Improved scaffolds for multiplexing inhibitory RNAs

Abstract

The present application relates to the field of RNA interference, more particularly to the field of RNA interference as applied in immunotherapy, such as adoptive cell therapy (ACT).Here, multiple shRNAs designed to downregulate multiple targets are proposed.Also proposed are polynucleotides, vectors encoding the shRNAs, and cells expressing such shRNAs alone or in combination with proteins of interest, such as chimeric antigen receptors (CARs) or T cell receptors (TCRs).These cells are particularly suitable for use in immunotherapy.

Description

[Technical Field]

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

[0002] Simultaneous downregulation of multiple targets in cells that are difficult to transduce in an efficient manner is a known challenge. Multiplexed genome engineering methods are often tedious. When considering solving the problems faced by multiplexed genome engineering, instead of gene knockout, systems that offer the possibility of knockdown can be considered, which allows greater flexibility (e.g., temporal control). Ideally, these systems are not too tedious (so there is no need to engineer a separate protein for each target, or downregulation can be achieved in a single transduction step), yet are 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] Because of the importance of the microRNA pathway in regulating gene activity, researchers are currently investigating the extent to which artificially designed molecules, small interfering RNAs ("siRNAs"), can mediate RNAi. siRNAs can cause cleavage of target molecules, such as mRNA, and like miRNAs, siRNAs rely on base complementarity to recognize said target molecules.

[0005] One class of molecules known as siRNAs is short hairpin RNAs ("shRNAs"). shRNAs are single strands containing a sense region and an antisense region that can hybridize to the sense region. shRNAs can form stem-loop structures, in which the sense and antisense regions form 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 multicomponent 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 interactions between multiple proteins. As a result, researchers are seeking effective methods for simultaneously delivering multiple siRNAs into a cell or organism.

[0007] One delivery option is the use of vector technology to express shRNAs, which are processed by the cell's 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 capable of expressing multiple shRNAs. Unfortunately, published literature describes several challenges when expressing multiple shRNAs from a single vector. These challenges include: (a) the risk of vector recombination and loss of shRNA expression; (b) reduced shRNA functionality due to position effects in multiplexed cassettes; (c) the complexity of shRNA cloning; (d) RNAi processing saturation; (e) cytotoxicity; and (f) undesirable nonspecific effects.

[0008] Furthermore, while siRNAs have been shown to be effective for short-term gene inhibition in certain transformed mammalian cell lines, their use in primary cell cultures or stable transcriptional knockdown has proven challenging. Knockdown efficacy is known to vary widely, ranging from <10% to >90% (e.g., Taxman et al., 2006), and therefore requires further optimization. This optimization is even more important in such settings, as efficacy typically decreases when multiple inhibitors are expressed. Thus, there remains a need to develop efficient cassettes and vectors for the delivery of multiplexed RNA interference molecules. While true for cellular applications in general, this has been much less explored in the field of ACT, and there is a great need for efficient systems in these cells. Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there is a need in the art to provide a system that enables cell therapy with multiplexed knockdown of targets that does not require multi-step manufacturing processes (and is therefore relatively easy and inexpensive to manufacture) and that provides flexibility (e.g., by making the changes reversible, attenuating the knockdown (e.g., to avoid toxicity), or swapping targets for one another). [Means for solving the problem]

[0010] (Summary of the Invention) Surprisingly, it is shown herein that not only can shRNAs be successfully multiplexed in cells, particularly in engineered immune cells, but multiple targets are also downregulated very efficiently using scaffolds, particularly multiplexed scaffolds, of naturally occurring miRNA clusters, particularly the miR-106a-363 ​​cluster.

[0011] Therefore, one object of the present invention is to provide a vector comprising a nucleic acid sequence encoding at least one RNA interference molecule having a scaffold selected from those present in the miR-106a-363 ​​cluster, particularly 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 certain embodiments, the vector is suitable for expression in eukaryotic cells, particularly immune cells. The RNA interference molecule also typically comprises a target sequence not present in the native scaffold sequence. This is typically achieved by replacing the 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 sequence of the mRNA encoding the target protein. Most particularly, the target sequence has a length of 18 to 23 nucleic acids. The complementary strand of the target sequence is typically referred to as the passenger sequence.

[0012] According to certain embodiments, at least one of the scaffolds of the one or more RNA interference molecules is a scaffold selected from the miR-106a scaffold, the miR-18b scaffold, and the miR-20b scaffold. In other words, according to these certain embodiments, there is provided a vector comprising a nucleic acid sequence encoding at least one RNA interference molecule having a scaffold selected from those present in the first three scaffolds of the miR-106a-363 ​​cluster, i.e., having a scaffold selected from the miR-106a scaffold, the miR-18b scaffold, and the miR-20b scaffold. For example, at least one RNA interference molecule may have a miR-106a scaffold, while other RNA interference molecules may have independently selected scaffolds, such as scaffolds 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.

[0013] According to certain embodiments, a plurality of RNA interference molecules are present in the vector. According to these embodiments, the at least one RNA interference molecule is at least two RNA interference molecules, particularly at least two multiplexed RNA interference molecules. Thus, according to these embodiments, a vector is provided comprising a nucleic acid sequence encoding at least two RNA interference molecules having a scaffold selected from those present in the miR-106a-363 ​​cluster, particularly having 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. When at least two multiplexed RNA interference molecules are present, these two or more molecules may have the same or different scaffolds, i.e., one or more scaffolds selected from miR-106a scaffold, miR-18b scaffold, miR-20b scaffold, miR-19b-2 scaffold, miR-92-2 scaffold, and miR-363 scaffold. However, it is particularly contemplated that no more than three of the scaffolds are the same, and even more particularly, that no more than two of the same scaffolds are used. This is to avoid recombination between the same scaffold sequences (see Example 5).

[0014] According to a particular embodiment, the scaffolds present in the vector are selected from only the six mentioned above (miR-106a scaffold, miR-18b scaffold, miR-20b scaffold, miR-19b-2 scaffold, miR-92-2 scaffold, and miR-363 scaffold), however, it is also envisaged that they may further be combined with different unrelated sequences, such as different scaffold sequences, in particular miR-196a2 sequences (to avoid recombination). According to certain of these embodiments, a vector is provided comprising a nucleic acid sequence encoding at least two RNA interference molecules, at least one of which has a scaffold selected from those present in the miR-106a-363 ​​cluster, in particular a scaffold 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.

[0015] According to certain embodiments, the scaffold sequence may be engineered 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 engineered (and modified compared to the native sequence) to reduce the number of mismatches and / or bulges in the stem region (see Example 3).

[0016] According to a further aspect, there is provided an engineered cell comprising a nucleic acid molecule encoding at least one RNA interference molecule having a scaffold selected from those present in the miR-106a-363 ​​cluster, particularly 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. The RNA interference molecule typically also includes a target sequence not present in the native scaffold sequence. To this end, the mature sequence of each miRNA scaffold is replaced with an optimal target sequence. The target sequence is typically 18 to 23 nucleic acids in length. It is particularly envisioned that the target sequence is directed against a sequence present in the engineered cell, particularly a target sequence. That is, the at least one RNA interference molecule has a sequence that targets (by base pair complementarity) a sequence in the engineered cell that encodes a protein to be downregulated.

[0017] According to certain embodiments, the engineered cells comprise at least two RNA interference molecules, in particular at least two multiplexed RNA interference molecules having a scaffold 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.

[0018] According to a further embodiment, the engineered cell comprises: a first exogenous nucleic acid molecule encoding a protein of interest; a second nucleic acid molecule encoding at least one RNA interference molecule having a scaffold 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; An engineered cell is provided, comprising:

[0019] It should be understood that the first and second exogenous nucleic acid molecules can be provided as a single vector, or they can be provided as separate nucleic acid molecules.

[0020] According to certain embodiments, the at least one RNA interference molecule comprises a target sequence within the scaffold that is different from the 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 engineered cell by base pair complementarity of the target sequence.

[0021] According to further particular embodiments, the engineered cells comprise: a first exogenous nucleic acid molecule encoding a protein of interest; a second nucleic acid molecule encoding at least two multiplexed RNA interference molecules having a scaffold 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; and An engineered cell comprising:

[0022] When at least two multiplexed RNA interference molecules are present, these two or more molecules may have the same or different scaffolds, i.e., one or more scaffolds selected from miR-106a scaffold, miR-18b scaffold, miR-20b scaffold, miR-19b-2 scaffold, miR-92-2 scaffold, and miR-363 scaffold. However, it is particularly contemplated that no more than three of the scaffolds are the same, and more particularly, it is contemplated that no more than two of the same scaffolds are used. This is to avoid recombination between the same scaffold sequences (see Example 5).

[0023] The engineered cells are in particular eukaryotic cells, more particularly engineered mammalian cells, even more particularly engineered human cells. According to particular embodiments, the cells are engineered immune cells. Exemplary immune cells are selected from T cells, NK cells, NKT cells, macrophages, stem cells, progenitor cells, and iPSC cells.

[0024] According to certain embodiments, the engineered cells further comprise a nucleic acid encoding a protein of interest. In particular, this protein of interest is a receptor, in particular a chimeric antigen receptor or TCR. The chimeric antigen receptor or engineered TCR can be directed against any target; typical examples include CD19, CD20, CD22, CD30, BCMA, B7H3, B7H6, NKG2D, HER2, HER3, GPC3, and MUC1, although many more exist and are suitable. According to certain embodiments, multiple proteins of interest can be present. In such cases, the second (or further) protein can be a receptor, or can be, for example, a cytokine, chemokine, hormone, antibody, histocompatibility antigen (e.g., HLA-E), tag, or any other protein of therapeutic or diagnostic value or that allows for detection.

[0025] 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 a lentivirus, retrovirus, adenovirus, adeno-associated virus, and Sendai virus).

[0026] 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, depending on the number of target molecules to be downregulated and practical considerations regarding co-expression of the multiplexed molecules. According to certain embodiments, at least three multiplexed RNA interference molecules are used. According to further particular embodiments, at least one of the at least three RNA interference molecules has a scaffold selected from a miR-106a scaffold and a miR-20b scaffold. According to another embodiment, at least one of the at least three RNA interference molecules has a scaffold selected from a miR-106a scaffold and a miR-18b scaffold.

[0027] According to certain embodiments, the scaffold sequence may be engineered 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 engineered (and modified compared to the native sequence) to reduce the number of mismatches and / or bulges in the stem region (see Example 3).

