WO2023152382A1

WO2023152382A1 - Artificial microrna construct

Info

Publication number: WO2023152382A1
Application number: PCT/EP2023/053529
Authority: WO
Inventors: David Marc DAVIES; Robert Page; Maya GLOVER
Original assignee: King's College London; Leucid Bio Ltd
Priority date: 2022-02-14
Filing date: 2023-02-13
Publication date: 2023-08-17
Also published as: GB202201927D0

Abstract

This invention relates to a polynucleotide construct comprising (a) an artificial microRNA (amiRNA) coding region comprising a polynucleotide encoding an amiRNA and (b) a protein-coding region comprising a polynucleotide encoding a chimeric antigen receptor (CAR). Also provided are vectors, host cells, pharmaceutical compositions, kits, methods of preparing host cells and uses thereof.

Description

ARTIFICIAL MICRORNA CONSTRUCT TECHNICAL FIELD This invention relates to a polynucleotide construct comprising (a) an artificial microRNA (amiRNA) coding region comprising a polynucleotide encoding an amiRNA and (b) a protein- coding region comprising a polynucleotide encoding a chimeric antigen receptor (CAR). Also provided are vectors, host cells, pharmaceutical compositions, kits, methods of preparing host cells and uses thereof. BACKGROUND Immunotherapy using chimeric antigen receptor (CAR)-engineered T-cells has proven transformative in the management of B-cell malignancy and multiple myeloma. Indeed, two CAR T-cell therapies (Axicabtagene ciloleucel and Tisagenlecleucel) have recently been approved for use in the UK through the Cancer Drugs Fund for the treatment of acute lymphoblastic leukaemia, diffuse large B-cell lymphoma and primary mediastinal large B-cell lymphoma. In addition, the CAR T-cell therapy Yescarta (axicabtagene ciloleucel) was recently approved by The National Institute for Health and Care Excellence (NICE) for routine use by the UK National Health Service (NHS) as third line treatment for patients with relapsing-remitting diffuse large B cell lymphoma (DLBCL) and primary mediastinal large B-cell lymphoma (PMBCL). Despite these successes, the large-scale adoption of CAR T-cells as a general treatment for all types of cancer is yet to be achieved. The complexity of the solid cancer microenvironment poses a particular challenge to the current CAR T-cell approaches. One potential reason for the reduced efficacy of CAR T-cells in the treatment of solid cancers is the presence of a highly immunosuppressive tumour microenvironment (TME) which can inhibit the function of infiltrating T-cells. Current research to address this involves combining CAR T-cells with inhibitors of various immunosuppressive pathways. Numerous different strategies have been employed to reduce the influence of these immunosuppressive pathways, including combining CAR T-cell therapy with checkpoint inhibitors, engineering the CAR T-cells to secrete checkpoint inhibitors themselves and replacing suppressive molecules in the T-cell with either signalling-null variants or so-called ‘switch receptors’ in which the signalling domain of the inhibitory molecule is replaced by a stimulatory motif. There remains a need for cellular immunotherapies which achieve improved efficacy. A further challenge for current CAR T-cell approaches is the necessity of autologous CAR T- cells, to reduce the risk of allorejection and graft versus host disease (GVHD). Consequently, the manufacturing process per patient is labour and resource intensive. This, in turn, is costly. In addition, patients have often received a variety of different therapeutic regimens prior to CAR T-cell therapy, which may impact upon the efficacy of the T-cells themselves. Coupled with their inherent biological variability, this makes it difficult to standardise the therapeutic product between patients. MicroRNA (miRNA) are small, non-coding RNAs that inhibit gene expression by binding to the 3’ untranslated region of target mRNA, thereby promoting degradation or translational repression. In some instances, miRNA have also been shown to bind any location of the coding sequence of target mRNA, including the majority or all of the coding sequence of target mRNA. MicroRNA are present within longer primary transcripts (pri-miRNA) that are processed into smaller precursor RNA (pre-miRNA) by the enzyme Drosha. These pre- miRNAs are then further processed into mature miRNA by the enzyme Dicer, before being loaded onto the RISC complex and mediating RNA interference. The present invention seeks to address one or more of the aforementioned issues. SUMMARY OF THE INVENTION The present invention provides a polynucleotide construct comprising (a) an artificial microRNA (amiRNA) coding region comprising a polynucleotide encoding an amiRNA and (b) a protein-coding region comprising a polynucleotide encoding a chimeric antigen receptor (CAR). As the skilled person will appreciate, a microRNA (miRNA) is a single-stranded non-coding RNA which is specific for a target mRNA. By “specific”, this will be understood to refer to being capable of specifically hybridising to the target mRNA. The miRNA “specifically hybridises” to its target mRNA when it hybridises with preferential or high affinity to the target mRNA but does not substantially hybridise, does not hybridise, or hybridises with only low affinity to other polynucleotides, especially other non-target mRNAs. Typically, hybridisation of a miRNA to a target mRNA is due to substantially complementary base- pairing between the miRNA and the target mRNA. The hybridisation of the miRNA to the target RNA induces the degradation and translational repression of the target mRNA, thereby reducing translation of the target mRNA and expression of the protein which it encodes. Therefore, miRNAs function to suppress target genes. Typically, in nature, a primary miRNA termed a pri-miRNA is processed intracellularly to form a precursor miRNA (pre-miRNA). Within the cell, the pre-miRNA is further processed to form the final mature miRNA. It will therefore be appreciated in the context of the invention that the polynucleotide construct may encode the “pri-amiRNA” and/or “pre-amiRNA” of the amiRNA. It will also be appreciated that pri-amiRNA can comprise the pre-amiRNA, and the pre-amiRNA can comprise the amiRNA. Thus, the pri-amiRNA comprises the amiRNA. In the context of the present invention, the term “artificial microRNA (amiRNA)” refers to a genetically engineered miRNA that doesn’t appear in nature. As will be clear from the examples, the inventors have taken the sequences of naturally occurring pri-miRNAs and modified them to create amiRNA by embedding the 5’ and/or 3’ stem sequences of previously validated short hairpin RNAs (shRNAs) or CRISPR guide RNAs specific for a target mRNA into the pri-miRNA. The inventors have also taken the sequences of naturally occurring pri-miRNAs and modified them to create amiRNA by embedding shRNAs generated by the inventors into the pri-miRNA. It will therefore be appreciated that the polynucleotide construct of the invention is recombinant. Chimeric antigen receptors are immune cell receptors which have been genetically engineered to confer the ability to target a specific antigen or antigens. Generally, chimeric antigen receptors are specific for one or more cancer-associated antigens. As such, chimeric antigen receptors are commonly used in the treatment of cancer. The present inventors have found that a polynucleotide construct encoding a CAR and an amiRNA can be successfully introduced into and expressed in a host cell. This is unexpected given the substantial size of such a construct. Advantageously, the provision of one construct encoding both the CAR and the amiRNA removes the need for a plurality of constructs each encoding the CAR and the amiRNA to be introduced to the cell. This reduces the time spent introducing and expressing the construct in the cell. In addition, this increases the efficiency and selectivity of engineering by ensuring expression of the amiRNA exclusively in those cells expressing the CAR. Without wishing to be bound by theory, the inventors believe this may improve the viability of the cell. The inventors have also found that the expression of this construct does not affect the viability, cytotoxicity or restimulation capacity of the host cells. This is surprising given the size of the construct. In addition, co-expression of the CAR and the amiRNA enables expression of the CAR to act as a marker for amiRNA expression. This provides a simple and effective construct. Without wishing to be bound by theory, the inventors believe that the amiRNA of the invention can function to suppress the expression of target genes which would otherwise reduce the effectiveness of the CAR-engineered cell. Thus, the amiRNA enables the CAR- expressing cell to have an increased and/or prolonged activity or viability, compared to cells expressing the CAR alone. For example, the amiRNA may be specific for a tumour microenvironment (TME) mRNA or an endogenous TCR or HLA mRNA. The inventors believe that this can increase the anti-cancer efficacy of the CAR-expressing host cell. The provision of the amiRNA and the CAR can also reduce potential detrimental side-effects associated with administration of