as cestodes and flukes), as well as ectoparasites such as ticks and mites. Parasitoids, i.e., parasitic organisms that kill the host organism, are considered to be included in the term parasite. According to certain embodiments, the infectious disease is caused by a microbial or viral organism.

[0163] As used herein, "microbial organism" may refer to bacteria such as gram-positive bacteria (e.g., Staphylococcus, Enterococcus, Bacillus), gram-negative bacteria (e.g., Escherichia, Yersinia), spirochetes (e.g., Treponema such as Treponema palladium, Leptospira, Borrelia such as Borrelia burgdorferi), mollicutes (i.e., bacteria without a cell wall such as Mycoplasma), acid-resistant bacteria (e.g., Mycobacterium such as Mycobacterium tuberculosis, Nocardia). "Microbacterial organisms" also include fungi (e.g., yeasts and molds, such as Candida, Aspergillus, Coccidioides, Cryptococcus, Histoplasma, Pneumocystis, or Trichophyton), protozoa (e.g., Plasmodium, Entamoeba, Giardia, Toxoplasma, Cryptosporidium, Trichomonas, Leishmania, Trypanosoma), and archaea. Further examples of infectious disease causing microorganisms that can be treated by the present method include, but are not limited to, Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus (MRSA)), Enterococcus (including vancomycin-resistant Enterococcus faecalis (VRE), the nosocomial pathogen Enterococcus faecalis), food pathogens, such as Bacillus subtilis, Bacillus cereus, Listeria monocytogenes, Salmonella, and Legionella pneumoniae.

[0164] "Viral organisms" or "viruses", as used interchangeably herein, are small infectious agents that can only replicate inside living cells of organisms. They include dsDNA viruses (e.g., adenoviruses, herpesviruses, poxviruses), ssDNA viruses (e.g., parvoviruses), dsRNA viruses (e.g., reoviruses), (+)ssRNA viruses (e.g., picornaviruses, togaviruses, coronaviruses), (-)ssRNA viruses (e.g., orthomyxoviruses, rhabdoviruses), ssRNA-RT (reverse transcription) viruses, i.e., viruses with (+)sense RNA that have a DNA intermediate in their life cycle (e.g., retroviruses), and dsDNA-RT viruses (e.g., hepadnaviruses).Examples of viruses that may infect human subjects include, but are not limited to, adenoviruses, astroviruses, hepadnaviruses (e.g., hepatitis B virus), herpesviruses (e.g., herpes simplex virus type 1, herpes simplex virus type 2, human cytomegalovirus, Epstein-Barr virus, varicella zoster virus, roseolovirus), papovaviruses (e.g., human papillomavirus and human polyomavirus), poxviruses (e.g., variola virus, vaccinia virus, smallpox virus), arenaviruses, bunyaviruses, calciviruses, coronaviruses (e.g., SARS coronavirus, MERS coronavirus, SARS-CoV-2 coronavirus (the etiological agent of COVID-19)), filoviruses (e.g., Ebola virus, Marburg virus), flaviviruses (e.g., yellow fever virus, Western blotting virus, Examples of the virus that can be treated include HIV, dengue virus, hepatitis C virus, tick-borne encephalitis virus, Japanese encephalitis virus, encephalitis virus), orthomyxoviruses (e.g., influenza A virus, influenza B virus, and influenza C virus), paramyxoviruses (e.g., parainfluenza virus, rubulavirus (mumps), morbillivirus (measles), pneumoviruses, such as human respiratory syncytial virus), picornaviruses (e.g., poliovirus, rhinovirus, coxsackie A virus, coxsackie B virus, hepatitis A virus, echovirus, and enterovirus), reovirus, retroviruses (e.g., lentiviruses, such as human immunodeficiency virus and human T-lymphotropic virus (HTLV)), rhabdoviruses (e.g., rabies virus), or togaviruses (e.g., rubella virus). According to a particular embodiment, the infectious disease to be treated is not HIV. According to another embodiment, the infectious disease to be treated is not a disease caused by a retrovirus. According to another embodiment, the infectious disease being treated is not a viral disease.

[0165] This is equivalent to saying that a method of treating an infectious disease is provided, comprising administering to a subject in need thereof an appropriate dose of the modified cells described herein (i.e., modified cells comprising exogenous nucleic acid molecules encoding two or more multiplexed RNA interference molecules, and optionally further nucleic acid molecules encoding a protein of interest), thereby ameliorating at least one symptom. Particularly contemplated microbial or viral infectious diseases are those caused by pathogens of. These cells provided for use as medicaments can be provided for use in allogenic therapy. That is, they are provided for use in a treatment (cells from another subject are provided to a subject in need thereof) where allogenic ACT is a possible treatment option. According to a specific embodiment, in an allogenic therapy, at least one of the RNA interference molecules is directed against TCR (most particularly against a subunit of the TCR complex). According to another embodiment, these cells are provided for use in autologous therapy, in particular autologies ACT therapy (i.e. cells obtained from a patient).