[0028] A "multiplex" is a polynucleotide encoding multiple molecules of the same type, such as multiple siRNAs, shRNAs, or miRNAs. Within a multiplex, when the molecules are of the same type (e.g., all shRNAs), they may contain the same or different sequences. Between the molecules of the same type, there may be intervening sequences, such as the linkers described herein. An example of a multiplex of the present invention is a polynucleotide encoding multiple tandem miRNA-based shRNAs. A multiplex may be single-stranded, double-stranded, or may have both single-stranded and double-stranded regions.

[0029] According to certain embodiments, the 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 levels of expression. For the same reason, it may be considered to exclude the H1 promoter (which is a weaker promoter than U6) or even Pol III promoters in general (although they may be preferred under certain conditions). According to certain embodiments, the promoter is selected from Pol II promoters and Pol III promoters. According to certain embodiments, the promoter is a natural or synthetic Pol II promoter. In certain embodiments, the promoter is a Pol II promoter selected from the group consisting of a cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EF1α) promoter (core or full-length), a phosphoglycerate kinase (PGK) promoter, a composite beta-actin promoter (CAG promoter) with an upstream CMV IV enhancer, a ubiquitin C (UbC) promoter, a spleen focus-forming virus (SFFV) promoter, a Rous sarcoma virus (RSV) promoter, an interleukin-2 promoter, a murine stem cell virus (MSCV) long terminal repeat (LTR), a gibbon ape leukemia virus (GALV) LTR, a simian virus 40 (SV40) promoter, and a tRNA promoter. These promoters are among the most commonly used polymerase II promoters driving mRNA expression, although generic housekeeping gene promoters can also be used.

[0030] According to certain embodiments, the 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, while conventional shRNA molecules are not (associated with toxicity, Grimm et al., Nature 441:537-541(2006)).

[0031] According to a particular embodiment, the different miRNA molecules are under the control of one promoter.

[0032] According to certain embodiments, at least two of the multiplexed RNA interference molecules are directed against the same target. However, the RNA interference molecules directed against the same target may still have different scaffold sequences and / or different target sequences. According to further certain embodiments, at least two of the multiplexed RNA interference molecules have the same scaffold but different target sequences. According to another certain embodiment, at least two of the multiplexed RNA interference molecules have different scaffolds but the same target sequence. According to certain embodiments, at least two of the multiplexed RNA interference molecules are the same.

[0033] According to another embodiment, all of the at least two multiplexed RNA interference molecules are different. According to a more specific embodiment, all of the at least two multiplexed RNA interference molecules are directed against different targets. However, the RNA interference molecules directed against different targets may still have the same scaffold (but have different target sequences).

[0034] Any suitable molecule present in the engineered cells can be targeted by the RNA interference molecule. Typical examples of possible targets include 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 cascades, regulatory receptors (e.g., NOTCH4), TAP, HLA-DM, HLA-DO, RING1, CD52, CD247, HCP5, DGKA, DGKZ, B2M, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, 2B4, A2AR, BAX, BLIMP1, C160 (POLR3A), CBL-B, and CCR6. , CD7, CD95, CD123, 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, LFA1, NEAT1, NFkB (including RELA, RELB, NFkB2, NFkB1, REL), NKG2A, NR4A (including NR4A1, NR4A2, NR4A3), PD1, PI3KCD, PPP2RD2, SHIP1, SOAT1, SOCS1, T-BET, TET2, TGFBR1, TGFBR2, TGFBR3, TIGIT, TIM3, TOX, and ZFP36L2.

[0035] Particularly preferred constructs have been identified that are miRNA-based. Accordingly, engineered cells are provided that comprise a polynucleotide that includes a microRNA-based shRNA coding region, said microRNA-based shRNA coding region comprising: one or more artificial miRNA-based shRNA nucleotide sequences, each of the artificial miRNA-based shRNA nucleotide sequences comprising miRNA scaffold sequence, the active or mature sequence; Passenger or star configuration wherein within each artificial miRNA-based shRNA nucleotide sequence, the active sequence comprises a sequence encoding one or more artificial miRNA-based shRNA nucleotide sequences that are at least 70% complementary to the passenger sequence.

[0036] 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.

[0037] Of particular advantage is that the miRNA-based shRNA nucleotide sequences of the present invention can be multiplexed. Accordingly, an engineered cell is provided that comprises a polynucleotide comprising a multiplexed microRNA-based shRNA coding region, wherein the multiplexed microRNA-based shRNA coding region comprises: two or more artificial miRNA-based shRNA nucleotide sequences, each of the artificial miRNA-based shRNA nucleotide sequences comprising: miRNA scaffold sequence, the active or mature sequence; Passenger or star configuration, and within each artificial miRNA-based shRNA nucleotide sequence, a sequence encoding two or more artificial miRNA-based shRNA nucleotide sequences, wherein the active sequence is at least 70% complementary to the passenger sequence.

[0038] Both the active sequence and the passenger sequence of each of the artificial miRNA-based shRNA nucleotide sequences are typically 18-40 nucleotides in length, more particularly 18-30 nucleotides in length, even more particularly 18-25 nucleotides in length, and most particularly 18-23 nucleotides in length. The active sequence can also be 18 or 19 nucleotides in length. Typically, the passenger sequence has the same length as the active sequence, although the possible presence of bulges means that they are not always the same length.

[0039] Typically, these microRNA scaffold sequences are separated by linkers. According to particular embodiments, at least a portion of the 5' and / or 3' linker sequences are used with their respective scaffolds.

[0040] The artificial sequence can be, for example, a naturally occurring scaffold (e.g., a miR cluster or fragment thereof, such as the miR-106a-363 ​​cluster) in which the endogenous miR sequence has been replaced by an engineered shRNA sequence directed against a specific target; a repeat of a single miR scaffold (such as the miR-20b scaffold) in which the endogenous miR sequence has been replaced by an engineered shRNA sequence directed against a specific target; an artificial miR-like sequence; or a combination thereof.

[0041] The engineered cell typically further comprises a nucleic acid molecule encoding a protein of interest, such as a chimeric antigen receptor or TCR, and may be an engineered immune cell, as described above.

[0042] 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 engineered cells, which can contribute to greater therapeutic efficacy.

[0043] The engineered cells described herein are also provided for use as a medicament. According to certain embodiments, the engineered cells are provided for use in the treatment of cancer.

[0044] This also provides a method of treating cancer, comprising administering to a subject in need thereof a suitable dose of engineered cells as described herein, thereby ameliorating at least one symptom.

[0045] The engineered cells may be autologous immune cells (cells obtained from the patient) or allogeneic immune cells (cells obtained from another subject). [Brief explanation of the drawings]

[0046] [Figure 1] FIG. 1 is a schematic diagram of a clustered scaffold, showing regions such as the target sequence, upper stem, lower stem, and scaffold. [Figure 2] Figure 2 shows the design of CAR expression vectors (e.g., CD19, BCMA, B7H3, B7H6, NKG2D, HER2, HER3, GPC3) without (top) or with (bottom) an miRNA scaffold, allowing the co-expression of a CAR and multiple shRNAs (e.g., 2, 4, 6, 8, ...) from the same vector. LTR: long terminal repeat; promoter (e.g., EF1a, PGK, SFFV, CAG, ...); marker protein (e.g., truncated CD34, CD19); multiplexed shRNA. [Figure 3]The use of native mRNA clusters increases the transduction efficiency compared to repetitively engineered single scaffolds. T cells were transduced with various vectors encoding CD19CAR and 3-6 multiplexed scaffolds according to the design shown in Figure 2. CD34 was used as the reporter gene, and the percentage of CD34+ T cells measured by FACS at day 4 post-transduction is shown in the bottom panel. The top panel shows the same results but after purification (the amount of cells eluted from the purification column divided by the amount of cells loaded onto the column). 1-2: scaffolds from the miR-17-92 cluster, 4 (miR-19a, miR-20a, miR-19b1, miR-92a1) and 3 scaffolds (miR-19a, miR-20a, miR-19b1), respectively; 3-5: scaffolds from the miR-106a-363 ​​cluster, 6 (all), 3 (last three), and 4 (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, respectively, of the miR-196a2 scaffold sequence; 10: a fake vector with only the CD34 tag. The target genes included in the constructs were B2M, CD52, and CD247 for the triplex scaffold, and an additional gene, TRAC, for the tetraplex scaffold. The hexaplex scaffold targeted each target gene twice, using two different target sequences for each target. [Figure 4]Comparison of CD247 (CD3ζ) 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 from the miR-23a-27a-24-2 cluster (CD247 target sequence in the miR-24-2 scaffold); 3-5: scaffolds from the miR-106a-363 ​​cluster, 6 (all), 3 (last three), and 4 (last four), respectively. The CD247 target sequence is in the miR-363 scaffold; in 3, an additional, different sequence is contained in the miR-20b scaffold. [Figure 5] The miRNA106a-363 ​​cluster and the design of the construct used are shown in FIG. [Figure 6] Figure 1 shows RNA expression in primary T cells from healthy donors transduced with a retroviral vector encoding a second-generation CD19-targeting CAR and a truncated CD34 selection marker, along with 3x or 6x shRNAs 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 expanded in IL-2 (100 IU / mL) for 6 days. mRNA expression of CD247, B2M, and CD52 was assessed by qRT-PCR using cyclophilin as a housekeeping gene. [Figure 7] Comparison of various shRNA target sequences allows fine tuning of knockdown levels. Twelve different target sequences, all directed against CD247, were evaluated in the miR-20b scaffold. T cells were harvested 12 days after activation (10 days after transduction). TCRab levels were measured by FACS; MFI is shown as a bar graph. All shRNAs achieved at least 50% knockdown, with some being much more efficient. [Figure 8]Knockdown of CD95 in the miR-18b scaffold. Sequences selected from 31 different target sequences, all directed against CD95, were evaluated in the miR-18b scaffold. T cells were harvested 16 days after activation (14 days after transduction). CD95 levels were measured by FACS; MFI is represented as a bar graph. The most efficient shRNAs achieved approximately 30% knockdown. [Figure 9] Comparison of the miR-106a, miR-18b, and miR-20b scaffold structures. The target sequence (here, 20 bp long) and passenger strand are shown as rectangles. While miR-106a and miR-20b have a mismatch at position 18 of the scaffold (position 14 of the target sequence), 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 to 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. Any construct with fewer mismatches or bulges than the native sequence achieves higher knockdown efficiency. Knockdown is measured in the same manner as in Figure 8. [Figure 10B] Modifications to 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. Any construct with fewer mismatches or bulges than the native sequence achieves higher knockdown efficiency. Knockdown is measured in the same manner as in Figure 8. [Figure 11]Figure 11 shows the evaluation of target sequence length. The effect of target sequence length on knockdown efficiency was evaluated for both B2M (left panel) and CD247 (right panel). Because the native scaffold sequence is identical to the target sequence at that position, constructs can be labeled with two lengths (19-20, 21-22, or 22-23). ​​Results shown are for the miR-106a scaffold; similar results were obtained for the miR-20b scaffold (not shown). Cluster: control with unrelated sequence; as further controls, the target sequences for CD247 and B2M, respectively, were used. [Figure 12A] Evaluation of simultaneous knockdown of different genes using different permutations of scaffolds. A: FACS data showing expression of B2M / HLA (left panel) and CD247 / CD3ζ (right panel) for the indicated duplex and triplex scaffolds. B: MFI of FACS data from panel A, now including expression of CD95 for the triplex scaffold. C: MFI of FACS data showing expression of B2M, CD247, and CD95 for the indicated constructs. [Figure 12B] Evaluation of simultaneous knockdown of different genes using different permutations of scaffolds. A: FACS data showing expression of B2M / HLA (left panel) and CD247 / CD3ζ (right panel) for the indicated duplex and triplex scaffolds. B: MFI of FACS data from panel A, now including expression of CD95 for the triplex scaffold. C: MFI of FACS data showing expression of B2M, CD247, and CD95 for the indicated constructs. [Figure 12C]Evaluation of simultaneous knockdown of different genes using different permutations of scaffolds. A: FACS data showing expression of B2M / HLA (left panel) and CD247 / CD3ζ (right panel) for the indicated duplex and triplex scaffolds. B: MFI of FACS data from panel A, now including expression of CD95 for the triplex scaffold. C: MFI of FACS data showing expression of B2M, CD247, and CD95 for the indicated constructs. DETAILED DESCRIPTION OF THE INVENTION