the CAR-expressing host cell, such as graft versus host disease or allorejection. amiRNA In some embodiments, the amiRNA coding region comprises a plurality of polynucleotides each encoding an amiRNA. Thus, in such embodiments, the plurality of polynucleotides encode a plurality of amiRNAs. Each amiRNA encoded by the plurality of polynucleotides may be specific for the same target site. In other words, each of the plurality of amiRNAs may be specific for the same target site. Alternatively, the plurality of amiRNAs may be specific for a plurality of different target sites. For example, the plurality of amiRNAs may be specific for at least two, at least three, at least four, at least five, at least six, at least seven or at least eight different target sites. In some embodiments, the plurality of amiRNAs is specific for at least two, at least three, at least four, at least five, at least six or at least seven different target sites. In some embodiments, the plurality of amiRNAs is specific for less than ten, less than nine or less than eight different target sites. Advantageously, this provides flexibility to target one or more different genes. In some embodiments, the plurality of amiRNAs is specific for a plurality of different target sites on different target mRNAs which encode polypeptides of different proteins. Alternatively, the plurality of amiRNAs may be specific for a plurality of different target sites on the same mRNAs which encode polypeptides of the same protein or protein complex. In some embodiments, the plurality of amiRNAs comprises a first subset of amiRNAs specific for a plurality of different target sites on one mRNAs which encodes polypeptides of a first protein or protein complex, and a second subset of amiRNAs specific for a plurality of different target sites on another mRNAs which encode polypeptides of a second protein or protein complex. In some embodiments, the plurality of amiRNAs comprises more than two subsets of amiRNAs, wherein each of the subsets comprises a plurality of amiRNAs specific for a plurality of different target sites on the same mRNAs which encode polypeptides of the same protein or protein complex, and each of the subsets target a protein or protein complex different from other subsets. In some embodiments the amiRNA coding region comprises at least two, at least three, at least four, at least five or at least six polynucleotides each encoding an amiRNA. In some embodiments the amiRNA coding region comprises less than 10, less than nine, less than eight or less than seven polynucleotides, each encoding an amiRNA. In some embodiments the amiRNA coding region comprises at least six polynucleotides each encoding an amiRNA. In some embodiments the amiRNA coding region comprises six polynucleotides each encoding an amiRNA. In such embodiments, the six polynucleotides encode six amiRNAs. The six amiRNAs may be specific for at least one, at least two, at least three, at least four, at least five or at least six different target mRNAs. The six amiRNAs may be specific for less than ten, less than nine, less than eight or less than seven different target mRNAs. In some embodiments, the six amiRNAs are specific for six different target mRNAs. In other embodiments, the six amiRNAs are specific for three different target mRNAs. In some embodiments the amiRNA coding region comprises eight polynucleotides each encoding an amiRNA. The eight amiRNAs may be specific for at least one, at least two, at least three, at least four, at least five, at least six, at least seven or at least eight different target mRNAs. The eight amiRNAs may be specific for less than eight or less than seven different target mRNAs. In some embodiments, the eight amiRNAs are specific for eight different target mRNAs. In other embodiments, the eight amiRNAs are specific for at least three different target mRNAs. The amiRNA is typically the same length as the naturally occurring miRNA from which it is derived, but may be longer or shorter. Once introduced into a cell, the amiRNA is typically processed intracellularly to form a final RNAi product which is capable of suppressing the target gene(s). The final RNAi is preferably at least 20 nucleotides in length, such as 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the final RNAi is of from 19 to 24 nucleotides in length. In some embodiments, the final RNAi is 20, 21, 22 or 23 nucleotides in length. The amiRNA is preferably at least 20 nucleotides in length, such as 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the amiRNA is of from 19 to 24 nucleotides in length. In some embodiments, the amiRNA is 20, 21, 22 or 23 nucleotides in length. As described above, the amiRNA of the invention typically comprises a 5’ or 3’ stem shRNA and/or CRISPR guide RNA sequence specific for a target mRNA. The 5’ or 3’ stem shRNA or CRISPR guide RNA sequence may be at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, or at least 23 nucleotides in length. The 5’ or 3’ stem shRNA or CRISPR guide RNA sequence may be no more than 30 nucleotides, no more than 28 nucleotides, no more than 26 nucleotides, no more than 24 nucleotides, no more than 23 nucleotides, no more than 22 nucleotides, no more than 21 nucleotides, no more than 20 nucleotides, no more than 19 nucleotides or no more than 18 nucleotides in length. The 5’ or 3’ stem shRNA or CRISPR guide RNA sequence may be no more than 24 nucleotides, no more than 23 nucleotides, no more than 22 nucleotides, no more than 21 nucleotides, no more than 20 nucleotides, no more than 19 nucleotides or no more than 18 nucleotides in length. In some embodiments, the amiRNA comprises a 5’ or 3’ stem shRNA sequence which is 18 or 19 nucleotides in length. In other embodiments, the amiRNA comprises a 5’ or 3’ stem CRISPR guide RNA sequence which is 22 nucleotides in length. In some embodiments the amiRNA is at least 20 nucleotides in length and comprises an 18 or 19 nucleotide 5’ or 3’ stem shRNA sequence. In some embodiments the amiRNA is at least 23 or 24 nucleotides in length and comprises a 22 nucleotide 5’ or 3’ stem CRISPR guide RNA sequence. Preferably, the amiRNA comprises at least one nucleotide from the naturally occurring miRNA from which it is derived. More preferably, the at least one nucleotide in the amiRNA is in the same position as for the naturally occurring miRNA from which it is derived. For example, the at least one nucleotide from the naturally occurring miRNA in the amiRNA may be at position 20, said nucleotide also being at position 20 in the naturally occurring miRNA from which it is derived. A 3’ terminal nucleotide from the naturally occurring miRNA may be the 3’ terminal nucleotide in the amiRNA. Alternatively, or in addition, a 5’ terminal nucleotide from the naturally occurring miRNA may be the 5’ terminal nucleotide in the amiRNA. In some embodiments, the amiRNA comprises a substitution of a naturally occurring miRNA sequence or a portion thereof with a sequence specific to a target site. When the sequence specific to a target site is shorter than the naturally occurring miRNA, the amiRNA may comprise the sequence specific to a target site and one or more nucleotides from the naturally occurring miRNA. The one or more nucleotides from the naturally occurring miRNA may be at the 5’-end, 3’-end or both 5’ and 3’-end of the sequence specific to a target site. In some embodiments, the sequence specific to a target site is one, two, three, four, five, six, seven or more nucleotides shorter than the naturally occurring miRNA. In some embodiments, a sequence specific to a target site has the same length as the naturally occurring miRNA. In such embodiments, the amiRNA comprises no nucleotides from the naturally occurring miRNA. In some embodiments, the at least one nucleotide from the naturally occurring miRNA comprises a uracil, adenine, guanine or cytosine. The at least one nucleotide from the naturally occurring miRNA may comprise an uracil, adenine or guanine. In some embodiments the at least one nucleotide comprises an adenine or an uracil. Preferably, the amiRNA comprises or consists of a 5’ or 3’ stem shRNA or CRISPR guide RNA sequence and at least one nucleotide from the naturally occurring miRNA from which it is derived. Preferably, the amiRNA comprises, from 5’ to 3’ (i) a 5’ or 3’ stem shRNA or CRISPR guide RNA sequence and (ii) nucleotide(s) from the naturally occurring miRNA from which it is derived. In some embodiments, the amiRNA comprises at least two, at least three or at least four nucleotides from the naturally occurring miRNA from which it is derived. Preferably, the amiRNA comprises an at least two, at least three or at least four nucleotide sequence from the naturally occurring miRNA from which it is derived. In some embodiments, the amiRNA comprises no more than five, no more than four, no more than three or no more than two nucleotides from the naturally occurring miRNA from which it is derived. In some embodiments, the amiRNA comprises a nucleotide sequence of no more than five, no more than four, no more than three or no more than two nucleotides in length from the naturally occurring miRNA from which it is derived. Preferably, the amiRNA comprises