[0166] Although specific embodiments, specific configurations, and materials and / or molecules have been discussed herein for the cells and methods according to the present invention, it should be understood that various changes and modifications in form and details can be made without departing from the scope and spirit of the present invention. Importantly, the vector variations discussed in the various vector embodiments also apply to modified cells (as the vector is suitable for expression in such cells), and vice versa. The various embodiments of the cells are typically associated with the vectors encoded in the cells. The following examples are provided to better illustrate certain embodiments and should not be considered as limiting the present application. The present application is limited only by the claims. EXAMPLES

[0167] Example 1. Multiplexing optimization Efficient processing of miRNAs from transcribed RNA by the DROSHA complex is crucial for efficient target knockdown. Our previous data showed that miRNA-based shRNAs can be efficiently co-expressed with CAR-encoding vectors and processed from the vector by the miRNA machinery. Furthermore, it is desirable to generate CAR expression vectors that can co-express multiple miRNA-based shRNAs (e.g., 2, 4, 6, 8...) from the same vector (Figure 2). However, previous studies have shown that co-expression of multiple miRNA-based shRNAs leads to loss of shRNA activity. Therefore, efficient miRNA processing is important for knockdown of multiple targets from a single expression vector.

[0168] We hypothesized that to achieve optimal multiplexing and avoid recombination, it would be best to start with miRNA clusters of natural origin, rather than multiplexing a single miRNA scaffold. Naturally occurring miRNA clusters vary significantly in size and the number of scaffolds present. Since the goal is to use multiplexed miRNA scaffolds to clone vectors, we sought to identify clusters with a promising ratio of size to number of scaffolds. The 13 clusters identified are listed in Table 1.

[0169] [Table 1]

[0170] Table 1. Identification of micro-RNA clusters with the indication of name, chromosomal location, size, location within the coding or non-coding sequence, and strand orientation. N indicates the number of microRNA scaffolds present in the cluster, size / N is the division of these two columns and indicates the average size of the miRNA scaffolds with interspersed sequences (linker + others) within the cluster. Light grey shading: high expression in T cells.

[0171] Two of these clusters (dark grey shading, Table 1) are included for illustrative purposes to show how sizes can vary. These clusters were immediately excluded as they were over 85000bp, too large for cloning. The most promising clusters were selected based on the size of the cluster and the number of miRNAs present in the cluster (N in Table 1). To get an idea of ​​the average miRNA scaffold + linker sequence, the total size as well as the size divided by the number of miRNA scaffolds was evaluated. As a first cutoff, we selected clusters with size / N lower than 250. This gave us enough clusters, and since the goal was to express the vector in engineered immune cells, we decided to focus on clusters that are highly expressed in immune cells such as T cells. This led to prioritization of four clusters (light grey shading, Table 1), all highly expressed in immune cells and with a total size of less than 1000bp. Moreover, they all contained at least three miRNA scaffolds (clusters with N at least 3 can allow multiplexing of more than two miRNAs) with an average size of less than 200 bp per scaffold, and were therefore highly suitable for cloning (see Table 1 ): miR17-92 cluster, miR106a-363 ​​cluster, miR106b-25 cluster (three paralogous microRNA clusters), and miR23a-27a-24-2 cluster.

[0172] 1.1 Selection of miRNA clusters suitable for multiplexing To evaluate whether the four miRNA clusters are suitable for multiplexed expression of shRNAs, we decided to transduce primary T cells from healthy donors with a retroviral vector encoding a second generation CD19-directed CAR with a truncated CD34 selection marker, together with various shRNAs introduced into the selected clusters. To allow comparison of the same number of shRNAs and the effect of truncation of the clusters, we also used fragments of the miR17-92 and miR106a-363 ​​clusters. The fragments were three or four consecutive miRNA scaffolds of the clusters, to allow comparison with the three miRNA scaffolds present in the other two clusters. The schematic design of such vectors is shown in Figure 2.

[0173] Three identical shRNA target sequences targeting CD247, B2M and CD52 were used for comparison. When four miRNA scaffolds were used, TRAC was additionally targeted. For six miRNA scaffolds, three targets were targeted twice, but with different target sequences. As a control, we used a repeat synthetic shRNA scaffold, the miR196a2 scaffold, which has previously been shown to be excellent for single shRNA knockdown and also suitable for multiplexed knockdown (WO2020 / 221939). This control was used with three and four shRNAs.

[0174] Despite the different sizes of the constructs, vector titers were only slightly affected by the amount of shRNA present (data not shown). However, as shown in Figure 3, in all groups, the use of different scaffolds derived from natural miRNA clusters increases transduction efficiency compared to the repeated use of the same scaffold (here, the miR-196a2 scaffold).