[0047] (Detailed Description of the Invention) definition The present invention will be described with respect to particular embodiments and with reference to certain drawings but the present invention is not limited thereto but only by the claims. Any reference signs in the claims should not be construed as limiting the scope. The drawings described are schematic only and are not limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn to scale for illustrative purposes. Where the term "comprising" is used in the present specification and claims, it does not exclude other elements or steps. Where an indefinite or definite article, e.g., "a," "an," or "the," is used when referring to a singular noun, the plural of that noun is also included unless specifically stated otherwise.

[0048] Furthermore, terms such as first, second, third, etc. in this specification and claims are used to distinguish between similar elements and not necessarily to describe an order or chronology. Terms so used are interchangeable under appropriate circumstances, and it should be understood that the embodiments of the invention described herein are capable of operation in orders other than those described or illustrated herein.

[0049] The following terms or definitions are provided solely to aid in the understanding of the invention.

[0050] Unless otherwise specified herein, all terms used herein have the same meaning as those of ordinary skill in the art. 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 Appendix 114), John Wiley & Sons, New York (2016) are intended for practitioners regarding definitions and terms in the art. The definitions provided herein should not be construed as having a narrower scope than understood by those skilled in the art.

[0051] As used herein, an "engineered cell" is a cell that has been modified through human intervention (as opposed to naturally occurring mutations).

[0052] As used herein, the term "nucleic acid molecule," which is synonymously referred to as "nucleotide" or "nucleic acid" or "polynucleotide," refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Nucleic acid molecules include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- 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 containing one or more modified bases and DNA or RNA with backbones 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 as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also encompasses relatively short nucleic acid strands often referred to as oligonucleotides.

[0053] A "vector" is a replicon, such as a plasmid, phage, cosmid, or virus, into which another nucleic acid segment is operatively inserted, bringing about the replication or expression of that segment. A "clone" is a population of cells derived by mitosis from a single cell or common ancestor. A "cell line" is a clone of a primary cell capable of stable growth in vitro for many generations. In some examples presented herein, cells are transformed by transfecting them with DNA.

[0054] The terms "express" and "produce" are used interchangeably herein and refer to the biosynthesis of a gene product. These terms include the transcription of a gene into RNA. These terms also include translation of RNA into one or more polypeptides, and further include all naturally occurring post-transcriptional and post-translational modifications.

[0055] 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 (as opposed to an endogenous factor) but that originates from outside that cell. Thus, the expression "exogenous nucleic acid molecule" refers to a nucleic acid molecule that has been introduced into said (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 that originates from inside that cell (and thus is typically also produced in non-transduced or non-transfected cells).

[0056] As used herein, "isolated" means that a biological component (such as a nucleic acid, peptide, or protein) has been substantially separated, produced away from, or purified away 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, and may still be isolated if such 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.

[0057] As used herein in the context of molecular biology, "multiplexing" refers to the simultaneous targeting of two or more (i.e., multiple) related or unrelated targets. The term "RNA interference molecule" as used herein 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 through base pair complementarity: within the RNA interference molecule is a target sequence (typically 18-23 nucleic acids) that can hybridize to the target nucleic acid molecule. Examples include siRNA (including shRNA) or miRNA molecules. Thus, as used herein, a "multiplexed RNA interference molecule" refers to 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, but two molecules may be directed against the same target (and may even be identical).

[0058] As used herein, a "promoter" is a regulatory region of nucleic acid that is usually located adjacent to a gene and provides a control point for regulated gene transcription.

[0059] A "multiplex" is a polynucleotide encoding multiple molecules of the same type, such as multiple siRNAs, shRNAs, or miRNAs. Within a multiplex, when the molecules are of the same type (e.g., all shRNAs), they may contain the same or different sequences. There may be intervening sequences between the molecules of the same type, such as the linkers described herein. An example of a multiplex of the present invention is a polynucleotide encoding multiple miRNA-based shRNAs. A multiplex may be single-stranded, double-stranded, or may have both single-stranded and double-stranded regions.

[0060] As used herein, a "chimeric antigen receptor" or "CAR" refers to a chimeric receptor (i.e., composed of portions from different sources) having at least a binding portion having specificity for an antigen (which can be derived, e.g., from an antibody, receptor, or its cognate ligand) and a signaling portion capable of transmitting a signal in an immune cell (e.g., the CD3ζ chain; other signaling or co-signaling portions can also be used, such as the FcεRIγ domain, the CD3ε domain, the recently described DAP10 / DAP12 signaling domain, or domains from CD28, 4-1BB, OX40, ICOS, DAP10, DAP12, CD27, and CD2 as costimulatory domains). A "chimeric NK receptor" is a CAR in which the binding portion is derived from or isolated from an NK receptor.

[0061] As used herein, "TCR" refers to a T cell receptor. In the context of adoptive cell transfer, this typically refers to an engineered TCR, i.e., a TCR engineered to recognize a specific antigen, most typically a tumor antigen. As used herein, "endogenous TCR" refers to a TCR endogenously present on an unmodified cell (typically a T cell). The TCR is typically a disulfide-linked, membrane-anchored heterodimeric protein composed of the 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 variable TCR receptor α and β chains, the CD3 co-receptor (comprising a CD3γ chain, a CD3δ chain, and two CD3ε chains), and two CD3ζ chains (also known as the CD247 molecule). As used herein, the term "functional TCR" refers to a TCR capable of signaling upon binding of its cognate ligand. Typically, for allogeneic therapy, manipulation is performed to reduce or impair TCR function, for example, by knocking out or knocking down at least one of the TCR chains. The endogenous TCR in the engineered 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) of a cell with an endogenous TCR without manipulation. Assays for assessing signaling capacity or T cell activation are known to those skilled in the art, and include, in particular, ELISAs measuring interferon-γ. According to another embodiment, the endogenous TCR is considered functional if it has not been manipulated to interfere with TCR function.

[0062] The term "immune cells" as used herein refers to cells that are part of the immune system (which may be either the adaptive or the innate immune system). Immune cells as used herein are typically immune cells prepared for adoptive cell transfer (either autologous or allogeneic). A wide variety of immune cells are used for adoptive therapy and are therefore contemplated for use in the methods used herein. Examples of immune cells include, but are not limited to, T cells, NK cells, NKT cells, lymphocytes, dendritic cells, myeloid cells, macrophages, stem cells, progenitor cells, or iPSCs. The latter three are not immune cells and therefore can be used in adoptive cell transfer for immunotherapy (see, e.g., Jiang et al., Cell Mol Immunol 2014; Themeli et al., Cell Stem Cell 2015). Typically, the preparation begins with stem cells or iPSCs (or can even begin with a dedifferentiation step from immune cells to iPSCs), but the preparation requires a differentiation step into immune cells before 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 step of destruction of a human embryo.

[0063] 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 particularly contemplated. It should be noted that in the context of adoptive transfer, the immune cells are typically primary cells (i.e., cells isolated directly from human or animal tissue and not cultured or cultured only briefly), rather than cell lines (i.e., cells that have been continuously passaged over an extended period of time to acquire homogeneous genetic and phenotypic characteristics). According to certain embodiments, the immune cells are primary cells (i.e., cells isolated directly from human or animal tissue and not cultured or cultured only briefly), rather than cell lines (i.e., cells that have been continuously passaged over an extended period of time to acquire homogeneous genetic and phenotypic characteristics). According to another particular embodiment, the immune cells are not from a cell line.

[0064] As used herein, "microRNA scaffold," "miRNA scaffold," or even "scaffold" refers to a well-characterized primary microRNA sequence containing specific microRNA processing requirements into which an RNA sequence can be inserted (typically to replace an existing miRNA sequence with an siRNA directed against a specific target). A microRNA scaffold minimally consists of a double-stranded upper stem region (typically 18-23 nucleotides), both sides of which are connected by a flexible loop sequence, and the upper stem region is typically processed by Dicer. Typically, the microRNA scaffold further comprises a lower stem region and, optionally, may further comprise 5' and 3' flanking sequences or base segments. The guide sequence or target sequence is inserted into the upper stem region and is a single-stranded sequence of 18-23 nucleotides. The target sequence recognizes its target through complementary base pairing, and therefore this sequence is typically the same as a sequence present in the target or its regulatory region. As used herein, "target" or "target protein" refers to a molecule (typically a protein, but may be a nucleic acid molecule) to be downregulated (i.e., the expression of which is decreased in a cell). Note that miRNAs act at the nucleic acid level, and therefore, even for proteins, the miRNA target sequence is the same as the sequence encoding the protein (e.g., mRNA sequence) or the sequence regulating the expression of the protein (e.g., 3'UTR region).