two, three, four or five nucleotides from the naturally occurring miRNA from which it is derived. More preferably, the amiRNA comprises a two, three, four or five nucleotide length sequence from the naturally occurring miRNA from which it is derived. Most preferably, the two, three, four or five nucleotide length sequence is at the 3’ end of the amiRNA, optionally in positions corresponding to the positions in which the sequence is found in the naturally occurring miRNA from which it is derived. In some embodiments, the amiRNA comprises a two-nucleotide sequence from the naturally occurring miRNA from which it is derived. The two-nucleotide sequence may consist of the sequence dd (SEQ ID NO:1), wherein d is selected from guanine, adenine and uracil. In some embodiments, the two-nucleotide sequence consists of the sequence dw (SEQ ID NO:2), wherein d is selected from guanine, adenine and uracil and w is selected from adenine and uracil. In some embodiments, the two nucleotide-sequence consists of the sequence gu (SEQ ID NO:3). In other embodiments, the two-nucleotide sequence consists of the sequence ua (SEQ ID NO:4). In some embodiments, the two-nucleotide sequence consists of the sequence ga (SEQ ID NO:5) or ag (SEQ ID NO:6). In other embodiments the amiRNA comprises a three-nucleotide sequence from the naturally occurring miRNA from which it is derived. The three-nucleotide sequence may consist of the sequence ddd (SEQ ID NO:7), wherein d is selected from guanine, adenine and uracil. In some embodiments, the three-nucleotide sequence consists of the sequence ddw (SEQ ID NO:8), wherein d is selected from guanine, adenine and uracil and w is selected from adenine and uracil. In other embodiments, the three-nucleotide sequence consists of the sequence dwd (SEQ ID NO:9). In other embodiments, the three nucleotide- sequence consists of the sequence wdd (SEQ ID NO:10). In yet other embodiments, the three-nucleotide sequence consists of the sequence wwd (SEQ ID NO:11), dww (SEQ ID NO:12) or wdw (SEQ ID NO:13). The three-nucleotide sequence may consist of the sequence www (SEQ ID NO:14). The three-nucleotide sequence may be selected from the following sequences: agu (SEQ ID NO:15), aua (SEQ ID NO:16), uga (SEQ ID NO:17), gua (SEQ ID NO:18) and uag (SEQ ID NO:19). In some embodiments the amiRNA comprises a four-nucleotide sequence from the naturally occurring miRNA from which it is derived. The four-nucleotide sequence may consist of the sequence nddd (SEQ ID NO:20), wherein n is selected from adenine, guanine, uracil and cytosine and d is selected from guanine, adenine and uracil. In some embodiments, the four-nucleotide sequence consists of the sequence nddw (SEQ ID NO:21). In other embodiments, the four-nucleotide sequence consists of the sequence ndwd (SEQ ID NO:22). In other embodiments, the four-nucleotide sequence consists of the sequence nwdd (SEQ ID NO:23). In yet other embodiments, the four-nucleotide sequence consists of the sequence nwwd (SEQ ID NO:24), ndww (SEQ ID NO:25) or nwdw (SEQ ID NO:26). The four-nucleotide sequence may consist of the sequence nwww (SEQ ID NO:27). In some embodiments, n consists of b, wherein b is selected from guanine, cytosine and uracil. In some embodiments, n consists of guanine or uracil. The four-nucleotide sequence may consist of the sequence uagu (SEQ ID NO:28). In some embodiments, the four-nucleotide sequence consists of the sequence gaua (SEQ ID NO:29). In other embodiments, the four-nucleotide sequence consists of the sequence cuga (SEQ ID NO:30). The four-nucleotide sequence may consist of the sequence ggua (SEQ ID NO:31). Alternatively, the four-nucleotide sequence may consist of the sequence uuag (SEQ ID NO:32). In other embodiments the amiRNA comprises a five-nucleotide sequence from the naturally occurring miRNA from which it is derived. The five-nucleotide sequence may consist of the sequence nnddd (SEQ ID NO:33), wherein n is selected from adenine, guanine, uracil and cytosine and d is selected from guanine, adenine and uracil. In some embodiments, the five-nucleotide sequence consists of the sequence dnddd (SEQ ID NO:34). In some embodiments, the five-nucleotide sequence consists of the sequence dndwd (SEQ ID NO:35). In other embodiments, the five-nucleotide sequence consists of the sequence dnwdd (SEQ ID NO:36). In yet other embodiments, the five-nucleotide sequence consists of the sequence dnwwd (SEQ ID NO:37), dndww (SEQ ID NO:38) or dnwdw (SEQ ID NO:39). The five-nucleotide sequence may consist of the sequence dnwww (SEQ ID NO:40). In some embodiments, the five-nucleotide sequence consists of the sequence guagu (SEQ ID NO:41). Alternatively, in some embodiments the amiRNA does not comprise any nucleotides from the naturally occurring miRNA from which it is derived. In such embodiments, it will be appreciated that the artificial microRNA (amiRNA) coding region comprises a polynucleotide encoding a pri-amiRNA, and so while the amiRNA does not comprise any nucleotides from the naturally occurring miRNA from which it is derived, the pri-amiRNA comprises nucleotides from the naturally occurring pri-miRNA from which it is derived. The amiRNA may be no more than about 40%, no more than about 35%, no more than about 30%, no more than about 25%, no more than about 24%, no more than about 23%, no more than about 22%, no more than about 21%, no more than about 20%, no more than about 19%, no more than about 18%, no more than about 17%, no more than about 16%, no more than about 15%, no more than about 14%, no more than about 13%, no more than about 12%, no more than about 11%, no more than about 10%, no more than about 9%, no more than about 8%, no more than about 7%, no more than about 6% or no more than about 5% homologous to the naturally occurring miRNA from which it is derived on nucleotide identity. In some embodiments the amiRNA is no more than about 25% homologous to the naturally occurring miRNA from which it is derived on nucleotide identity. In some embodiments, the amiRNA is no more than about 22%, optionally no more than about 20% homologous to the naturally occurring miRNA from which it is derived on nucleotide identity. Homology based on sequence identity and identity are interchangeable herein. This allows for variation, deletion, addition, or a combination thereof of nucleotides within the amiRNA. When referring to homology of the amiRNA to the naturally occurring miRNA, it will be appreciated that this refers to homology of the amiRNA to the final mature naturally occurring miRNA sequence, not to the precursor naturally occurring pre-miRNA or pri- miRNA sequences. For the amiRNA, homology based on sequence identity is measured over the entire length of the naturally occurring miRNA from which the amiRNA is derived. This may also be referred to as global homology based on sequence identity or global sequence identity. In embodiments comprising pri-amiRNAs, which are discussed further below, homology based on sequence identity is measured over the entire length of the naturally occurring pri- miRNA from which the pri-amiRNA is derived. In embodiments comprising pri-amiRNAs, the pri-amiRNA may have at least about 75% homology or at least about 80% homology to the naturally occurring pri-miRNA from which it is derived. In some embodiments, the pri- amiRNA may have at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94% or at least about 95% homology to the naturally occurring pri-miRNA from which it is derived. In some embodiments, the pri- amiRNA is no more than about 99%, no more than about 98%, no more than about 97%, no more than about 96% or no more than about 95% homologous to the naturally occurring pri-miRNA from which it is derived. In some embodiments, the pri-amiRNA has of from about 85% to about 95% homology to the naturally occurring pri-miRNA from which it is derived. Methods of measuring homology based on sequence identity, including global homology based on sequence identity or global sequence identity, are known in the art. For example, the UWGCG Package provides the BESTFIT program which can be used to calculate homology or identity (e.g., used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can also be used to calculate identity, homology, or line up sequences (typically on their default settings), for example as described in Altschul S.F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. In embodiments comprising a plurality of amiRNAs, the plurality of amiRNAs may be derived from a miRNA cluster. In some embodiments, the plurality of amiRNAs comprises amiRNAs derived from a miRNA cluster. The plurality of amiRNAs may further comprise at least one amiRNA derived from a miRNA which may not be associated with a miRNA cluster. An “miRNA cluster” is a defined unit known in the art which comprises two or more miRNAs known to be transcribed from adjacent polynucleotides in an organism, for example a human, from a single promoter. The miRNAs in a miRNA cluster are typically transcribed together in the same orientation and are not separated from each other by a transcription unit. Most miRNA clusters comprise two to eight miRNAs. Various suitable miRNA clusters will be known to the skilled person. In the context