[0175] The fold increase in T cells from transduction to harvest was not significantly different between constructs (neither between clustered scaffolds nor between clustered and repeated single scaffolds). However, the knockdown efficiency differed between constructs. All clusters achieved knockdown to some extent, but there were clear differences between the clustered scaffolds, with scaffolds from the miR-106a-363 ​​cluster achieving the best and most consistent knockdown, and those from the miR23a-27a-24-2 cluster being the least effective. In Figure 4, we show an example comparing TCR expression in miR23a-27a-24-2 clustered scaffolds, or miR106a-363 ​​clustered scaffolds or fragments thereof, with a control without shRNA, or with shRNA. The increased knockdown observed in the full scaffold can be explained by the fact that CD247 is targeted twice in this construct. As a result of these experiments, the miR-106a-363 ​​cluster scaffold was selected for further evaluation.

[0176] Example 2. Multiplexing with the miR-106a-363 ​​cluster scaffold The feasibility of multiplexing up to six shRNAs was evaluated in difficult-to-transduce primary immune cells. To assess this, primary T cells were transduced with a retroviral vector encoding a second-generation CD19 CAR containing either 3x shRNA or 6x shRNA targeting CD247, β2m, and CD52 introduced into the miR-106a-363 ​​cluster. The vector design is shown in Figure 5.

[0177] Briefly, primary T cells from healthy donors were transduced with a retroviral vector encoding a second-generation CD19-directed CAR, a truncated CD34 selection marker, and three shRNAs targeting CD247, B2M, and CD52 introduced into the last three miRs (miR-19b2, miR-92a2, and miR-363) of the 106a-363 ​​miRNA cluster, or six shRNAs targeting the same three genes in the six miR scaffolds of the cluster (in this case, the two shRNAs targeting CD247 were different). Briefly, shRNAs were expressed as 6-plex, 3-plex, or no shRNA (tCD34) as a control. Two days after transduction, cells were enriched using CD34-specific magnetic beads and further expanded with IL-2 (100 IU / mL) for 6 days. The mRNA expression of CD247, B2M, and CD52 was assessed by qRT-PCR using cyclophilin as a housekeeping gene.

[0178] The results are shown in Figure 6. Multiplexed shRNAs provided efficient RNA knockdown levels for all target genes. Incorporation of six multiplexed shRNAs (two shRNAs for each protein target) provided higher RNA knockdown levels compared to three multiplexed shRNAs (one shRNA for each protein target) (Figure 6).

[0179] Example 3 Optimization of individual scaffolds of the miR-106a-363 ​​cluster Although initial data were already promising and showed that multiplexing could be achieved using scaffolds derived from the miR-106a-363 ​​cluster, further studies were carried out to see if individual scaffolds could be modified to improve knockdown of selected targets. As it stands that the natural scaffolds have already been under evolutionary selective pressure to accommodate knockdown (meaning that the lower and upper stem regions have been at least partially optimized by evolution), it was decided to first evaluate different target sequences to improve target downregulation, as they had not yet been optimized. In the first example, the same target protein was chosen.

[0180] Since it has been previously described that the processivity of each miRNA / shRNA can depend on and be influenced by others in the cluster ( Bofill-De Ros and Gu, 2016 ), we decided to test scaffolds with various target sequences as part of the overall cluster, but unrelated sequences in other scaffold sequences (which would not affect target downregulation).

[0181] The results of CD247 downregulation in the miR-20b scaffold are shown in Figure 7. The initial scaffold sequence already resulted in about 50% downregulation. All other target sequences tested also successfully knocked down the target, with some achieving knockdown far exceeding 50%. In other words, by selecting the target sequence, the most effective knockdown could be achieved, and no further modification of the miR-20b scaffold was required.

[0182] Similar results were obtained with the miR-106a scaffold sequences using different sequences against the B2M target (data not shown). To exclude that the effect was related to a specific target sequence-scaffold combination, the B2M target sequences were also tested with the miR-20b scaffold. Although there was some variability in terms of knockdown efficiency, the three target sequences that achieved the highest knockdown in the miR-106a scaffold also achieved the highest knockdown when used with the miR-20b scaffold. This means that once an effective target sequence is identified, it can be used across scaffolds.

[0183] Optimization of shRNA against CD95 was performed on the miR-18b scaffold. However, after testing 31 target sequences, the best knockdown achieved was about 30% (see FIG. 8). Although this knockdown is not negligible, it was significantly less effective than the >75% knockdown consistently obtained with other scaffolds. Comparing the miR-18b scaffold to the miR-106a or miR-20b scaffolds (FIG. 9), it is clear that this scaffold contains more mismatches in the target sequence / upper stem region (3 vs. 1) and also contains a bulge near the end of the upper stem. Since high knockdown was achieved with other scaffold sequences, we hypothesized that reducing the number of mismatches and / or removing the bulge may improve knockdown efficiency.