[0065] Examples of miRNA scaffolds include those present in 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 the SMARTvector™ micro-RNA adaptive scaffold (Horizon Discovery, Lafayette, CO, USA). As used herein, "miR-106a" corresponds to Gene ID 406899 in humans, "miR-18b" corresponds to Gene ID 574033 in humans, "miR-20b" corresponds to Gene ID 574032 in humans, "miR-19b-2" corresponds to Gene ID 406981 in humans, "miR-92-2," also known as "miR-92a-2," corresponds to Gene ID 407049 in humans, and "miR-363" corresponds to Gene ID 574031 in humans.

[0066] As used herein, a "microRNA cluster" or "miRNA cluster" refers to a collection of microRNA scaffolds that function together. Naturally occurring microRNA clusters have been well described, including the miR-106a-363 ​​cluster, the miR-17-92, miR-106b-25, and miR-23a-27a-24-2 clusters. A miRNA cluster can be considered a combined scaffold. As used herein, a "combined miRNA scaffold" refers to the combination of multiple miRNA scaffolds that function under the control of a single promoter. The multiple miRNA scaffolds may be the same or different and may have target sequences for the same or different target proteins, and, if the target is the same, may have the same or different target sequences for that target. When such combined scaffolds are under the control of a single promoter, they are also referred to as "multiplexed scaffolds," "multiplexed scaffolds," or "multiplexed miRNA scaffolds." In some cases, when the number of scaffolds is fixed, this can be used in place of the prefix "multiple." 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 having six different miRNA scaffolds (e.g., the miR-106a-363 ​​cluster) can be considered a hexaplex miRNA scaffold.

[0067] FIG. 1 shows a schematic example of a multiplexed scaffold sequence, displaying the upper and lower stem regions, the target sequence, and the individual scaffolds used herein.

[0068] The term "subject" refers to humans and non-human animals, including all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In the most particular embodiments of the methods, the subject is a human.

[0069] The term "treat" or "treatment" refers to any success or indication of success in the attenuation or amelioration of injury, pathology, or condition, including any objective or subjective parameter such as alleviation, remission, or reduction of symptoms or making the condition more tolerable to the patient, slowing the rate of degeneration or decline, reducing the debilitating end point of degeneration, improving the physical or mental well-being of a subject, or extending the survival time. The treatment can be evaluated by objective or subjective parameters, including the results of a physical examination, neurological examination, or psychiatric evaluation.

[0070] As used herein, the phrases "adoptive cell therapy," "adoptive cell transfer," or "ACT" refer to the transfer of cells, most typically immune cells, into a subject (e.g., a patient). These cells can be derived from the subject (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; in cancer immunotherapy, to generate an immune response against the cancer. T cells are most often used for ACT, but applications also include the use of other types of immune cells, such as NK cells, lymphocytes (e.g., tumor-infiltrating lymphocytes (TILs)), dendritic cells, and myeloid cells.

[0071] 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 medical condition, age, sex, and weight of the individual, and 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.

[0072] The term "graft-versus-host disease" or "GvHD" refers to a condition that can occur after allogeneic transplantation. In GvHD, the donated bone marrow, peripheral blood (stem) cells, or other immune cells view the recipient's body as foreign, and the donor cells attack the body. Because donor immunocompetent immune cells, e.g., T cells, are the primary driver of GvHD, one strategy for preventing GvHD is by reducing (TCR-based) signaling in these immunocompetent cells, e.g., by directly or indirectly inhibiting the function of the TCR complex.

[0073] To assess whether this targeting of 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.

[0074] The fundamental approach is based on the transcription of RNA from a specific vector, which is processed by the endogenous RNA processing machinery to generate active shRNA that can target the mRNA of choice through base recognition and the resulting destruction of that specific mRNA by the RISC complex.The specific destruction of the targeted mRNA results in a decrease in the expression of the associated protein.Although RNA oligonucleotides can be transfected into the target cells of choice to achieve temporary knockdown of gene expression, the expression of the desired shRNA from an integrating vector allows for the stable knockdown of gene expression.

[0075] The efficient expression of shRNAs is highly dependent on coupling to a polymerase III (Pol III) promoter (e.g., H1, U6), which generates RNA species lacking a 5' cap and 3' polyadenylation, allowing processing of the shRNA duplex. Once transcribed, the shRNA is processed, exported from the nucleus, further processed, and loaded into the RNA-induced silencing complex (RISC) complex, leading to the targeted degradation of the appropriate mRNA (Moore et al., 2010). While effective, the efficiency of transcription driven by a Pol III promoter can lead to cytotoxicity due to the saturation of the endogenous microRNA pathway resulting from the excessively high expression of shRNAs from a Pol III promoter (Fowler et al., 2016). Furthermore, expression of both a therapeutic gene and an shRNA from a single vector is typically achieved by using a polymerase II (Pol II) promoter to drive the therapeutic gene and a Pol III promoter to drive the shRNA of interest. While this is functional, it comes at the expense of vector space and therefore fewer options for including a therapeutic gene (Chumakov et al., 2010; Moore et al., 2010).

[0076] Embedding the shRNA within a microRNA (mir) framework allows the shRNA to be processed under the control of a Pol II promoter (Giering et al., 2008). Importantly, the expression level of embedded shRNAs tends to be low, thereby avoiding the toxicity observed when using other systems, such as the U6 promoter (Fowler et al., 2015). In fact, mice administered shRNAs driven by a liver-specific Pol II promoter showed stable gene knockdown for over a year without tolerance issues (Giering et al., 2008). However, this was only for a single shRNA administered in liver cells, and the reduction in protein levels was only 15% (Giering et al., 2008). Therefore, it is unclear whether higher efficiency can be achieved with multiple targets, especially in immune cells (which are more difficult to manipulate).

[0077] Surprisingly, it has been demonstrated herein that elements of the miR106a-363 ​​cluster are unexpectedly efficient at downregulating targets, and in particular at multiplexed downregulation of targets: the expression of multiple microRNA-based shRNAs against different targets (based on the individual scaffolds occurring in the miR106a-363 ​​cluster) was feasible in T cells without recombination, without toxicity, and while simultaneously achieving efficient downregulation of multiple targets.

[0078] Therefore, one object of the invention is to provide a vector comprising a nucleic acid sequence encoding at least one RNA interference molecule having a scaffold selected from those present in the miR-106a-363 ​​cluster, particularly 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 specific embodiment, the vector is suitable for expression in eukaryotic cells, particularly immune cells. The RNA interference molecule also typically comprises a target sequence not present in the native scaffold sequence. Most particularly, the target sequence has a length of 18 to 23 nucleic acids.

[0079] According to certain embodiments, at least one of the scaffolds of the one or more RNA interference molecules is a scaffold selected from the miR-106a scaffold, the miR-18b scaffold, and the miR-20b scaffold. In other words, according to these specific embodiments, there is provided a vector comprising a nucleic acid sequence encoding at least one RNA interference molecule having a scaffold selected from those present in the first three scaffolds of the miR-106a-363 ​​cluster, i.e., a scaffold selected from the miR-106a scaffold, the miR-18b scaffold, and the miR-20b scaffold. For example, at least one RNA interference molecule may have a miR-106a scaffold, while other RNA interference molecules may have independently selected scaffolds, such as scaffolds 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.

[0080] According to certain embodiments, the at least one RNA interference molecule present in the vector is at least two RNA interference molecules, particularly at least two multiplexed RNA interference molecules. When at least two multiplexed RNA interference molecules are present, these two or more molecules may have the same or different scaffolds, i.e., may have one or more scaffolds selected from miR-106a scaffold, miR-18b scaffold, miR-20b scaffold, miR-19b-2 scaffold, miR-92-2 scaffold, and miR-363 scaffold. However, it is particularly envisaged that no more than three of the scaffolds are the same, and more particularly that no more than two of the same scaffolds are used. This is to avoid recombination between the same scaffold sequences or other factors that reduce the miRNA processing (see Example 5).

[0081] According to a particular embodiment, the scaffolds present in the vector are selected from only the six mentioned above (miR-106a scaffold, miR-18b scaffold, miR-20b scaffold, miR-19b-2 scaffold, miR-92-2 scaffold, and miR-363 scaffold). However, it is also envisaged that they may be further combined with different scaffold sequences, in particular different unrelated sequences (to avoid recombination), such as the miR-196a2 sequence. Alternatively, they may be combined with other miRNA cluster sequences, in particular with scaffolds from the miR-17-92 cluster, the miR-106b-25 cluster, and / or the miR-23a-27a-24-2 cluster.

[0082] According to certain embodiments, the scaffold sequence may be engineered 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 complementary Watson-Crick base pair. As used herein, "bulge" refers to an unpaired stretch of nucleotides (typically 1 to 5, particularly 1 to 3) located in one strand of a nucleic acid duplex. More specifically, when one of the scaffold sequences used is a miR-18b scaffold, the scaffold can be engineered (modified compared to the 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 to the target strand, or by removing the excess unpaired nucleotides in the case of a bulge.

[0083] The vectors disclosed herein are particularly suitable for use in cells used for ACT. Accordingly, an object of the invention is to provide engineered cells containing a nucleic acid molecule encoding at least one RNA interference molecule having a scaffold selected from those present in the miR-106a-363 ​​cluster, particularly 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. The RNA interference molecule typically also contains a target sequence not present in the native scaffold sequence. The target sequence typically has a length of 18 to 23 nucleic acids. It is particularly envisioned that the target sequence is directed against a sequence present in the engineered cell, particularly a target sequence. That is, the at least one RNA interference molecule has a sequence in the engineered cell that encodes a protein to be downregulated or a sequence that targets (by base pair complementarity) a regulatory region of the target protein.

[0084] According to certain embodiments, the engineered cells comprise at least two RNA interference molecules, in particular at least two multiplexed RNA interference molecules having a scaffold 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.

[0085] Cells containing at least one RNA interference molecule or at least two RNA interference molecules may have advantages, particularly therapeutic benefits. The RNA interference molecule may actually be directed against a target whose (over)expression is undesirable. However, typically, the engineered cells provided herein further contain at least one protein of interest.