of the present invention, “derived from a miRNA cluster” will be understood to mean that the sequence of amiRNAs has been generated by modification of a sequence of the naturally occurring miRNA cluster. It will therefore be appreciated that in the context of the present invention, the phrase “derived from a miRNA cluster” refers to a genetically modified miRNA cluster. Therefore, “derived from a miRNA cluster” means recombinant and non-naturally occurring. In some embodiments, amiRNAs derived from a miRNA cluster are generated by substituting one or more of a pri-miRNA 5’ stem and 3’ stem sequences with sequences specific for a target site. In some embodiments, amiRNAs derived from a miRNA cluster are generated by substituting one or more of a pri-miRNA 5’ stem and 3’ stem sequences or a portion thereof with sequences specific for a target site. In some embodiments, amiRNAs derived from a miRNA cluster comprise a miRNA cluster modified to remove at least one miRNA from the cluster and further modified to substitute one or more of a pri-miRNA 5’ stem and 3’ stem sequences with sequences specific for a target site. In some embodiments, amiRNAs derived from a miRNA cluster are generated by modifying all the pri-miRNA 5’ stem and 3’ stem sequences within the miRNA cluster. In some embodiments, amiRNAs derived from a miRNA cluster are generated by modifying some but not all of the pri-miRNA 5’ stem and 3’ stem sequences within the miRNA cluster. In some embodiments, amiRNAs derived from a miRNA cluster comprise a miRNA cluster modified to reorder the miRNA in the cluster and further modified to substitute one or more of a pri-miRNA 5’ stem and 3’ stem sequences with sequences specific for a target site. In some embodiments, amiRNAs derived from a miRNA cluster comprise at least one miRNA from one miRNA cluster linked to at least one miRNA from a second, different miRNA cluster. In such embodiments it will be appreciated that at least one of a pri-miRNA 5’ stem and 3’ stem sequences will be substituted with sequences specific for a target site. amiRNAs derived from a miRNA cluster may comprise at least two miRNA derived from one miRNA cluster linked to at least one miRNA derived from a second, different miRNA cluster. In some embodiments amiRNAs derived from a miRNA cluster comprise at least two miRNA derived from one miRNA cluster linked to at least two miRNA derived from a second, different miRNA cluster. In some embodiments, amiRNAs are not derived from a naturally occurring miRNA cluster. In some embodiments, amiRNAs are derived from an miRNA cluster, which is not naturally occurring. In some cases, the miRNA cluster is generated by linking a plurality of miRNAs, wherein each of the miRNAs is not from a naturally occurring miRNA cluster. In some cases, the miRNA cluster is generated by linking one or more miRNAs, each from a naturally occurring miRNA cluster, and one or more miRNAs, each not from a naturally occurring miRNA cluster. amiRNAs derived from a miRNA cluster may comprise at least three miRNA derived from one miRNA cluster linked to at least one miRNA derived from a second, different miRNA cluster. In some embodiments amiRNAs derived from a miRNA cluster comprise at least three miRNA derived from one miRNA cluster linked to at least two miRNA derived from a second, different miRNA cluster. In some embodiments amiRNAs derived from a miRNA cluster comprise at least three miRNA derived from one miRNA cluster linked to at least three miRNA derived from a second, different miRNA cluster. amiRNAs derived from a miRNA cluster may comprise at least four miRNA derived from one miRNA cluster linked to at least one miRNA derived from a second, different miRNA cluster. In some embodiments amiRNAs derived from a miRNA cluster comprise at least four miRNA derived from one miRNA cluster linked to at least two miRNA derived from a second, different miRNA cluster. amiRNAs derived from a miRNA cluster may comprise at least four miRNA derived from one miRNA cluster linked to at least three miRNA derived from a second, different miRNA cluster. In some embodiments amiRNAs derived from a miRNA cluster comprise at least four miRNA derived from one miRNA cluster linked to at least four miRNA derived from a second, different miRNA cluster. amiRNAs derived from a miRNA cluster may comprise at least five miRNA derived from one miRNA cluster linked to at least one miRNA derived from a second, different miRNA cluster. In some embodiments amiRNAs derived from a miRNA cluster comprise at least five miRNA derived from one miRNA cluster linked to at least two miRNA derived from a second, different miRNA cluster. amiRNAs derived from a miRNA cluster may comprise at least five miRNA derived from one miRNA cluster linked to at least three miRNA derived from a second, different miRNA cluster. In some embodiments amiRNAs derived from a miRNA cluster comprise at least five miRNA derived from one miRNA cluster linked to at least four miRNA derived from a second, different miRNA cluster. In some embodiments amiRNAs derived from a miRNA cluster comprise at least five miRNA derived from one miRNA cluster linked to at least five miRNA derived from a second, different miRNA cluster. In some embodiments amiRNAs derived from a miRNA cluster comprise at least six miRNA derived from one miRNA cluster linked to at least one miRNA derived from a second, different miRNA cluster. In some embodiments amiRNAs derived from a miRNA cluster comprise at least six miRNA derived from one miRNA cluster linked to at least two miRNA derived from a second, different miRNA cluster. In some embodiments amiRNAs derived from a miRNA cluster comprise at least six miRNA derived from one miRNA cluster linked to at least three miRNA derived from a second, different miRNA cluster. In some embodiments amiRNAs derived from a miRNA cluster comprise at least six miRNA derived from one miRNA cluster linked to at least four miRNA derived from a second, different miRNA cluster. Without wishing to be bound by theory, the present inventors believe that the modification of a naturally occurring miRNA cluster, as described above, may improve the efficacy of RNA silencing by the cluster. The plurality of amiRNAs may have no more than about 75%, no more than about 74%, no more than about 73%, no more than about 72%, no more than about 71%, no more than about 70%, no more than about 69%, no more than about 68%, no more than about 67%, no more than about 66%, no more than about 65%, no more than about 64%, no more than about 63%, no more than about 62%, no more than about 61%, or no more than about 60% identity to the naturally occurring miRNA cluster. In some embodiments, the plurality of amiRNAs has at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58% or at least about 59% identity to the naturally occurring miRNA cluster. Preferably, the plurality of amiRNAs has of from about 55% to about 75% identity to the naturally occurring miRNA cluster. More preferably, the plurality of amiRNAs has of from about 60% to about 75% identity to the naturally occurring miRNA cluster. Most preferably, the plurality of amiRNAs has of from about 65% to about 71% identity to the naturally occurring miRNA cluster. The miRNA cluster may be a miR-17-92 or miR-106a-363 cluster. The miR-17-92 cluster typically comprises SEQ ID NO:42. The miR-106a-363 cluster may comprise SEQ ID NO:43. Alternatively, the miRNA cluster may be any of the miRNA clusters A-T specified in Table 1, below. It will be appreciated that each miRNA cluster in Table 1 comprises the miRNAs in the same row. For example, miRNA cluster A comprises miRNAs miR-101-1 and miR-3671, while miRNA cluster B comprises miRNAs miR-29b-2 and miR-29c.

Table 1: Table adapted from Weber MJ, (2005), New human and mouse microRNA gene found by homology search FEBS J, 272(1); 59-73. The miR-17-92 cluster contains the miRNAs miR-17, miR-18a, miR-19a, miR-20a, miR-19b- 1 and miR-92a-1. The naturally occurring miRNA miR-17 typically comprises SEQ ID NO:44, while the naturally occurring miR-18a typically comprises SEQ ID NO:45. The naturally occurring miRNA miR-19a typically comprises SEQ ID NO:46. The naturally occurring miRNA miR-20a typically comprises SEQ ID NO:47, while the naturally occurring miRNA miR-92a-1 typically comprises SEQ ID NO:48. The miR-106a-363 cluster contains the miRNAs miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2 and miR-363. The naturally occurring miRNA miR-106a typically comprises SEQ ID NO:49. The naturally occurring miRNA miR-18b typically comprises SEQ ID NO:50. The naturally occurring miRNA miR-20b typically comprises SEQ ID NO:51, while the naturally occurring miRNA miR-19b-2 typically comprises SEQ ID NO:52. The naturally occurring miRNA miR-92a-2 typically comprises SEQ ID NO:53, while the naturally occurring miRNA miR-363 typically comprises SEQ ID NO:54. In some embodiments, the amiRNA is derived from miR-155, miR-17, miR-18a, miR-19a, miR-19b, miR-20a and/or miR-92a-1. The amiRNA may be derived from miR-17, miR-18a, miR-19a, miR-19b, miR-20a and/or miR-92a-1. In some embodiments the amiRNA is derived from miR-155. The naturally occurring miRNA miR-155 typically comprises SEQ ID NO:55. In