[0184] The five different constructs evaluated are shown in FIG. 10A, and the results are shown in FIG. 10B. Surprisingly, the deletion of even one mismatch or bulge significantly increases the knockdown efficiency. Retaining only one mismatch, which also occurs in the miR-106a or miR-20b scaffolds, increases the knockdown efficiency from about 30% to more than 60% for the same target sequence. Thus, the miR-18b scaffold sequence can be used as is, but the knockdown efficiency can be significantly increased by reducing the number of mismatches or bulges. As a further example in the present invention, we use construct 28.5, in which the bulge at positions 15-16 and the mismatches at positions 20 and 25 have been removed, but the mismatch at position 29 has been retained. Of note, the removal of the mismatch is by adapting the passenger sequence, since the target sequence needs to be matched.

[0185] Example 4 Evaluation of target sequence length The native target sequences found in the miR-106a-363 ​​cluster are typically quite long (22-23 bp). To assess whether these could be shortened, target sequences of various lengths (one against CD247 and one against B2M) were inserted into the scaffolds and assessed for knockdown efficiency. Sequence shortening was achieved by replacing the nucleotides at the 3´ end of the target sequence with those found in the native scaffold. Results for the miR-106a scaffold are shown in Figure 11. It can be seen that sequences as short as 18 bp function as well as the maximum length, and in some cases even better. Similar results were obtained for the miR-20b scaffold (not shown). For most experiments, we decided to work with a 20 bp target sequence (as shown in Figure 9).

[0186] Example 5 Evaluation of combinations of individual scaffolds outside of cluster association It is generally accepted that in miRNA clusters, the presence of multiple flanking sequence determinants, as well as other clusters, is likely important to achieve downregulation, however, our previous experiments showed that this is not always the case.

[0187] Indeed, to optimize the activity of two co-expressed shRNAs, we previously hypothesized that not only the size but also the sequence of the linker between the two miRNA-based shRNAs, as well as the miRNA scaffold, would affect shRNA activity. To optimize shRNA processing, we evaluated the effect of various shRNA linkers on the knockdown of two target genes, CD247 (CD3ζ) and CD52. Linkers from 0 to 92 bp were used, but the linker did not seem to affect knockdown efficacy, apart from a construct lacking a spacer between the two hairpins, which showed slightly lower knockdown activity for TCR (but not for CD52) compared to the other constructs. Importantly, a construct without a linker also functioned well to reduce the expression of both shRNAs (data not shown). Although these experiments were performed with the miR-196a2 scaffold, initial experiments showed that the linker of the miR-106a-363 ​​cluster could also be significantly reduced.

[0188] To assess whether the processing capacity and activity of individual scaffolds is affected by the presence of others in the cluster, we decided to test the scaffolds in different permutations. For this purpose, we chose non-contiguous scaffolds (to eliminate the influence of neighboring scaffolds in the cluster): miR-106a and miR-20b. Furthermore, rather than using all six miRNA scaffolds in the cluster, duplexes and triplexes were generated (in contrast to Example 2). We also generated a miR-106a-miR-18b-miR-20b triplex, corresponding to the first three scaffolds in the miR-106a-363 ​​cluster, to assess whether there are cluster-related effects. In the case of the duplex, the targeted genes were B2M and CD247. In the case of the triplex, CD95 was added.

[0189] In summary, the following constructs were made:

[0190] Duplex: miR-106a (targets B2M)-miR-20b (targets CD247) miR-20b (targets CD247)-miR-106a (targets B2M) miR-20b (targets B2M)-miR-20b (targets CD247) miR-106a (targets B2M)-miR-106a (targets CD247)

[0191] Triplex: miR-20b (targets B2M)-miR-20b (targets CD95)-miR-20b (targets CD247) miR-106a (targets B2M)-miR-106a (targets CD95)-miR-106a (targets CD247) miR-106a (targets B2M)-miR-18b (targets CD95)-miR-20b (targets CD247)

[0192] The results are shown in Figures 12A-C. As shown in Figure 12, all duplexes evaluated were highly efficient at downregulating both CD247 and B2M. In particular, CD247 knockdown was shown to be highly efficient, resulting in barely detectable levels of CD3Z. Since B2M is highly abundant, knockdown was not expected to be complete, but >80% reduction in B2M levels was consistently achieved. Remarkably, the level of downregulation is identical regardless of the order of the scaffolds in the duplex.