[0086] According to these embodiments, a first exogenous nucleic acid molecule encoding a protein of interest; a second nucleic acid molecule encoding at least one RNA interference molecule having a scaffold 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; An engineered cell is provided, comprising:

[0087] According to a further particular embodiment, a first exogenous nucleic acid molecule encoding a protein of interest; a second nucleic acid molecule encoding at least two multiplexed RNA interference molecules having a scaffold 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; An engineered cell is provided, comprising:

[0088] When at least two multiplexed RNA interference molecules are present, these two or more molecules may have the same or different scaffolds, i.e., one or more scaffolds 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. However, it is particularly contemplated that no more than three of the scaffolds are the same, and more particularly, that no more than two of the same scaffolds are used. This is to avoid recombination between the same scaffold sequences or overloading the miRNA processing capacity of the cell (see Example 5). For the same reason, when there are multiple target sequences for the same target, it is particularly contemplated that either different target sequences are used, or the same target sequence is used in different scaffolds. It is particularly contemplated that the same target sequence may occur in the same scaffold, but that they will occur no more than twice.

[0089] The optional additional protein of interest can, for example, provide an additive, supportive, or even synergistic effect or can be used for various purposes. For example, the protein of interest can be a CAR for a tumor, and the RNA interference molecule can interfere with tumor function, for example, by targeting an immune checkpoint, directly downregulating tumor targets, or targeting the tumor microenvironment. Alternatively or additionally, one or more of the RNA interference molecules can prolong the persistence of the therapeutic cells or otherwise alter a physiological response (e.g., interfere with GvHD or host-versus-graft response).

[0090] The protein of interest can in principle be any protein, depending on the setting. However, typically, they are proteins with a therapeutic function. These may include, for example, secreted therapeutic proteins such as interleukins, cytokines or hormones. However, according to particular embodiments, the protein of interest is not secreted. Instead of a therapeutic protein, the protein of interest may perform a different function, such as a diagnostic or detection function. Thus, the protein of interest may be a tag or a reporter gene. Typically, the protein of interest is a receptor. According to further particular embodiments, the receptor is a chimeric antigen receptor or a TCR. The chimeric antigen receptor can be for any target expressed on the surface of a target cell, and typical 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 , BCMA, B7H3, B7H6, CEACAM5, EGFRvIII, EPHA2, mesothelin, NKG2D, HER2, HER3, GPC3, Flt3, DLL3, IL1RAP, KDR, MET, mucin1, IL13Ra2, FOLH1, FAP, CA9, FOLR1, ROR1, GD2, PSCA, GPNMB, CSPG4, ULBP1, ULBP2, although 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 part of a receptor, and the CAR is typically named after the receptor). An example of the latter is an NKG2D-CAR.

[0091] The engineered TCR can be directed against any cellular target, including intracellular targets. In addition to the cell surface targets listed above, 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.

[0092] According to these particular embodiments in which an additional protein of interest is present, the first and second nucleic acid molecules in the engineered cell are typically present in a single vector, such as a eukaryotic expression plasmid, a minicircle DNA, or a viral vector (e.g., derived from a lentivirus, retrovirus, adenovirus, adeno-associated virus, or Sendai virus). According to further particular embodiments, the viral vector is selected from a lentiviral vector and a retroviral vector. In particular for the latter, the vector load (i.e., the overall size of the construct) is important, making the use of a compact multiplex cassette particularly advantageous.

[0093] Notably, the cells described herein may contain multiple proteins of interest: for example, a receptor protein and a reporter protein (see Figure 2), or may contain a receptor protein, an interleukin, and a tag protein.

[0094] The engineered cells are in particular eukaryotic cells, more particularly engineered mammalian cells, and even more particularly engineered human cells. According to particular embodiments, the cells are engineered immune cells. Exemplary immune cells are selected from T cells, NK cells, NKT cells, macrophages, stem cells, progenitor cells, and iPSC cells.

[0095] 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 considerations regarding co-expression of the multiplexed molecules. As shown herein, the miR-106a-363 ​​cluster has six scaffolds (Figures 5-6), and scaffolds can be replicated without loss of knockdown activity (Example 5), so up to 12 scaffolds can in principle be multiplexed, although in practice fewer numbers are used.

[0096] A "multiplex" is a polynucleotide encoding multiple molecules of the same type, such as multiple siRNAs, shRNAs, or miRNAs. Within a multiplex, when molecules are of the same type (e.g., all shRNAs), they may contain the same or different sequences. Intervening sequences, such as linkers, may be present between molecules of the same type, as described herein. An example of a multiplex of the present invention is a polynucleotide encoding multiple tandem miRNA-based shRNAs. A multiplex may be single-stranded, double-stranded, or have both single-stranded and double-stranded regions.

[0097] According to certain embodiments, the at least two multiplexed RNA interference molecules are under the control of a single promoter. Typically, when multiple RNA interference molecules are expressed, this is achieved by incorporating multiple copies of an shRNA-expression cassette. These typically have the same promoter sequence, resulting in frequent recombination events that remove the repetitive sequence fragments. As a solution, several different promoters are typically used in the expression cassette (e.g., Chumakov et al., 2010). However, according to this embodiment, recombination is avoided by the use of a single promoter. Although expression is typically lower, this is advantageous in terms of toxicity, since too much siRNA can be harmful to cells (e.g., by interfering with the endogenous siRNA pathway). The use of a single promoter has the additional advantage that all shRNAs are co-regulated and expressed at similar levels. Notably, as shown in the examples, multiple shRNAs can be transcribed from a single promoter without a significant decrease in efficacy.

[0098] According to a further specific embodiment, both the at least two multiplexed RNA interference molecules and the protein of interest are under the control of a single promoter. This also reduces vector load (because a separate promoter is not 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 molecules are intended to have an additive or synergistic effect in tumor eradication. Examples of useful RNA targets include (without limitation): CD247, TRAC (both of which downregulate the TCR complex, making the cells more suitable for allogeneic therapy), B2M (to expand tissue compatibility), CD52 (allowing the cells to survive CD52-directed chemotherapy), CD95 (making the cells insensitive to CD95-induced cell death), checkpoint molecules (e.g., PD-1, PD-L1, CTLA4), and many more.

[0099] Typically, the promoter used to express the RNA interference molecule is not a U6 promoter, as this promoter is associated with toxicity, especially at high expression levels. For the same reason, one can consider excluding the H1 promoter (a weaker promoter than U6) or even Pol III promoters in general (although they may be preferable in certain situations). Thus, according to certain embodiments, the promoter used to express the RNA interference molecule is not an RNA Pol III promoter. RNA Pol III promoters lack temporal and spatial control and do not allow for controlled expression of miRNA inhibitors. In contrast, many RNA Pol II promoters allow for tissue-specific expression, and both inducible and repressible RNA Pol II promoters exist. While tissue-specific expression is often not necessary in the context of the invention (because cells are selected prior to manipulation), it is still advantageous to have a specific promoter for, for example, immune cells, since it has been shown that differences in RNAi efficacy from various promoters are particularly pronounced in immune cells (Lebbink et al., 2011). In certain embodiments, the promoter is selected from a Pol II promoter and a Pol III promoter. In certain embodiments, the promoter is a natural or synthetic Pol II promoter. Suitable promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, an elongation factor 1α (EF1α) promoter (core or full-length), a phosphoglycerate kinase (PGK) promoter, a composite beta-actin promoter with an upstream CMV IV enhancer (CAG promoter), a ubiquitin C (UbC) promoter, a spleen focus-forming virus (SFFV) promoter, a Rous sarcoma virus (RSV) promoter, an interleukin-2 promoter, a murine stem cell virus (MSCV) long terminal repeat (LTR), a gibbon ape leukemia virus (GALV) LTR, a simian virus 40 (SV40) promoter, and a tRNA promoter. These promoters are among the most commonly used polymerase II promoters for driving mRNA expression.

[0100] According to certain embodiments, the 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, while conventional shRNA molecules are not (related to toxicity, Grimm et al., Nature 441:537-541(2006)).

[0101] According to certain embodiments, the miRNA molecules can be provided as individual miRNA scaffolds under the control of a single promoter. Each scaffold selected typically corresponds to one miRNA (FIG. 1), and the scaffolds can be repeated or combined with other scaffolds to obtain the expression of multiple RNA interference molecules (FIGS. 1-2). However, when combined with repeated or additional scaffolds, it is typically assumed that all of the multiplexed RNA interference molecules are under the control of a single promoter (i.e., the promoter is not repeated when individual scaffolds are repeated or when other scaffolds are added).

[0102] Scaffold sequences particularly suitable for miRNA multiplexing are those found in bona fide polycistronic miRNA clusters or portions thereof, in which 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; the miR-106a-363 ​​cluster and fragments thereof (i.e., one or more individual scaffolds) are particularly contemplated. Of note, it is also specifically contemplated to use portions of such natural clusters, rather than the entire sequences, to secure vector payloads (this is particularly useful, since not all miRNAs are evenly spaced and not all linker sequences may be required). Indeed, it has been shown herein (Example 5) that scaffolds can be used outside the context of the clusters and can be combined in different ways. Other considerations can also be taken into account, such as employing the miRNAs that are most efficiently processed in cells. For example, the miR-17-92 cluster consists of (in order) 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 miR-92a1) scaffold, 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) or miR-19a through miR-19b-1 (three of the six miRNAs) with their linkers. Similarly, the 106a-363 ​​cluster consists 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 cluster are the scaffold sequences from miR-106a to miR-20b (i.e., three of the six miRNAs) (see Example 5), miR-20b to miR-363 (i.e., four of the six miRNAs), or miR-19b-2 to miR-363 (i.e., three of the six miRNAs) (see Figure 6). Both the natural linker sequences can be used, as well as fragments thereof or artificial linkers (again, reducing the payload of the vector).

[0103] Since miRNA scaffolds from the miR-106a-363 ​​cluster are particularly contemplated, particularly contemplated linkers are the 5' and 3' sequences of each scaffold (see Figure 1). Linker sequences can be, for example, 150 bp, 140 bp, 130 bp, 120 bp, 110 bp, 100 bp, 90 bp, 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, 30 bp, 20 bp, 10 bp, or less on either side of the scaffold. When two non-adjacent scaffolds are used in the cluster (e.g., as in Example 5), the linker, by definition, is not the same as that found in the cluster. Nevertheless, hybrid linkers can be created, for example, by using the 30, 60, or 90 bp present at the 3' end of one scaffold in the cluster and fusing it to a linker consisting of 30, 60, or 90 bp at the 5' end of the next selected scaffold.