some embodiments the amiRNA is derived from miR-17. In some embodiments the amiRNA is derived from miR-18a. In some embodiments the amiRNA is derived from miR-19a. In some embodiments the amiRNA is derived from miR-19b. In some embodiments the amiRNA is derived from miR-20a. In some embodiments the amiRNA is derived from miR-92a-1. As defined above, “derived from” in the context of a miRNA will be understood to mean that the miRNA has been genetically engineered to form the amiRNA, preferably wherein the amiRNA retains at least one nucleotide from the original miRNA. Alternatively, in some embodiments the amiRNA does not comprise any nucleotides from the naturally occurring miRNA from which it is derived. In such embodiments, it will be appreciated that the artificial microRNA (amiRNA) coding region comprises a polynucleotide encoding a pri-amiRNA, and so while the amiRNA does not comprise any nucleotides from the naturally occurring miRNA from which it is derived, the pri-amiRNA comprise nucleotides from the naturally occurring pri-miRNA from which it is derived. The amiRNA (or each amiRNA, in a plurality of amiRNAs), may comprise at least two, at least three, at least four or at least five nucleotides from miR-155, miR-17, miR-18a, miR- 19a, miR-19b, miR-20a and/or miR-92a-1. Preferably, the amiRNA comprises an at least two, at least three or at least four nucleotide sequence from miR-155, miR-17, miR-18, miR-19a, miR-19b, miR-20a and/or miR-92a-1. More preferably, the amiRNA comprises an at least two, at least three or at least four nucleotide sequence from miR-155, miR-17, miR- 18a, miR-19a, miR-19b, miR-20a and/or miR-92a-1 at the 3’ terminus of the amiRNA. In some embodiments, the amiRNA comprises no more than six, five, four, three or two nucleotides from miR-155, miR-17, miR-18a, miR-19a, miR-19b, miR-20a and/or miR-92a- 1. In some embodiments, the amiRNA comprises a nucleotide sequence of no more than five, no more than four, no more than three or no more than two nucleotides in length from miR-155, miR-17, miR-18a, miR-19a, miR-19b, miR-20a and/or miR-92a-1. In other embodiments the amiRNA is derived from miR-106a, miR-18b, miR-20b, miR-19b- 2, miR-92a-2 and/or miR-363. In some embodiments the amiRNA is derived from miR- 106a. In some embodiments the amiRNA is derived from miR-18b. In some embodiments the amiRNA is derived from miR-20b. In some embodiments the amiRNA is derived from miR-19b-2. In some embodiments the amiRNA is derived from miR-92a-2. In some embodiments the amiRNA is derived from miR-363.The amiRNA (or each amiRNA, in a plurality of amiRNAs), may comprise at least two, at least three, at least four or at least five nucleotides from miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2 and/or miR-363. Preferably, the amiRNA comprises an at least two, at least three or at least four nucleotide sequence from miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2 and/or miR-363. More preferably, the amiRNA comprises an at least two, at least three or at least four nucleotide sequence from miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2 and/or miR-363 at the 3’ terminus of the amiRNA. In some embodiments, the amiRNA comprises no more than six, five, four, three or two nucleotides from miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2 and/or miR-363. In some embodiments, the amiRNA comprises a nucleotide sequence of no more than five, no more than four, no more than three or no more than two nucleotides in length from miR- 106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2 and/or miR-363. In some embodiments, the amiRNA is derived from miR-30a, miR-30b, miR-30c-1, miR- 30c-2, miR-30d and/or miR-30e. In some embodiments the amiRNA is derived from miR- 30a. In some embodiments the amiRNA is derived from miR-30b. In some embodiments the amiRNA is derived from miR-30c-1. In some embodiments the amiRNA is derived from miR- 30c-2. In some embodiments the amiRNA is derived from miR-30d. In some embodiments the amiRNA is derived from miR-30e. The naturally occurring miRNA miR-30a typically comprises SEQ ID NO:220. The naturally occurring miRNA miR-30b typically comprises SEQ ID NO:221. The naturally occurring miRNA-30c-1 typically comprises SEQ ID NO:222. The naturally occurring miRNA-30c-2 typically comprises SEQ ID NO:223. The naturally occurring miR-30d typically comprises SEQ ID NO:224, while the naturally occurring miRNA miR-30e typically comprises SEQ ID NO:225. The amiRNA (or each amiRNA, in a plurality of amiRNAs), may comprise at least two, at least three, at least four or at least five nucleotides from miR-30a, miR-30b, miR-30c-1, miR-30c-2, miR-30d and/or miR-30e. Preferably, the amiRNA comprises an at least two, at least three or at least four nucleotide sequence from miR-30a, miR-30b, miR-30c-1, miR- 30c-2, miR-30d and/or miR-30e. More preferably, the amiRNA comprises an at least two, at least three or at least four nucleotide sequence from miR-30a, miR-30b, miR-30c-1, miR- 30c-2, miR-30d and/or miR-30e at the 3’ terminus of the amiRNA. In some embodiments, the amiRNA is derived from any miRNA provided in Table 1 above. The amiRNA (or each amiRNA, in a plurality of amiRNAs), may comprise at least 1, at least two, at least three, at least four or at least five nucleotides from any miRNA provided in Table 1 above. Preferably, the amiRNA comprises an at least two, at least three or at least four nucleotide sequence from any of the miRNAs provided in Table 1. More preferably, the amiRNA comprises an at least two, at least three or at least four nucleotide sequence from any of the miRNAs provided in Table 1 at the 3’ terminus of the amiRNA. In some embodiments, the amiRNA comprises no more than six, five, four, three or two nucleotides from any of the miRNAs provided in Table 1. In some embodiments, the amiRNA comprises a nucleotide sequence of no more than five, no more than four, no more than three or no more than two nucleotides in length from any of the miRNAs provided in Table 1. In some embodiments, the plurality of amiRNAs is derived from one or more of miR-155, miR-17, miR-18a, miR-19a, miR-19b, miR-20a and miR-92a-1. Thus, the plurality of polynucleotides may each encode an amiRNA derived from one or more of miR-155, miR- 17, miR-18a, miR-19a, miR-19b, miR-20a and miR-92a-1. The plurality of amiRNAs may be derived from one or more of the miRNAs miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1. In some embodiments, the plurality of amiRNAs may be derived from miR- 17, miR-18a, miR-19a, miR-20a and miR-19b-1. The plurality of amiRNAs may be derived from miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1. In some embodiments, the plurality of amiRNAs is derived from miR-19b and at least one other miRNA. The plurality of amiRNAs may be derived from miR-19b and at least one other miRNA selected from miR-17, miR-18a, miR-19a, miR-20a and miR-92a-1. In some embodiments, the plurality of amiRNAs is derived from miR-17 and at least one other miRNA. In some embodiments, the plurality of amiRNAs is derived from miR-17 and miR-19b. In some embodiments the plurality of amiRNAs is derived from miR-17, miR-19b and miR-20a. In some embodiments the plurality of amiRNAs is derived from miR-19b and miR-20a. In some embodiments the plurality of amiRNAs is derived from miR-17 and miR- 20a. In some embodiments the plurality of amiRNAs is derived from miR-17 and at least one other miRNA. The at least one other miRNA may not be from the miR-17-92 cluster. In some embodiments the at least one other miRNA may be from a different miRNA cluster (i.e a cluster which is not the miR-17-92 cluster). In some embodiments the plurality of amiRNAs is derived from miR-17, miR-19b and at least one other miRNA. In some embodiments the plurality of amiRNAs is derived from miR-17, miR-19b, miR-20a and at least one other miRNA. In some embodiments the plurality of amiRNAs is derived from miR- 19b, miR-20a and at least one other miRNA. In some embodiments the plurality of amiRNAs is derived from miR-17, miR-20a and at least one other miRNA. In some embodiments, the plurality of amiRNAs comprises from 5’ to 3’ an amiRNA derived from miR-17, an amiRNA derived from miR-18a, an amiRNA derived from miR-19a, and an amiRNA derived from miR-19b-1. In some embodiments, the plurality of amiRNAs comprises from 5’ to 3’ an amiRNA derived from miR-17, an amiRNA derived from miR-18a, an amiRNA derived from miR-19a, an amiRNA derived from miR-19b-1 and an amiRNA derived from miR-92a-1. The plurality of amiRNAs may comprise an amiRNA derived from miR-19b-1. In some embodiments, the plurality of amiRNAs comprises from 5’ to 3’, an amiRNA not derived from the miR-17-92 cluster and an amiRNA derived from miR-17. The amiRNA not derived from the miR-17-92 cluster may be derived from a miRNA cluster which is not the miR-17-92 cluster. In other embodiments, the plurality of amiRNAs comprises from 5’ to 3’, an amiRNA derived from miR-17 and an amiRNA not derived from the miR-17-92 cluster. The plurality of amiRNAs may comprise, from 5’ to 3’, an amiRNA derived from miR-17 and an amiRNA derived from miR-19b. In some embodiments the plurality of amiRNAs comprises, from 5’ to 3’, an