[0193] It is well known that when multiplexing identical shRNAs, recombination can cause problems, leading to much lower expression and ultimately lower knockdown levels. This was exactly the reason for evaluating combinations of different scaffolds. Nevertheless, to see if this is feasible in practice, two and three identical scaffolds were tested. All of the duplexes with identical scaffolds, as well as the miR-20b triplex scaffold, achieved transduction levels comparable to duplexes or triplexes with different scaffolds, both above 15%. However, the miR-106a triplex scaffold led to very low transduction levels (less than 2%) and was not evaluated further. Duplexes with miR-20b scaffolds achieved the same level of target knockdown as duplexes with non-identical scaffolds (Figure 12A-B). Duplexes of the miR-106a scaffold achieved the same downregulation of CD3Z, but were slightly less effective at knocking down B2M, with levels reduced by approximately 50%, indicating that these scaffolds can be replicated and still achieve high knockdown (Figure 12C). Surprisingly, the miR-20b triplex scaffold achieved knockdown levels comparable to triplexes with three different scaffolds, but slightly better knockdown was obtained for each target gene using the three different scaffolds, indicating some loss of efficacy (Figures 12A-B). The triplex scaffold with three different miRNA scaffolds achieves downregulation of the same targets as the duplex. Additionally, CD95 was downregulated by over 50% (Figures 12B-C), consistent with the results of this target sequence when used in a clustered context (Figure 10B).

[0194] These experiments show that scaffolds can be used independently and well outside the context of a cluster. The order of the scaffolds does not seem to be important to achieve the desired knockdown, and scaffolds from all clusters do not need to be present to achieve knockdown. Indeed, a single scaffold is sufficient and can be replicated without loss of activity. It was shown that miR-20b can be used as a triplex, but this appears to be slightly less efficient than using different scaffolds. Nevertheless, considering that there are six different scaffold sequences in the miR-106a-363 ​​cluster, which can be replicated without loss of effect, multiplexed downregulation of up to 12 targets is in principle feasible.

[0195] Example 6 Optimization of individual scaffolds by creating chimeric scaffolds As an alternative to removing mismatches or bulges in individual scaffolds (see Example 3), it was reasoned that the knockdown efficiency of individual scaffolds with mismatches and / or bulges (e.g., miR-18b) could be improved not only by mutating the scaffolds by removing mismatches, but also by replacing the upper stem / loop region with that of a scaffold with high knockdown efficiency. Indeed, when the upper stem / loop region of either the miR-106a scaffold or the miR-20b scaffold was used to replace the upper stem / loop region of the miR-18b scaffold, the knockdown efficiency of selected targets was significantly increased (data not shown). Since there are several conserved scaffolds within the three miR-17 paralog clusters, we decided to evaluate this for other scaffold sequences as well. The results showed that indeed, scaffolds with few mismatches and bulges similar to the miR-106a, mir-20b or miR-17 scaffolds could be used. That is, the upper stem / loop region of scaffolds from the miR-17 family (miR-17 scaffold, miR-20a scaffold, miR-106a scaffold, miR-20b scaffold, miR-106b scaffold, and miR-93 scaffold) could be fused to the lower stem of any of the scaffolds of the miR106a-363, miR17-92, and miR106b-25 clusters to maintain or increase knockdown efficiency. As an example, Figure 13 shows the effect of changing the scaffold on knockdown efficacy. For this purpose, four different constructs were tested.A negative control including only CAR T without shRNA, and three CAR T cells with shRNA: one duplex with miR-106a scaffold (targeting B2M) and miR-20b scaffold (targeting CD247), a triplex with miR-106a scaffold (targeting B2M)-miR-18b scaffold (targeting CD95)-miR-20b scaffold (targeting CD247) in which the loop region of miR-18 has been altered (see Example 3), and a triplex with miR-106a scaffold (targeting B2M)-miR-18b scaffold (targeting CD95)-miR-20b scaffold (targeting CD247) in which the upper stem / loop region of scaffold miR-18b has been replaced with the upper stem / loop region of miR-17 scaffold. As seen in Figure 13, knockdown of HLA-I and TCR is achieved with all three constructs, while knockdown of CD95 is achieved with the triplex construct. However, the chimeric scaffold construct achieved a higher degree of knockdown of CD95. Concurrently, alterations in the scaffold targeting CD95 were also found to induce changes in the expression of HLA-I, while neither the scaffold nor the target sequence was altered (Figure 13, left panel), thus indicating changes in the microprocessing of the cluster.

[0196] To further optimize this, we decided to keep microprocessing constant by retaining the scaffold sequence of the latter construct (i.e., triplex with miR-106a scaffold (targeting B2M)-miR-18b scaffold (targeting CD95)-miR-20b scaffold (targeting CD247), where the upper stem / loop region of scaffold miR-18b was replaced with the upper stem / loop region of miR-17 scaffold). To ensure that microprocessing is independent of the target sequence, different target sequences for B2M were evaluated. The results are shown in FIG. 14. Four different B2M target sequences in triplex scaffolds were tested against duplexes with miR-106a scaffold (targeting B2M) and miR-20b scaffold (targeting CD247) as well as negative controls including CAR T cells without shRNA. All triplex constructs achieved good knockdown of all three targets (Figure 14A,B). HLA-I expression showed some variability due to the different target sequences, but knockdown of the other two targets remained constant. All four sequences tested achieved at least 50% knockdown while maintaining TCR knockdown, and also achieved high knockdown of CD95.