[0104] The miRNA scaffolds are particularly used as is, i.e., without any modifications to the scaffold sequence. In particular, the lower stem sequence is kept the same as that found in each miRNA scaffold. Preferably, the loop sequences in the upper stem are also unchanged, although experiments have shown that these are primarily flexible structures, and their length and sequence can be adapted as long as the upper stem structure is not affected. While not preferred, those skilled in the art will understand that scaffolds with such modified loops are within the scope of the present application. The target sequence is found within the upper stem of the scaffold. The natural target sequence of the miR-106a-363 ​​cluster is 22-23 bp in length. As shown in Example 4, the target sequence can be shortened in size without adverse effects. The target sequence can be 18-23 bp in length, with 18-21 bp sequences being particularly contemplated, and 18-20 bp sequences being particularly contemplated. If shorter sequences are required, target sequences of 18 or 19 bp may be used without issue.

[0105] As is evident for targeting, the target sequence is the part of the scaffold that clearly needs to be applied to the target. Because the miRNA scaffolds have some mismatches in their structure, the question is whether these mismatches should be retained. As shown in Example 3 (and Figure 9), the mismatch found at position 14 of the target sequence in miR-106a and miR-20b can be retained without negative effects on downregulation of the target, meaning that the passenger strand is not perfectly complementary to the guide strand. Also, as shown in Example 3 (and Figure 10), when multiple mismatches are present (e.g., in the miR-18b scaffold), more efficient knockdown can be achieved (if necessary) by making the passenger strand more complementary to the guide strand. Note that, although this modification is not required to achieve significant levels of knockdown, removing mismatches at positions 6, 11, and 15 of the target sequence (corresponding to bp 20, 70, 25, 65, and 29, 61 of the scaffold (see Figure 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 similarly improves the downregulation in other scaffolds, but is not yet necessary, as testing of different target sequences consistently yielded satisfactory knockdown levels.

[0106] The cells disclosed herein typically contain multiplexed RNA interference molecules. These may be directed against one or more targets that need to be downregulated (either targets within the cell if the shRNA is secreted, or targets outside the cell). Each RNA interference molecule may target a different molecule, and they may target the same molecule or a combination thereof (i.e., multiple RNA molecules directed against one target, while a single RNA interference molecule is directed against a different target). If the RNA interference molecules are directed against the same target, they may target the same region or different regions. In other words, if the RNA interference molecules are directed against the same target, they may be the same or different. Examples of such combinations of RNA interference molecules are provided in the Examples section.

[0107] Thus, according to certain embodiments, at least two of the multiplexed RNA interference molecules are directed against the same target. According to further certain embodiments, these at least two RNA interference molecules use the same miRNA scaffold. They can be directed against the same target by using the same target sequence (according to these certain embodiments, at least two of the multiplexed RNA interference molecules are the same) or by using different target sequences (according to these certain 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 against the same target have different miRNA scaffold sequences. In that case, they may have the same target sequence or different target sequences directed against the same target.

[0108] According to another embodiment, all of the at least two multiplexed RNA interference molecules are different. According to a further particular embodiment, all of the at least two multiplexed RNA interference molecules are directed against different targets.

[0109] Any suitable molecule present in the engineered cell can be targeted by the RNA interference molecule of the present invention.Typical examples of possible targets include: MHC class I gene, MHC class II gene, MHC co-receptor gene (for example, HLA-F, HLA-G), TCR chain, CD3 chain, NKBBiL, LTA, TNF, LTB, LST1, NCR3, AIF1, LY6, heat shock protein (for example, HSPA1L, HSPA1A, HSPA1B), complement cascade, regulatory receptor (for example, NOTCH4), TAP, HLA-DM, HLA-DO, RING1, CD52, CD247, HCP5, DGKA, DGKZ, B2M, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, 2B4, A2AR, BAX, BLIMP1, C160(POLR3A), CBL-B, CCR6, CD7, CD95, CD123, 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, LFA1, NEAT 1, NFkB (including RELA, RELB, NFkB2, NFkB1, and REL), NKG2A, NR4A (including NR4A1, NR4A2, and NR4A3), PD1, PI3KCD, PPP2RD2, SHIP1, SOAT1, SOCS1, T-BET, TET2, TGFBR1, TGFBR2, TGFBR3, TIGIT, TIM3, TOX, and ZFP36L2.

[0110] Another way of expressing the invention disclosed herein is that particularly preferred constructs have been identified that are miRNA-based. Accordingly, there is provided an engineered cell comprising a polynucleotide comprising a microRNA-based shRNA coding region, said microRNA-based shRNA coding region comprising: one or more artificial miRNA-based shRNA nucleotide sequences, each of the artificial miRNA-based shRNA nucleotide sequences comprising miRNA scaffold sequence, the active or mature sequence; 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; The invention further comprises a sequence encoding one or more artificial miRNA-based shRNA nucleotide sequences, including:

[0111] 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.

[0112] A particular advantage of the miRNA-based shRNA nucleotide sequences of the present invention is that they can be multiplexed. Accordingly, engineered cells are provided that contain polynucleotides comprising multiplexed microRNA-based shRNA coding regions, wherein the multiplexed microRNA-based shRNA coding regions are: two or more artificial miRNA-based shRNA nucleotide sequences, each of the artificial miRNA-based shRNA nucleotide sequences comprising: miRNA scaffold sequence, the active or mature sequence; Passenger or star configuration, and within each artificial miRNA-based shRNA nucleotide sequence, a sequence encoding two or more artificial miRNA-based shRNA nucleotide sequences, wherein the active sequence is at least 70% complementary to the passenger sequence.

[0113] The miRNA-based shRNA nucleotide sequence is particularly selected from the group consisting of miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92-2, and miR-363 sequences. Both the active sequence and the passenger sequence of each of the artificial miRNA-based shRNA nucleotide sequences are typically 18 to 40 nucleotides in length, more particularly 18 to 30 nucleotides in length, even more particularly 18 to 25 nucleotides in length, and most particularly 18 to 23 nucleotides in length. The active sequence may also be 18 or 19 nucleotides in length. 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.

[0114] Typically, these microRNA scaffold sequences are separated by linkers. In microRNA clusters, linkers can be up to 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 150 nucleotides, or 100 nucleotides in length. When multiplexing scaffold sequences, the goal may be to use natural linker sequences (those 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 goal may be to reduce vector payload and linker length; linker sequences can therefore be, for example, 30-60 nucleotides long, although even shorter stretches will also work. Indeed, surprisingly, it has been found that linker length does not play a critical role; they can be very short (less than 10 nucleotides) or even absent and do not interfere with shRNA function. According to certain embodiments, at least a portion of the 5' and / or 3' linker sequence is used with the 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.

[0115] The miRNA-based shRNA nucleotide sequence is considered an artificial sequence because, even though the scaffold sequence may exist in nature, the endogenous miR sequence has been replaced with an engineered shRNA sequence directed against a specific target. The artificial sequence may be, for example, a naturally occurring scaffold (e.g., a miR cluster or fragment thereof, such as the miR-106a-363 ​​cluster) in which the endogenous miR sequence has been replaced with an engineered shRNA sequence directed against a specific target; a repeat of a single miR scaffold (e.g., the miR-20b scaffold) in which the endogenous miR sequence has been replaced with an engineered shRNA sequence directed against a specific target; an artificial miR-like sequence; or a combination thereof.

[0116] The engineered cell typically further comprises a nucleic acid molecule encoding a protein of interest, such as a chimeric antigen receptor or TCR, and may be an engineered immune cell, as described above.

[0117] 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 engineered cell, which can contribute to greater therapeutic efficacy.

[0118] The engineered cells described herein are also provided for use as a medicament. According to certain embodiments, the engineered cells are provided for use in the treatment of cancer. Examples of cancers that can be treated include, but are not limited to, adenocarcinoma, adrenocortical 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.

[0119] According to certain embodiments, the cells may be provided for the treatment of liquid or hematological cancers, such as leukemias (including acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL), among others), lymphomas (including Hodgkin's lymphoma and non-Hodgkin's lymphomas, such as B-cell lymphomas (including DLBCL), T-cell lymphomas, Burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, and small lymphocytic lymphoma, among others), multiple myeloma, or myelodysplastic syndromes (MDS).

[0120] This is equivalent to providing a method for treating cancer, comprising administering to a subject in need thereof a suitable dose of engineered cells (i.e., engineered cells 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) as described herein, thereby ameliorating at least one symptom associated with said cancer. Cancers contemplated for treatment include, but are not limited to, adenocarcinoma, adrenocortical 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 for treating a hematological cancer, comprising administering to a subject in need thereof a suitable dose of engineered cells described herein, thereby ameliorating at least one symptom of said cancer.

[0121] According to another embodiment, the cells can be provided for use in the treatment of an autoimmune disease, including, but 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-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, psoriatic arthritis, Addison's disease, ankylosing spondylitis, Behçet's disease, celiac disease, Coxsackie myocarditis, endometriosis, fibromyalgia, Graves' disease, Hashimoto's thyroiditis, Kawasaki disease, Meniere's disease, myasthenia gravis, sarcoidosis, scleroderma, Sjögren's syndrome, thrombocytopenic purpura (TTP), ulcerative colitis, vasculitis, and vitiligo.

[0122] This is equivalent to providing a method of treating an autoimmune disease, comprising administering to a subject in need thereof a suitable dose of engineered cells as described herein, thereby ameliorating at least one symptom associated with said autoimmune disease. Examples of autoimmune diseases that can be treated are listed above.

[0123] According to a further embodiment, the cells can be provided for use in the treatment of an infectious disease. "Infectious disease" is used herein to refer to any type of disease caused by the presence of a foreign organism (pathogen) in or on the subject or organism with the disease. Infections are usually caused by microorganisms or parasitic microorganisms such as viruses, prions, bacteria, and viroids, although it is believed that larger organisms such as macroparasites and fungi can also be infectious. Such 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 in the absence of overt disease), 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 such as helminths (e.g., nematodes such as roundworms, filariae, hookworms, pinworms, and whipworms, or flatworms such as cestodes and trematodes), but also ectoparasites such as ticks and mites. Parasitoids, i.e., parasites that kill the host organism, are contemplated to be included within the term parasite. According to certain embodiments, the infectious disease is caused by a microbial or viral organism.