amiRNA derived from miR-17, an amiRNA derived from miR- 19b and an amiRNA derived from miR-20a. In some embodiments the plurality of amiRNAs comprises, from 5’ to 3’, an amiRNA derived from miR-19b and an amiRNA derived from miR-20a. In some embodiments the plurality of amiRNAs comprises, from 5’ to 3’, an amiRNA derived from miR-17 and an amiRNA derived from miR-20a. The plurality of amiRNAs may comprise from 5’ to 3’ an amiRNA derived from miR-17, an amiRNA derived from miR-19b and at least one amiRNA not derived from the miR-17-92 cluster. In some embodiments the plurality of amiRNAs comprises, from 5’ to 3’, an amiRNA derived from miR-17, an amiRNA derived from miR-19b, an amiRNA derived from miR-20a and at least one amiRNA not derived from the miR-17-92 cluster. In some embodiments the plurality of amiRNAs comprises, from 5’ to 3’, an amiRNA derived from miR-19b, an amiRNA derived from miR-20a and at least one amiRNA not derived from the miR-17-92 cluster. In some embodiments the plurality of amiRNAs comprises, from 5’ to 3’, an amiRNA derived from miR-17, an amiRNA derived from miR-20a and at least one amiRNA not derived from the miR-17-92 cluster. The plurality of amiRNAs may comprise from 5’ to 3’ at least one amiRNA not derived from the miR-17-92 cluster, an amiRNA derived from miR-17 and an amiRNA derived from miR- 19b. In some embodiments the plurality of amiRNAs comprises, from 5’ to 3’, at least one amiRNA not derived from the miR-17-92 cluster, an amiRNA derived from miR-17, an amiRNA derived from miR-19b and an amiRNA derived from miR-20a. In some embodiments the plurality of amiRNAs comprises, from 5’ to 3’, at least one amiRNA not derived from the miR-17-92 cluster, an amiRNA derived from miR-19b and an amiRNA derived from miR-20a. In some embodiments the plurality of amiRNAs comprises, from 5’ to 3’, at least one amiRNA not derived from the miR-17-92 cluster, an amiRNA derived from miR-17 and an amiRNA derived from miR-20a. The plurality of amiRNAs may be derived from one or more of the miRNAs miR-106a, miR- 18b, miR-20b, miR-19b-2, miR-92a-2 and miR-363. For example, the plurality of amiRNAs may be derived from miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2 and miR-363. In some embodiments, the plurality of amiRNAs is derived from miR-106a, miR-18b, miR-20b, miR-19b-2 and miR-92a-2. Thus, the plurality of polynucleotides may each encode an amiRNA derived from one or more of the miRNAs miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2 and miR-363. In some embodiments, the plurality of amiRNAs is derived from miR-106a and at least one other miRNA. The plurality of amiRNAs may be derived from miR-106a and at least one other miRNA selected from miR-18b, miR-20b, miR-19b-2, miR-92a-2 and miR-363. In some embodiments the plurality of amiRNAs is derived from miR-106a and at least one other miRNA. The at least one other miRNA may be from a different miRNA cluster (i.e a cluster which is not the miR-106a-363 cluster). In some embodiments, the plurality of amiRNAs comprises from 5’ to 3’ an amiRNA derived from miR-106a, an amiRNA derived from miR-18b, an amiRNA derived from miR-20b, an amiRNA derived from miR-19b-2 and an amiRNA derived from miR-92a-2. In some embodiments, the plurality of amiRNAs comprises from 5’ to 3’ an amiRNA derived from miR-106a, an amiRNA derived from miR-18b, an amiRNA derived from miR-20b, an amiRNA derived from miR-19b-2, an amiRNA derived from miR-92a-2 and an amiRNA derived from miR-363. In some embodiments, the plurality of amiRNAs comprises from 5’ to 3’ an amiRNA derived from miR-92a-2, an amiRNA derived from miR-19b-2, an amiRNA derived from miR-20b, an amiRNA derived from miR-18b and an amiRNA derived from miR-106a. In some embodiments, the plurality of amiRNAs comprises from 5’ to 3’ an amiRNA derived from miR-363, amiRNA derived from miR-92a-2, an amiRNA derived from miR-19b-2, an amiRNA derived from miR-20b, an amiRNA derived from miR-18b and an amiRNA derived from miR- 106a. In some embodiments, the plurality of amiRNAs comprises from 5’ to 3’, an amiRNA not derived from the miR-106a-363 cluster and an amiRNA derived from miR-106a. In other embodiments, the plurality of amiRNAs comprises from 5’ to 3’, an amiRNA derived from miR-106a and an amiRNA not derived from the miR-106a-363 cluster. The plurality of amiRNAs may be derived from one or more of the miRNAs miR-30a, miR- 30b, miR-30c-1, miR-30c-2, miR-30d and miR-30e. For example, the plurality of amiRNAs may be derived from miR-30a, miR-30b, miR-30c-1, miR-30c-2, miR-30d and miR-30e. Thus, the plurality of polynucleotides may each encode an amiRNA derived from one or more of the miRNAs miR-30a, miR-30b, miR-30c-1, miR-30c-2, miR-30d and miR-30e. In some embodiments, the plurality of amiRNAs is derived from miR-30a and at least one other miRNA. The plurality of amiRNAs may be derived from miR-30a and at least one other miRNA selected from miR-30b, miR-30c-1, miR-30c-2, miR-30d and miR-30e. In some embodiments the plurality of amiRNAs comprises at least one amiRNA derived from miR-30a and at least one other amiRNA. The at least one other amiRNA may be derived from a miRNA cluster. The at least one other amiRNA may be derived from the miR-106a- 363 cluster or the miR-17-92 cluster. In some embodiments, the plurality of amiRNAs comprises from 5’ to 3’ an amiRNA derived from miR-30a, an amiRNA derived from miR-30b, an amiRNA derived from miR-30c-1, an amiRNA derived from miR-30c-2 and an amiRNA derived from miR-30d. In some embodiments, the plurality of amiRNAs comprises from 5’ to 3’ an amiRNA derived from miR-30a, an amiRNA derived from miR-30b, an amiRNA derived from miR-30c-1, an amiRNA derived from miR-30c-2, an amiRNA derived from miR-30d and an amiRNA derived from miR-30e. In some embodiments, the plurality of amiRNAs comprises from 5’ to 3’, an amiRNA derived from a miRNA which is not miR30a and an amiRNA derived from miR-30a. In other embodiments, the plurality of amiRNAs comprises from 5’ to 3’, an amiRNA derived from miR-30a and an amiRNA derived from a miRNA which is not miR30a. In some embodiments, the plurality of amiRNAs comprises an amiRNA derived from miR-17 and an amiRNA derived from miR-30a. In some embodiments, the plurality of amiRNAs comprises an amiRNA derived from miR-17, an amiRNA derived from miR-19b and an amiRNA derived from miR-30a. In some embodiments, the plurality of amiRNAs comprises an amiRNA derived from miR-17, an amiRNA derived from miR-19b, an amiRNA derived from miR-20a and an amiRNA derived from miR-30a. In some embodiments, the plurality of amiRNAs comprises an amiRNA derived from miR-19b, an amiRNA derived from miR-20a, and an amiRNA derived from miR-30a. In some embodiments the plurality of amiRNAs comprises an amiRNA derived from miR-17, an amiRNA derived from miR-20a and an amiRNA derived from miR-30a. The plurality of amiRNAs may comprise, from 5’ to 3’, an amiRNA derived from miR-30a and an amiRNA derived from miR-17. Alternatively, the plurality of amiRNAs may comprise, from 5’ to 3’, an amiRNA derived from miR-17 and an amiRNA derived from miR-30a. In some embodiments, the plurality of amiRNAs comprises, from 5’ to 3’, an amiRNA derived from miR-30a, an amiRNA derived from miR-17 and an amiRNA derived from miR- 19b. In some embodiments, the plurality of amiRNAs comprises, from 5’ to 3’, an amiRNA derived from miR-30a, an amiRNA derived from miR-17, an amiRNA derived from miR-19b and an amiRNA derived from miR-20a. In some embodiments, the plurality of amiRNAs comprises at least six amiRNAs, wherein at least one of the amiRNAs is derived from miR-30a. In some embodiments, the plurality of amiRNAs comprises at least six amiRNAs, wherein at least one of the amiRNAs is derived from miR-17. In some embodiments, the plurality of amiRNAs comprises at least six amiRNAs, wherein at least one of the amiRNAs is derived from miR-19b. In some embodiments, the plurality of amiRNAs comprises at least six amiRNAs, wherein at least one of the amiRNAs is derived from miR-20a. In some embodiments, the plurality of amiRNAs comprises at least six amiRNAs, wherein one of the amiRNAs is derived from miR-30a and another amiRNA is derived from miR-17. In some embodiments, the plurality of amiRNAs comprises at least six amiRNAs, wherein one amiRNA is derived from miR-30a, one amiRNA is derived from miR-17 and one amiRNA is derived from miR-19b. In some embodiments, the plurality of amiRNAs comprises at least six amiRNAs, wherein one amiRNA is derived from miR-30a, one amiRNA is derived from miR-17, one amiRNA is derived from miR-19b and one amiRNA is derived from miR-20a. Pri-amiRNAs Preferably, the amiRNA coding region comprises a polynucleotide encoding a pri-amiRNA which comprises an amiRNA. Preferably, the pri-amiRNA forms a stem-loop structure comprising a 5’ strand, a loop region and a 3’ strand. The 5’ strand may otherwise be referred to herein as the 5’ stem sequence, and the 3’ strand as the 3’ stem sequence. As described above in relation to the amiRNA, the pri-amiRNA is a genetically engineered pri-miRNA that doesn’t appear in nature. As will be clear from the examples, the inventors have taken the sequences of naturally occurring pri-miRNAs