[0197] To check whether knockdown of sequences also has functional consequences, various functional assays were performed with BCMA CAR T cells further transduced with triplex scaffold. The results are shown in Figure 15. Targeting B2M in adoptive T cells should downregulate HLA-I, thereby inhibiting T cell allo-recognition and preventing host-versus-graft reactions. However, complete depletion of HLA renders cells prey to cell killing by NK, which is why gene-edited cell therapy typically requires co-expression of HLA-E. We speculated that knockdown of B2M would be beneficial in this regard, since HLA-I is downregulated, but not to the extent that cells are completely eliminated by cytolysis by NK. As shown in Figure 15A, downregulation of B2M achieved with triplex shRNA scaffold protects BCMA CAR T cells from killing by NK when cells are co-cultured with NK cells. As a comparison, BCMA CAR-Ts with B2M knocked out with Crispr / Cas9 were used and were completely lysed by allogeneic NK cells. Importantly, as shown in Figure 15B, shRNA against B2M inhibits T cell non-self recognition similarly to B2M knockout, with 80% of cells remaining viable versus 50% with CARs without shRNA. Finally, to test the effect of shRNA against CD95 (Fas receptor), cells were incubated with 100 ng / ml Fas ligand. As seen in Figure 15C, shRNA against CD95 (in triplex scaffolds) protects against FasL-mediated apoptosis.

[0198] These results indicate that shRNAs can be successfully multiplexed when the chimeric scaffold is contained in a cluster.

[0199] Example 7 Optimization of clusters by creating chimeric clusters Since the upper stem and loop regions of the miR-17 family scaffold proved to be beneficial in optimizing the knockdown efficiency of the miR-106a-363 ​​cluster, we evaluated whether multiplexed clusters could be created using only the miR-17 family scaffold. For this purpose, a fourplexed shRNA cluster was designed, including four shRNA scaffolds from three different miR17-92 paralog clusters: miR-106a and miR-20b from the miR-106a-363 ​​cluster; miR-93 from the miR-106b-25 cluster, and miR-20a from the miR-17-92 cluster. The target genes were B2M in miR-106a, CD3 zeta (as above) in miR-20b, MICA (NKG2D ligand) in miR-93, and CD28 in miR-20a. A schematic diagram of the construct is shown in Figure 16A. Wild-type / native flanking sequences were used as linkers (see Example 5) and no additional restriction sites were inserted between the scaffolds to minimize the risk of altered microprocessing. Knockdown was compared to a negative control without shRNA and to a duplex of identical miR-106a (targeting B2M) and miR-20b (targeting CD247) scaffolds. As shown in Figure 16B, left and right panels, knockdown of TCR and HLA class I is similar for duplex and fourplex constructs. Furthermore, the fourplex succeeds in knockdown of CD28 expression (Figure 16B, middle panel) and MICA expression (Figure 16C). Thus, it is clear that miR-17 family scaffolds can be combined to achieve multiplex knockdown, although the association (and therefore processing) of the scaffolds is altered compared to the wild-type cluster. Moreover, this does not encounter recombination, as shown by the high level of knockdown achieved.

[0200] To further support this, we decided to add an additional scaffold. A fiveplex scaffold was created by adding the miR-17 scaffold to the fourplex scaffold shown in Figure 16A. The target sequence of this scaffold was CD95.

[0201] This fiveplex scaffold was compared to two other fiveplex scaffolds: one in which the target sequences remained identical, but two (MICA and CD28) were swapped out of the scaffold to assess position effects, and one in which different B2M and CD3 zeta sequences were used that were optimized for a different unrelated scaffold. Since the microprocessing of the unrelated scaffolds may be different, we sought to check whether the use of non-optimized sequences would be a valid strategy.

[0202] Fiveplex 1: miR-106a (targets B2M with a non-optimized sequence)-miR-20b (targets CD247 with a non-optimized sequence)-miR-93b (targets CD28)-miR-20a (targets MICA)-miR-17 (targets CD95) Fiveplex 2: miR-106a (targets B2M)-miR-20b (targets CD247)-miR-93b (targets CD28)-miR-20a (targets MICA)-miR-17 (targets CD95) Fiveplex 3: miR-106a (targets B2M)-miR-20b (targets CD247)-miR-93b (targets MICA)-miR-20a (targets CD28)-miR-17 (targets CD95)