[0124] 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 pallidum, Leptospira, Borrelia such as Borrelia burgdorferi), mollicutes (i.e., bacteria without a cell wall such as Mycoplasma), acid-fast bacteria (e.g., Mycobacterium such as Mycobacterium tuberculosis, Nocardia). "Microbacterial organisms" also encompasses 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 microbial organisms that can be treated by the methods include, but are not limited to, Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus (MRSA)), Enterococcus (including vancomycin-resistant enterococci (VRE), the nosocomial pathogen Enterococcus faecalis), food pathogens such as Bacillus subtilis, Bacillus cereus, Listeria monocytogenes, Salmonella, and Legionella pneumophila.

[0125] " Viral organisms " or " virus ", as used herein as an equivalent, are small infectious agents that can only replicate inside the living cells of organisms. They include dsDNA viruses (such as adenoviruses, herpesviruses, poxviruses), ssDNA viruses (such as parvoviruses), dsRNA viruses (such as reoviruses), (+)ssRNA viruses (such as picornaviruses, togaviruses, coronaviruses), (-)ssRNA viruses (such as orthomyxoviruses, rhabdoviruses), ssRNA-RT (reverse transcription) viruses, i.e., viruses with (+)sense RNA and DNA intermediates in their life cycle (such as retroviruses), and dsDNA-RT viruses (such as hepatitis viruses).Examples of viruses that can also infect human subjects include, but are not limited to, adenoviruses, astroviruses, hepatitis viruses (e.g., hepatitis B virus), herpes viruses (e.g., herpes simplex virus type I, herpes simplex virus type II, human cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, roseolovirus), papovaviruses (e.g., human papillomavirus and human polyomavirus), poxviruses (e.g., smallpox virus, cowpox virus, smallpox virus), arenaviruses, buniaviruses, 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, West Nile virus, Examples of the infectious disease to be treated include HIV, dengue virus, hepatitis C virus, tick-borne encephalitis virus, Japanese encephalitis virus, and encephalitis virus), orthomyxoviruses (e.g., influenza A virus, influenza B virus, and influenza C virus), paramyxoviruses (e.g., parainfluenza virus, rubulavirus (mumps), measles virus, and pneumoviruses such as human respiratory syncytial virus), picornaviruses (e.g., poliovirus, rhinovirus, coxsackie A virus, coxsackie B virus, hepatitis A virus, ecovirus, 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 certain embodiments, 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.

[0126] This is equivalent to providing a method for treating an infectious disease, comprising administering to a subject in need thereof a suitable dose of the engineered cells described herein (i.e., engineered cells comprising exogenous nucleic acid molecules encoding two or more multiplexed RNA interference molecules, and optionally comprising an additional nucleic acid molecule encoding a protein of interest), thereby ameliorating at least one symptom. Particularly contemplated microbial or viral infectious diseases are those caused by the pathogens listed above.

[0127] These cells provided for use as a drug can be provided for use in allogeneic therapy. That is, they are provided for use in treatments where allogeneic ACT is considered a treatment option (cells from another subject are provided to a subject in need thereof). According to certain embodiments, in allogeneic therapy, at least one of the RNA interference molecules is directed against the TCR (most particularly, directed against a subunit of the TCR complex). According to another embodiment, these cells are provided for use in autologous therapy, particularly autologous ACT therapy (i.e., using cells obtained from the patient).

[0128] While specific embodiments, particular configurations, and materials and / or molecules have been discussed herein for the cells and methods according to the invention, it will be understood that various changes or modifications in form and detail can be made without departing from the scope and spirit of the invention. Importantly, the vector variations discussed in the various vector embodiments also apply to the engineered cells (as the vectors are suitable for expression in such cells), and vice versa: the various cell embodiments are typically associated with the vectors encoded in the cells. The following examples are presented to better illustrate certain embodiments, and should not be construed as limiting the present application. The present application is limited only by the claims. [Example]

[0129] Example 1. Multiplexing optimization Efficient processing of the miRNA from the transcribed RNA by the DROSHA complex is crucial for efficient target knockdown. Our previous data demonstrated that miRNA-based shRNAs can be efficiently coexpressed with CAR-encoding vectors and processed from the vector by the miRNA machinery. It would be even more desirable to generate CAR expression vectors that can coexpress multiple miRNA-based shRNAs (e.g., 2, 4, 6, 8, etc.) from the same vector (Figure 2). However, previous studies have shown that coexpression of multiple miRNA-based shRNAs leads to a loss of shRNA activity. Therefore, efficient miRNA processing is important for knockdown of multiple targets from a single expression vector.

[0130] To achieve optimal multiplexing and avoid recombination, we hypothesized that it would be best to start with naturally occurring miRNA clusters rather than propagating a single miRNA scaffold. Naturally occurring miRNA clusters vary greatly in the size and number of scaffolds present. Because the goal was to use the multiplexed miRNA scaffolds for cloning vectors, we sought to identify clusters with promising scaffold number-to-size ratios. The 13 identified clusters are listed in Table 1.

[0131] [Table 1]

[0132] Table 1. Identification of micro-RNA clusters by name, chromosomal location, size, location within coding or non-coding sequences, 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 microRNA scaffolds with interspersed sequences (linker + others) in the cluster. Light grey shading: high expression in T cells.

[0133] To illustrate how the sizes can vary, two of these clusters (dark gray shading, Table 1) are shown for illustrative purposes. These clusters exceeded 85,000 bp and were immediately eliminated as too large for cloning. The most promising clusters were selected based on the size and number of miRNAs present in the cluster (the N in Table 1). To capture the average miRNA scaffold plus linker sequence, we evaluated the size divided by the number of miRNA scaffolds, rather than just the total size. As a first cutoff, we selected clusters with a size / N of less than 250. This yielded sufficient clusters, and because the goal was to express the vector in engineered immune cells, we decided to focus on clusters highly expressed in immune cells, such as T cells. This led to the prioritization of four clusters (light gray shading, Table 1), all highly expressed in immune cells and with a total size less than 1,000 bp. Furthermore, they all contain at least three miRNA scaffolds (clusters with N at least 3 are expected to allow multiplexing of more than two miRNAs) with an average size of less than 200 bp per scaffold, making them highly suitable for cloning (see Table 1): the miR17-92 cluster, the miR106a-363 ​​cluster, the miR106b-25 cluster (three paralogous microRNA clusters), and the miR23a-27a-24-2 cluster.

[0134] 1.1 Selection of suitable miRNA clusters for multiplexing To assess whether the four miRNA clusters are suitable for multiplexed expression of shRNAs, primary T cells from healthy donors were transduced with a retroviral vector encoding a second-generation CD19-targeting CAR and a truncated CD34 selectable marker, along with different shRNAs introduced into the selected clusters. To allow for comparison of the effects of the same number of shRNAs and cluster truncations, fragments of the miR17-92 and miR106a-363 ​​clusters were also used. These fragments were three or four consecutive miRNA scaffolds from the clusters, allowing for comparison of the three miRNA scaffolds present in the other two clusters. The schematic design of such vectors is shown in Figure 2.

[0135] Three identical shRNA target sequences targeting CD247, B2M, and CD52 were used for comparison. When four miRNA scaffolds were used, TRAC was also targeted. For six miRNA scaffolds, the three targets were targeted twice, but with different target sequences. As a control, the miR196a2 scaffold, a repetitive synthetic shRNA scaffold, was used, which has previously been shown to be superior for single shRNA knockdown and suitable for multiplexed knockdown (WO 2020 / 221939). This control was used with three and four shRNAs.

[0136] Despite the different sizes of the constructs, vector titers were only slightly affected by the amount of shRNA present (data not shown). However, in both populations, the use of a different scaffold from the native miRNA cluster increases the transduction efficiency compared to the same scaffold repeated (here, the miR-196a2 scaffold), as shown in Figure 3.

[0137] The fold increase in T cell numbers from transduction to harvest did not differ significantly between the constructs (neither between the clustered scaffolds nor between the clustered scaffolds and the repeated single scaffolds). However, the knockdown efficiency differed between the constructs. While all clusters achieved knockdown to some extent, there were clear differences between the clustered scaffolds, with the scaffolds from the miR-106a-363 ​​cluster achieving the best and most consistent knockdown, and the miR23a-27a-24-2 cluster scaffolds being the least effective. Figure 4 shows an example comparing TCR expression with no shRNA control or shRNA in the miR23a-27a-24-2 clustered scaffold or in the miR106a-363 ​​clustered scaffold or its fragment. The increased knockdown observed with the complete scaffold can be explained by the fact that CD247 is targeted twice in this construct. As a result of these experiments, the scaffold of the miR-106a-363 ​​cluster was selected for further evaluation.

[0138] Example 2. Multiplexing of the miR-106a-363 ​​cluster using the 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 or 6x shRNAs targeting CD247, β2m, and CD52 integrated into the miR-106a-363 ​​cluster. The vector design is shown in Figure 5. Briefly, primary T cells from healthy donors were transduced with a retroviral vector encoding a second-generation CD19-targeting CAR, a truncated CD34 selection marker, and three shRNAs targeting CD247, B2M, and CD52 integrated into the last three miRs of the 106a-363 ​​miRNA cluster (miR-19b2, miR-92a2, and miR-363), or six shRNAs targeting the same three genes in the six-miR scaffold of the cluster (in this case, the two shRNAs targeting CD247 were different). Briefly, shRNAs were expressed as a 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 in IL-2 (100 IU / mL) for six days. The mRNA expression of CD247, B2M, and CD52 was assessed by qRT-PCR using cyclophilin as a housekeeping gene.

[0139] The results are shown in Figure 6. Multiplexed shRNAs achieved efficient RNA knockdown levels for all target genes. Incorporating six multiplexed shRNAs (two shRNAs for each protein target) resulted in higher RNA knockdown levels compared to three multiplexed shRNAs (one shRNA for each protein target) (Figure 6).

[0140] Example 3. Optimization of individual scaffolds of the miR-106a-363 ​​cluster Although the initial data were already promising, demonstrating that multiplexing could be achieved using scaffolds from the miR-106a-363 ​​cluster, further investigations were undertaken to determine whether individual scaffolds could be modified to improve knockdown of the selected targets. Given that the native scaffolds have naturally been under evolutionary selection pressure to tailor knockdown (meaning that the lower and upper stem regions have been at least partially optimized by evolution), we first evaluated different target sequences to improve target downregulation, since they have not yet been optimized. In our first example, we selected the same target protein.

[0141] Since it has previously been described that the processivity of each miRNA / shRNA can depend on and be affected by others in the cluster (Bofill-De Ros and Gu, 2016), we decided to test the scaffolds with different target sequences as part of the overall cluster, but with unrelated sequences in the other scaffold sequences (to not affect target downregulation).