and used them to create pri- amiRNA by embedding the 5’ or 3’ stem sequences of previously validated shRNAs or CRISPR guide RNAs specific for a target mRNA into the pri-miRNA. The inventors have also taken the sequences of naturally occurring pri-miRNAs and modified them to create amiRNA by embedding shRNAs generated by the inventors into the pri-miRNA. In some embodiments, the pri-miRNA comprises a substitution of a 5’ stem strand or a portion thereof or a 3’ stem strand or a portion thereof of a naturally occurring pri-miRNA with a sequence specific to a target. In some embodiments, the pri-amiRNA comprises (i) a substitution of a 5’ stem strand or a portion thereof of the naturally occurring pri-miRNA with a sequence specific to a target site, and (ii) a substitution of a 3’ stem strand or a portion thereof of the naturally occurring pri-miRNA with a sequence complementary to the sequence specific to the target site. In some embodiments, the 5’ stem strand and 3’ stem strand are modified to maintain a stem-loop structure of the naturally occurring pri-miRNA. In some embodiments, the 5’ stem strand and the 3’ stem strand are completely complementary to each other. In some embodiments, the 5’ stem strand and the 3’ stem strand have one or more mismatch. In some embodiments, the pri-miRNA and the naturally occurring pri-miRNA have a same sequence except the substitution within the 5’ stem strand and/or the 3’ stem strand. In some embodiments, the pri-miRNA and the naturally occurring pri-miRNA have an identical sequence in the loop region. In some embodiments, the pri-miRNA and the naturally occurring pri-miRNA have an identical sequence in the 5’ flanking sequence. In some embodiments, the pri-miRNA and the naturally occurring pri-miRNA have an identical sequence in the 3’ flanking sequence. In some embodiments, the pri-miRNA and the naturally occurring pri-miRNA have one, two, three, four, five, or more nucleotide difference in the loop region. In some embodiments, the pri-miRNA and the naturally occurring pri- miRNA have one, two, three, four, five, or more nucleotide difference in the 5’ flanking sequence. In some embodiments, the pri-miRNA and the naturally occurring pri-miRNA have one, two, three, four, five, or more nucleotide difference in the 3’ flanking sequence. The pri-amiRNA may have at least about 75% homology to the naturally occurring pri- miRNA from which it is derived. The pri-amiRNA may have at least about 80% homology to the naturally occurring pri miRNA from which it is derived. In some embodiments, the pri- amiRNA may have at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94% or at least about 95% homology to the naturally occurring pri-miRNA from which it is derived. In some embodiments, the pri-amiRNA is no more than about 99%, no more than about 98%, no more than about 97%, no more than about 96% or no more than about 95% homologous to the naturally occurring pri-miRNA from which it is derived. In some embodiments, the pri-amiRNA has of from about 85% to about 95% homology to the naturally occurring pri-miRNA from which it is derived. In some embodiments, the pri- amiRNA has of from about 75% to about 95% homology to the naturally occurring pri- miRNA from which it is derived. The pri-amiRNA may be no more than about 75%, no more than about 74%, no more than about 73%, no more than about 72%, no more than about 71%, no more than about 70%, no more than about 69%, no more than about 68%, no more than about 67%, no more than about 66%, no more than about 65%, no more than about 64%, no more than about 63%, no more than about 62%, no more than about 61%, or no more than about 60% homologous to the naturally occurring pri-miRNA from which it is derived on nucleotide identity. In some embodiments, the pri-amiRNA is at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58% or at least about 59% homologous to the naturally occurring pri-miRNA from which it is derived on nucleotide identity. Preferably, the pri-amiRNA has of from about 55% to about 75% identity to the naturally occurring pri-miRNA from which it is derived on nucleotide identity. More preferably, the pri-amiRNA has of from about 60% to about 75% identity to the naturally occurring pri-miRNA from which it is derived on nucleotide identity. Most preferably, the pri-amiRNA has of from about 65% to about 71% identity to the naturally occurring pri-miRNA from which it is derived on nucleotide identity. Stem-loop structures, which may otherwise be referred to as hairpin structures, are well known to the skilled person. Typically, stem-loop structures are formed by a single stranded RNA. In the stem-loop structure, the 5’ stem and 3’ stem strands are generally substantially complementary to one another. This enables base pairing between the 5’ stem strand and 3’ stem strand to form the stem structure (Figure 1). Generally, the loop region is positioned between the 5’ stem strand and the 3’ stem strand. Hence, the pri-amiRNA preferably comprises, from 5’ to 3’, the 5’ stem strand, the loop region and the 3’ stem strand. Thus, a pri-amiRNA 5’ stem strand is 5’ to the pri-amiRNA loop region and a pri-amiRNA 3’ stem strand is 3’ to the pri-amiRNA loop region. In some embodiments the 5’ stem strand or 3’ stem strand (which may otherwise be referred to as the 5’ stem sequence and 3’ stem sequence, respectively), comprises a sequence mismatch with the opposing strand. Thus, in some embodiments the 5’ stem strand comprises a sequence mismatch with the 3’ stem strand. In other embodiments the 3’ stem strand comprises a sequence mismatch with the 5’ stem strand. By “sequence mismatch” this will be understood to mean at least one ribonucleotide of the 5’ stem strand or 3’ stem strand not being complementary to the corresponding ribonucleotide of the opposing strand. The sequence mismatch may comprise a point mutation, insertion or deletion. The inclusion of one or more sequence mismatches into the 5’ or 3’ stem strand may facilitate processing of the pri-amiRNA into the mature amiRNA. Without wishing to be bound by theory, the present inventors believe that a sequence mismatch may mimic structural bulges present in naturally occurring pri-miRNA strands. Preferably, the 5’ stem strand or 3’ stem strand comprises the amiRNA. In some embodiments, the 5’ stem strand comprises the amiRNA. In other embodiments the 3’ stem strand comprises the amiRNA. Preferably, the pri-amiRNA retains the naturally occurring pri-miRNA sequences flanking the 5’ stem strand and/or 3’ stem strand. Thus, in some embodiments, the pri-amiRNA comprises pri-miRNA flanking sequences. By “flanking sequences” these will be understood to refer to the nucleotide sequence 5’ to the 5’ stem strand and the nucleotide sequence 3’ to the 3’ stem strand in the naturally occurring pri-miRNA. Expression from these pri-miRNA unmodified flanking sequences can advantageously be driven by standard RNA polymerase II-dependent promoters, which can remove the need for alternative promoters to be included in the construct. A pri-miRNA flanking sequence may be at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides or at least about 80 nucleotides. The pri-miRNA flanking sequence may be no more than about 100 nucleotides, no more than about 90 nucleotides or no more than about 85 nucleotides. In some embodiments the pri-miRNA flanking sequence is of from about 50 nucleotides to about 100 nucleotides, optionally of from about 70 nucleotides to about 90 nucleotides. In some embodiments the pri-miRNA flanking sequence is of from about 80 nucleotides to about 86 nucleotides. Optionally, the pri-miRNA flanking sequence is 84 or 85 nucleotides. Exemplary flanking sequences include, but are not limited to SEQ ID NOs 56 and 57. In some embodiments, the pri-amiRNA comprises SEQ ID NO:56 as a 5’ flanking sequence and SEQ ID NO:57 as a 3’ flanking sequence. In some embodiments the pri-amiRNA is derived from pri-miR-155. In some embodiments the pri-amiRNA is derived from pri-miR-155 and the 5’ stem strand comprises the amiRNA. “Derived from a pri-miRNA” will be understood to mean that the pri-miRNA has been genetically engineered to form the pri-amiRNA. In some embodiments, the pri-amiRNA is derived from pri-miR-155, pri-miR-17, pri-miR- 18a, pri-miR-19a, pri-miR-19b, pri-miR-20a and/or pri-miR-92a-1. Pri-miR-155 typically comprises SEQ ID NO:58, while Pri-miR-17 typically comprises SEQ ID NO:59. Pri-miR-18a typically comprises SEQ ID NO:60. Pri-miR-19a typically comprises SEQ ID NO:61, while pri-miR-19b typically comprises SEQ ID NO:62. In some embodiments Pri-miR-20a comprises SEQ ID NO:63 or SEQ ID NO:212. Pri-miR-20a typically comprises SEQ ID NO:63. In some embodiments Pri-miR-92a-1 comprises SEQ ID NO:64 or SEQ ID NO:213. Pri-miR-92a-1 typically comprises SEQ ID NO:64. The pri-amiRNA may be derived from pri-miR-17, pri-miR-18a, pri-miR-19a, pri-miR-19b, pri-miR-20a and/or pri-miR-92a-1. In some embodiments the pri-amiRNA is derived from pri-miR-155. In some embodiments the pri-amiRNA is derived from pri-miR-17. In some embodiments the pri-amiRNA is derived from pri-miR-18a. In some embodiments the pri- amiRNA is derived from pri-miR-19a. In some embodiments the