[0203] The results are shown in Figure 17. Fiveplex 2 and 3 achieved knockdown in all five target genes (Figures 17A and B), indicating that target sequences can be switched within the relevant scaffold without affecting the knockdown or microprocessing of the cluster. However, this appears to be true only for target sequences optimized for the relevant scaffold. Knockdown of TCR is less efficient in fiveplex 1, and knockdown of HLA-I is almost nonexistent. Furthermore, knockdown of CD95 and CD28 appears to be less efficient (Figure 17A), and knockdown of MICA is also unsuccessful (Figure 17B), even though the scaffolds and sequences used for these four targets were identical to those in fiveplex 2 and 3. This also indicates changes in microprocessing that affect the knockdown of multiple sequences (see also Figure 13). Thus, sequences that work in scaffolds from the miR-17 family cluster can be used in a multiplex situation with different scaffolds from the miR-17 family. However, sequences available in other scaffolds cannot automatically be used in miR-17 family clusters.

[0204] Example 8. Optimization of clusters by combining chimeric clusters with chimeric scaffolds. As shown in Examples 6 and 7, optimal multiplexing results are achieved when the suboptimal scaffold is modified by chimerizing it with the miR-17 family upper stem / loop region (Example 6) or by combining it with several miR-17 family scaffolds from various miR-17-92 paralog clusters (Example 7). We next assessed whether these two strategies could be combined to achieve even higher multiplexing.

[0205] First, a fiveplex chimeric construct was designed as follows:

[0206] Chimeric miR-92a2 with the upper stem / loop region from miR-106a (targeting B2M)-optimized miR-18b (targeting CD95)-miR-20b (targeting CD247)-miR-93b (targeting MICA)-miR-17 (targeting CD28). See scheme in Figure 18A.

[0207] Essentially, this is the triplex construct described in Example 6 fused to the miR-93b scaffold (as done in Example 7) with an additional chimeric scaffold (a scaffold derived from the miR-106a-363 ​​cluster) with the lower stem from miR-92a2 and the upper stem and loop from miR-17.

[0208] When this fiveplex construct is compared to a triplex construct (consisting only of miR-106a (targeting B2M)-optimized miR-18b (targeting CD95)-miR-20b (targeting CD247)), it is found that the fiveplex is at least as efficient as the triplex in achieving knockdown of the target genes. The top and middle histograms in FIG. 18B show a clear reduction compared to the control histogram below. FIG. 18C is the same as FIG. 18B but shows the results as relative MFI. The triplex successfully knocks down three target genes, while the fiveplex downregulates all five genes to the same extent.

[0209] Finally, by adding an additional chimeric scaffold, a sixplex cluster was designed, this time using the miR-363 lower stem and the miR-20a upper stem and loop.

[0210] The design is as follows:

[0211] miR-106a (targets CD38)-optimized miR-18b (targets CD95)-miR-20b (targets CD247)-miR-93b (targets B2M)-chimera with upper stem / loop region from miR-17; miR-92a2 (targets CD28)-chimera with upper stem / loop region from miR-20a (targets CD27).

[0212] As can be seen, the B2M target sequence was tested in different non-adjacent scaffolds. As shown in Figure 19A, all six genes were knocked down compared to the control without shRNA. Figure 19B is the same as Figure 19A, but shows the results as relative MFI. With the exception of B2M, all targets achieved more than 50% knockdown, likely due to the fact that the targets are abundant.

[0213] We consistently demonstrated multiplexed knockdown when using miRNA scaffolds derived from the miR-17 family cluster, knockdown that could be further improved by creating chimeric scaffolds using the upper stem / loop region from the miR-17 family scaffold and / or by creating chimeric clusters and using miR-17 family scaffolds derived from different paralog clusters.

[0214] It will be appreciated that the invention is not limited to the particular details described herein merely by way of example, and that various modifications and variations are possible within the scope of the invention.

[0215] References BofiN-De Ros X, Gu S. Guidelines for the optimal design of miRNA-based shRNAs. Methods. 2016 Jul l;103:157-66. Chumakov SP, Kravchenko JE, Prassolov VS, Frolova El, Chumakov PM. Efficient downregulation of multiple mRNA targets with a single shRNA-expressing lentiviral vector. Plasmid. 2010 May;63(3):143- 9. Fowler DK, Williams C, Gerritsen AT, Washbourne P. Improved knockdown from artificial microRNAs in an enhanced miR-155 backbone: a designer's guide to potent multi-target RNAi. Nucleic Acids Res.2016 Mar 18;44(5):e48. Giering JC, Grimm D, Storm TA, Kay MA. Expression of shRNA from a tissue-specific pol II promoter is an effective and safe RNAi therapeutic. Mol Ther. 2008 Sep;16(9):1630-6. Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR, Marion P, Salazar F, Kay MA. Fatality in mice due to oversaturation of cellular microRNA / short hairpin RNA pathways. Nature. 2006 May 25;441(7092):537-41. Jiang Z, Han Y, Cao X. Induced pluripotent stem cell (iPSCs) and their application in immunotherapy. Cell Mol Immunol. 2014 Jan;ll(l):17-24. 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. Moore CB, Guthrie EH, Huang MT, Taxman DJ. Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown. Methods Mol Biol. 2010;629:141-58. Taxman DJ, Livingstone LR, Zhang J, Conti BJ, locca HA, Williams KL, Lich JD, Ting JP, Reed W. Criteria for effective design, construction, and gene knockdown by shRNA vectors. BMC Biotechnol. 2006 Jan 24;6:7. Themeli M, Riviere I, Sadelain M. New cell sources for T cell engineering and adoptive immunotherapy. Cell Stem Cell. 2015 Apr 2;16(4):357-66.