[0142] The results of CD247 downregulation in the miR-20b scaffold are shown in Figure 7. The initial scaffold sequence already resulted in approximately 50% downregulation. All other target sequences tested successfully knocked down the target, with some achieving knockdown much higher than 50%. In other words, by selecting the target sequence, maximally effective knockdown could be achieved, and no further manipulation of the miR-20b scaffold was required.

[0143] Similar results were obtained for the miR-106a scaffold sequence using different sequences for the B2M target (data not shown). To rule out that the effect was related to the specific target sequence-scaffold combination, the B2M target sequence was also tested in the miR-20b scaffold. Although there were some minor variations in knockdown efficiency, the three target sequences that achieved the highest knockdown in the miR-106a scaffold also achieved the highest knockdown when used in the miR-20b scaffold. This means that once an effective target sequence is identified, it can be used across scaffolds.

[0144] The miR-18b scaffold was used to optimize shRNAs against CD95. However, after testing 31 target sequences, the best knockdown achieved was approximately 30% (see Figure 8). While this knockdown is not negligible, it is significantly less effective than the >75% knockdown consistently observed with other scaffolds. When comparing the miR-18b scaffold with the miR-106a or miR-20b scaffolds (Figure 9), it is clear that this scaffold contains more mismatches in the target sequence / upper stem region (3 vs. 1) and a bulge near the end of the upper stem. Because high knockdown was achieved with the other scaffold sequences, we hypothesized that reducing the number of mismatches and / or removing the bulge could potentially improve the knockdown efficiency.

[0145] The five different constructs evaluated are shown in Figure 10A, and the results are shown in Figure 10B. Surprisingly, deleting even one mismatch or bulge significantly improved the knockdown efficiency. When only the single mismatch that also occurs in the miR-106a or miR-20b scaffolds is retained, the knockdown efficiency increases from approximately 30% to over 60% for the same target sequence. Thus, while the miR-18b scaffold sequence can be used as is, knockdown efficiency can be significantly increased by reducing the number of mismatches or bulges.

[0146] 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 they could be shortened, target sequences of different lengths (one for CD247 and one for B2M) were inserted into the scaffold and evaluated for knockdown efficiency. Sequence shortening was achieved by replacing the 3'-terminal nucleotide of the target sequence with that 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, and possibly even better than, the maximum length. Similar results were obtained for the miR-20b scaffold (not shown). For most experiments, the standard 20 bp sequence was deemed functional (as shown in Figure 9).

[0147] Example 5. Evaluation of combinations of individual scaffolds outside the cluster association It is generally accepted that in miRNA clusters, the presence of many flanking sequence determinants, as well as other clusters, is thought to be important for achieving downregulation, however, our previous experiments have shown that this is not always the case.

[0148] 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 two miRNA-based shRNAs, as well as the miRNA scaffold, would affect shRNA activity. To optimize shRNA processing, we evaluated the effect of different shRNA linkers on the knockdown of two target genes, CD247 (CD3ζ) and CD52. Linkers ranging from 0 to 92 bp were used, but the linker did not appear to affect the knockdown efficacy, except for the 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, the linker-less construct also performed very well in reducing the expression of both shRNAs (data not shown). Although these experiments were performed using the miR-196a2 scaffold, initial experiments indicated that the linker of the miR-106a-363 ​​cluster could also be significantly reduced.

[0149] To assess whether the processivity and activity of each individual scaffold was affected by the presence of others in the cluster, we decided to test the scaffolds in different permutations. To this end, we selected non-contiguous scaffolds: miR-106a and miR-20b (to eliminate the effects of adjacent scaffolds in the cluster). Furthermore, rather than using all six miRNA scaffolds in the cluster, we created duplexes and triplexes (the opposite of Example 2). We also created the miR-106a-miR-18b-miR-20b triplex, corresponding to the first three scaffolds in the miR-106a-363 ​​cluster, to assess whether there were cluster-related effects. For duplexes, the targeted genes were B2M and CD247. For triplexes, CD95 was added.

[0150] In summary, the following constructs were made: 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) 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)

[0151] The results are shown in Figures 12A-C. As shown in Figure 12, all of the duplexes evaluated were highly efficient at downregulating both CD247 and B2M. The CD247 knockdown proved particularly efficient, leading to almost undetectable levels of CD3Z. Because B2M is much more abundant, the knockdown was not expected to be complete, but a greater than 80% reduction in B2M levels was consistently achieved. Surprisingly, the level of downregulation was the same regardless of the order of the scaffolds in the duplex.

[0152] It is well known that multiplexing the same shRNA can cause recombination problems, resulting in much lower expression and ultimately lower knockdown levels. This was precisely the reason for evaluating combinations of different scaffolds. Nevertheless, to examine whether this is actually feasible, we tested two and three identical scaffolds. All of the duplexes with the same scaffold, as well as the miR-20b triplex scaffold, achieved transduction levels comparable to those of duplexes or triplexes with different scaffolds, all above 15%. However, the miR-106a triplex scaffold achieved very low transduction levels (<2%) and was not further evaluated. Duplexes with the miR-20b scaffold achieved the same level of target knockdown as duplexes with non-identical scaffolds (Figures 12A-B). The miR-106a scaffold duplex achieved the same downregulation of CD3Z but was slightly less effective at knocking down B2M, albeit at a reduction of approximately 50%, demonstrating that these scaffolds can be replicated and still achieve high knockdown (Figure 12C). Notably, the miR-20b triplex scaffold achieved knockdown levels comparable to triplexes with three different scaffolds, but the use of three different scaffolds achieved slightly better knockdown for each target gene, indicating some loss of efficacy (Figures 12A-B). The triplex scaffold with three different miRNA scaffolds achieved the same downregulation of the targets as the duplex. Furthermore, CD95 was downregulated by more than 50% (Figures 12B-C), consistent with the results of this target sequence when used in the cluster setting (Figure 10B).

[0153] These experiments demonstrate that scaffolds can be used independently and outside of the cluster context. The order of the scaffolds is important to achieve the desired knockdown; not all scaffolds in the cluster need to be present to achieve knockdown. In fact, a single scaffold is sufficient and can be replicated without loss of activity. While it has been shown that the miR-20b can be used as a triplex, this appears to be slightly less efficient than using different scaffolds. Nevertheless, given that the miR-106a-363 ​​cluster contains six different scaffold sequences, which can be replicated without loss of efficacy, multiplexed downregulation of up to 12 targets is in principle feasible.

[0154] References Bofill-De Ros X, Gu S. Guidelines for the optimal design of miRNA-based shRNAs. Methods. 2016 Jul 1;103:157-66. Chumakov SP, Kravchenko JE, Prassolov VS, Frolova EI, 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;11(1):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, Iocca 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 first exogenous nucleic acid molecule encoding a protein of interest; a second nucleic acid molecule encoding at least one RNA interference molecule having a scaffold 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, The at least one RNA interference molecule is located within the scaffold and contains its natural target sequence. engineered cells containing a target sequence different from that of the

2. 2. The engineered cell of claim 1, wherein the target sequence is 18 to 23 nucleotides.

3. The RNA interference molecule binds to the target sequence in the engineered cells by base pair complementarity.

3. The engineered cell of claim 1 or 2, which is directed against a target.

4. The engineered cell according to any one of claims 1 to 3, which is an engineered immune cell.

5. The engineered cell of claim 4 , wherein the immune cell is selected from a T cell, an NK cell, an NKT cell, and a macrophage.

6. The engineered cell of any one of claims 1 to 5, wherein the protein of interest is a receptor.

7. The engineered cell described in claim 6, wherein the receptor is a chimeric antigen receptor or a TCR.

8. The engineered cell of any one of claims 1 to 7, wherein the at least one RNA interference molecule is a multiplexed RNA interference molecule comprising at least two scaffolds.

9. 9. The engineered cell of claim 8, wherein the multiplexed RNA interference molecule comprises at least three scaffolds.

10. The engineered cell of claim 8 or 9, wherein at least one of the scaffolds is selected from a miR-106a scaffold and a miR-20b scaffold.

11. 10. The engineered cell of claim 8 or 9, wherein at least one of the scaffolds is a miR-18b scaffold, and the scaffold has been modified to reduce mismatches and / or bulges in the stem region.

12. 12. The engineered cell of any one of claims 8 to 11, wherein all of the multiplexed RNA interference molecules comprise a miR-scaffold 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.

13. The engineered cell of any one of claims 8 to 12, wherein at least two of the multiplexed RNA interference molecules are directed against the same target.

14. The engineered cell of any one of claims 8 to 12, wherein the at least two multiplexed RNA interference molecules are all directed against different targets.

15. 15. The engineered cell of any one of claims 8 to 14, wherein at least two of the multiplexed RNA interference molecules have the same scaffold.

16. The molecule targeted by the at least one RNA interference molecule is selected from the group consisting of MHC class I and Ib. I genes, MHC class II genes, MHC co-receptor genes, HLA-F, HLA-G, TCR chains, NKBBiL, LTA, TNF, LTB, LST1, NCR3, AIF1, LY6, heat shock proteins, HSPA1L, HSPA1A, HSPA1B, complement cascade, regulatory receptors, NOTCH4, TAP, HLA-DM, HLA-DO, RING1, CD52, CD247, HCP5, DGKA, DGKZ, B2M, MICA, MICB, ULBP1, ULBP2, U LBP3, ULBP4, ULBP5, ULBP6, 2B4, A2AR, BAX, BLIMP1, C160, POLR3A, CBL-B, CCR6, CD7, CD95, CD123, DGK, DGKA, DGKB, DGKD, DGKE, DKG G, DGKH, DGKI, DGKK, DGKQ, DGKZ, DNMT3A, DR4, DR5, EGR2, FABP4, FABP5, FASN, GMCSF, HPK1, IL-10R, IL10RA, IL10RB, IL2, LFA1, NEAT 16. The engineered cell of any one of claims 1 to 15, wherein the target gene is selected from: 1, NFkB, RELA, RELB, NFkB2, NFkB1, REL, NKG2A, NR4A, NR4A1, NR4A2, NR4A3, PD1, PI3KCD, PPP2RD2, SHIP1, SOAT1, SOCS1, T-BET, TET2, TGFBR1, TGFBR2, TGFBR3, TIGIT, TIM3, TOX, and ZFP36L2.

17. The engineered cell of any one of claims 1 to 16 for use as a medicament.

18. 17. The engineered cell of any one of claims 1 to 16 for use in the treatment of cancer.

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