Claims

1. A nucleic acid molecule comprising at least one RNA interference molecule having a modified scaffold, the modified scaffold comprising a lower stem region and an upper stem / loop region, the lower stem region of the scaffold being of a miR scaffold from the miR-17 family cluster, and at least a portion of the upper stem / loop region of the scaffold being modified to differ from a wild-type sequence.

2. 2. The nucleic acid molecule of claim 1, wherein the lower stem region of the modified scaffold is selected from a miR-17 scaffold, a miR-18a scaffold, a miR-19a scaffold, a miR-20a scaffold, a miR-19b-1 scaffold, a miR-92-1 scaffold, a miR-106a scaffold, a miR-18b scaffold, a miR-20b scaffold, a miR-19b-2 scaffold, a miR-92-2 scaffold, a miR-363 scaffold, a miR-106b scaffold, a miR-25 scaffold, and a miR-93 scaffold.

3. 2. The nucleic acid molecule of claim 1, wherein the modified scaffold is a chimeric scaffold, and wherein at least a portion of the upper stem / loop region is not from the same miR scaffold as the lower stem region, and wherein the at least a portion of the upper stem / loop region is selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold.

4. The nucleic acid molecule of claim 1 , wherein the at least one RNA interference molecule is at least two multiplexed RNA interference molecules.

5. A nucleic acid molecule comprising at least two RNA interference molecules having different scaffolds, the at least two different scaffolds having a lower stem region of a miR scaffold from a miR-17 family cluster; At least one RNA interference molecule has a chimeric scaffold; at least a portion of the upper stem / loop region is not from the same miR scaffold as the lower stem region; said at least a portion of said upper stem / loop region is selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold; and / or A nucleic acid molecule, wherein said scaffolds derived from said at least two RNA interference molecules are miR-17 family scaffolds derived from different miR-17 family clusters.

6. 13. A vector suitable for expression in a modified immune cell comprising the nucleic acid molecule of claim 1.

7. A modified cell comprising a first exogenous nucleic acid molecule encoding a protein of interest and a second nucleic acid molecule comprising at least one RNA interference molecule having a modified scaffold, wherein a lower stem region of the scaffold is that of a miR scaffold from the miR-17 family cluster, and at least a portion of an upper stem / loop region of the scaffold is modified to differ from a wild-type sequence.

8. A modified cell comprising a first exogenous nucleic acid molecule encoding a protein of interest and a second nucleic acid molecule comprising at least two RNA interference molecules having different scaffolds, wherein the at least two different scaffolds comprise a lower stem region of a miR scaffold from a miR-17 family cluster; at least one RNA interference molecule comprises a chimeric scaffold, wherein at least a portion of the upper stem / loop region is not from the same miR scaffold as the lower stem region, and wherein said at least a portion of the upper stem / loop region is selected from a miR-17 scaffold, a miR-20a scaffold, a miR-106a scaffold, a miR-20b scaffold, a miR-106b scaffold, and a miR-93 scaffold; and / or The modified cell, wherein said scaffolds derived from said at least two RNA interference molecules are miR-17 family scaffolds derived from different miR-17 family clusters.

9. The modified cell of claim 7 or 8, which is a modified immune cell.

10. 10. The modified cell of claim 9, wherein the modified immune cell is selected from a T cell, a NK cell, a NKT cell, a macrophage, a stem cell, a progenitor cell, and an iPSC cell.

11. The modified cell according to claim 7 or 8, wherein the protein of interest is a receptor, in particular a chimeric antigen receptor or a TCR.

12. The modified cell of claim 7 or 8, wherein the at least one RNA interference molecule is at least two multiplexed RNA interference molecules under the control of one promoter.

13. The modified cell of claim 12, wherein the at least two multiplexed RNA interference molecules are at least three multiplexed RNA interference molecules.

14. A nucleic acid molecule according to claim 1, a vector according to claim 6 or a modified cell according to any one of claims 7 or 8 for use as a medicament.

15. 10. A nucleic acid molecule according to claim 1, a vector according to claim 6, or a modified cell according to claim 7 or 8 for use in the treatment of cancer.

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