pri-amiRNA is derived from pri-miR-19b. In some embodiments the pri-amiRNA is derived from pri-miR-20a. In some embodiments the pri-amiRNA is derived from pri-miR-92a-1. In other embodiments the pri-amiRNA is derived from pri-miR-106a, pri-miR-18b, pri-miR- 20b, pri-miR-19b-2, pri-miR-92a-2 and pri-miR-363. Pri-miR-106a typically comprises SEQ ID NO:65, while pri-miR-18b typically comprises SEQ ID NO:66. Pri-miR-20b typically comprises SEQ ID NO:67. Pri-miR-19b-2 typically comprises SEQ ID NO:68. Pri-miR-92a-2 typically comprises SEQ ID NO:69. Pri-miR-363 typically comprises SEQ ID NO:70. In other embodiments the pri-amiRNA is derived from pri-miR-106a, pri-miR-18b, pri-miR- 20b, pri-miR-19b-2, pri-miR-92a-2 or pri-miR-363. In some embodiments the pri-amiRNA is derived from pri-miR-106a. In some embodiments the pri-amiRNA is derived from pri-miR- 18b. In some embodiments the pri-amiRNA is derived from pri-miR-20b. In some embodiments the pri-amiRNA is derived from pri-miR-19b-2. In some embodiments the pri- amiRNA is derived from pri-miR-92a-2. In some embodiments the pri-amiRNA is derived from pri-miR-363. In some embodiments the pri-amiRNA is derived from pri-miR-30a, pri-miR-30b, pri-miR- 30c-1, pri-miR-30c-2, pri-miR-30d or pri-miR-30e. In some embodiments the pri-amiRNA is derived from pri-miR-30a. Pri-miR30a may comprise SEQ ID NO:226. In some embodiments the pri-amiRNA is derived from pri-miR-30b. Pri-miR30b may comprise SEQ ID NO:227. In some embodiments the pri-amiRNA is derived from pri-miR-30c-1. Pri-miR30c-1 may comprise SEQ ID NO:228. In some embodiments the pri-amiRNA is derived from pri-miR- 30c-2. Pri-miR30c-2 may comprise SEQ ID NO:229. In some embodiments the pri-amiRNA is derived from pri-miR-30d. Pri-miR30d may comprise SEQ ID NO:230. In some embodiments the pri-amiRNA is derived from pri-miR-30e. Pri-miR30e may comprise SEQ ID NO:231. The pri-amiRNA may comprise a pri-miR-155 (SEQ ID NO:71), pri-miR-17 (SEQ ID NO:72), pri-miR-18a (SEQ ID NO:73), pri-miR-19a (SEQ ID NO:74), pri-miR-19b (SEQ ID NO:75), pri-miR-20a (SEQ ID NO:76) or pri-miR-92a-1 (SEQ ID NO:77) loop region. In some embodiments, the pri-amiRNA comprises a pri-miR-155 loop region (SEQ ID NO:71). In some embodiments the pri-amiRNA comprises a pri-miR-17 loop region (SEQ ID NO:72). In some embodiments the pri-amiRNA comprises a pri-miR-18a loop region (SEQ ID NO:73). In some embodiments, the pri-amiRNA comprises a pri-miR-19a loop region (SEQ ID NO:74). In some embodiments, the pri-amiRNA comprises a pri-miR-19b loop region (SEQ ID NO:75). In some embodiments the pri-amiRNA comprises a pri-miR-20a loop region (SEQ ID NO:76). In some embodiments the pri-amiRNA comprises a pri-miR-92a-1 loop region (SEQ ID NO:77). The pri-amiRNA may comprise a 5’ stem strand which comprises the amiRNA, a pri-miR-155 loop region (SEQ ID NO:71) and a 3’ stem strand. In some embodiments, the pri-amiRNA comprises a 5’ stem strand which comprises the amiRNA, a pri-miR-19b loop region (SEQ ID NO:75) and a 3’ stem strand. In some embodiments the pri-amiRNA comprises a 5’ stem strand, a pri-miR-155 loop region (SEQ ID NO:71) and a 3’ stem strand which comprises the amiRNA. In some embodiments, the pri-amiRNA comprises a 5’ stem strand, a pri-miR-19b loop region (SEQ ID NO:75) and a 3’ stem strand which comprises the amiRNA. In some embodiments, the pri-amiRNA comprises a pri-miR-17 (SEQ ID NO:72), pri-miR- 18a (SEQ ID NO:73), pri-miR-19a (SEQ ID NO:74), pri-miR-19b (SEQ ID NO:75), pri-miR- 20a (SEQ ID NO:76) or pri-miR-92a-1 loop region (SEQ ID NO:77). In other embodiments, the pri-amiRNA comprises a pri-miR-106a (SEQ ID NO:78), pri-miR- 18b (SEQ ID NO:79), pri-miR-20b (SEQ ID NO:80), pri-miR-19b-2 (SEQ ID NO:81), pri- miR-92a-2 (SEQ ID NO:82) or pri-miR-363 loop region (SEQ ID NO:83). In some embodiments the pri-amiRNA comprises a pri-miR-106a loop region (SEQ ID NO:78). In some embodiments the pri-amiRNA comprises a pri-miR-18b loop region (SEQ ID NO:79). In some embodiments the pri-amiRNA comprises a pri-miR-20b loop region (SEQ ID NO:80). In some embodiments the pri-amiRNA comprises a pri-miR-19b-2 loop region (SEQ ID NO:81). In some embodiments the pri-amiRNA comprises a pri-miR-92a-2 loop region (SEQ ID NO:82). In some embodiments the pri-amiRNA comprises a pri-miR-363 loop region (SEQ ID NO:83). The pri-amiRNA may comprise a pri-miR-30a, pri-miR-30b, pri-miR-30c-1, pri-miR-30c-2, pri-miR-30d or pri-miR-30e loop region. In some embodiments, the pri-amiRNA comprises a pri-miR-30a loop region (SEQ ID NO:232). In some embodiments the pri-amiRNA comprises a pri-miR-30b loop region (SEQ ID NO:233). In some embodiments the pri-amiRNA comprises a pri-miR-30c-1 loop region (SEQ ID NO:234). In some embodiments, the pri- amiRNA comprises a pri-miR-30c-2 loop region (SEQ ID NO:235). In some embodiments, the pri-amiRNA comprises a pri-miR-30d loop region (SEQ ID NO:236). In some embodiments the pri-amiRNA comprises a pri-miR-30e loop region (SEQ ID NO:237). The pri-amiRNA may comprise a 5’ stem strand which comprises the amiRNA, a pri-miR-30a loop region and a 3’ stem strand. In some embodiments, the pri-amiRNA comprises a 5’ stem strand, a pri-miR-30a loop region and a 3’ stem strand which comprises the amiRNA. The pri-amiRNA may comprise a pri-miR-155, pri-miR-17, pri-miR-18a, pri-miR-19a, pri- miR-19b, pri-miR-20a and/or pri-miR-92a-1 flanking sequence. The pri-amiRNA may comprise a pri-miR-17, pri-miR-18a, pri-miR-19a, pri-miR-19b, pri-miR-20a and/or pri-miR- 92a-1 flanking sequence. In other embodiments the pri-amiRNA comprises a pri-miR-106a, pri-miR-18b, pri-miR-20b, pri-miR-19b-2, pri-miR-92a-2 and/or pri-miR-363 flanking sequence. In some embodiments the pri-amiRNA comprises a pri-miR-30a, pri-miR-30b, pri-miR-30c-1, pri-miR-30c-2, pri-miR-30d or pri-miR-30e flanking sequence. In some embodiments, the amiRNA coding region comprises a plurality of polynucleotides each encoding a pri-amiRNA. Thus, the amiRNA coding region may comprise a plurality of polynucleotides encoding a plurality of pri-amiRNAs. For example, the amiRNA coding region may comprise at least two, at least three, at least four, at least five or at least six polynucleotides each encoding a pri-amiRNA. The amiRNA coding region may comprise less than 10, less than nine, less than eight or less than seven polynucleotides each encoding an a pri-amiRNA. For example, the amiRNA coding region may comprise six polynucleotides each encoding a pri-amiRNA. In some embodiments the amiRNA coding region comprises seven polynucleotides each encoding a pri-amiRNA. In some embodiments the amiRNA coding region comprises eight polynucleotides each encoding a pri-amiRNA. A plurality of pri-amiRNAs may comprise at least two, at least three, at least four, at least five or at least six pri-amiRNAs. In some embodiments, a plurality of pri-amiRNAs may comprise less than 10, less than nine, less than eight or less than seven pri-amiRNAs. For example, the plurality of pri-amiRNAs may comprise six pri-amiRNAs. The plurality of pri-amiRNAs may comprise seven or eight pri-amiRNAs. Each pri-amiRNA of the plurality of pri-amiRNAs may be as defined above. For example, the plurality of pri-amiRNAs may be derived from a pri-miRNA cluster. The pri-miRNA cluster may be a pri-miR-17-92 or pri-miR-106a-363 cluster. In some embodiments, the plurality of pri-amiRNAs is derived from one or more of pri-miR- 155, pri-miR-17, pri-miR-18a, pri-miR-19a, pri-miR-19b, pri-miR-20a and pri-miR-92a-1. The plurality of pri-amiRNAs may be derived from one or more of the pri-miRNAs pri-miR- 17, pri-miR-18a, pri-miR-19a, pri-miR-20a, pri-miR-19b-1 and pri-miR-92a-1. In some embodiments, the plurality of pri-amiRNAs may be derived from pri-miR-17, pri-miR-18a, pri-miR-19a, pri-miR-20a and pri-miR-19b-1. The plurality of pri-amiRNAs may be derived from pri-miR-17, pri-miR-18a, pri-miR-19a, pri-miR-20a, pri-miR-19b-1 and pri-miR-92a-1. In some embodiments, the plurality of pri-amiRNAs is derived from pri-miR-17 and pri-miR- 19b. In some embodiments the plurality of pri-amiRNAs is derived from pri-miR-17, pri- miR-19b and pri-miR-20a. In some embodiments the plurality of pri-amiRNAs is derived from pri-miR-19b and pri-miR-20a. In some embodiments the plurality of pri-amiRNAs is derived from pri-miR-17 and pri-miR-20a. In some embodiments the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-17 and at least one other pri-amiRNA. The at least one other pri-amiRNA may not be derived from the pri-miR-17-92 cluster. In some embodiments, the at least one other pri-amiRNA may be derived from a different pri-miRNA cluster (i.e. which is not a pri-miR- 17-92 cluster). In some embodiments the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-17, a pri-amiRNA derived from pri-miR-19b and at least one other pri- amiRNA. In some embodiments the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-17, a pri-amiRNA derived from pri-miR-19b, a pri-amiRNA derived from pri-miR-20a and at least one other pri-amiRNA. In some embodiments the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-19b, a pri-amiRNA derived from pri-miR-20a and at least one other pri-amiRNA. In some embodiments the plurality of pri- amiRNAs comprises a pri-amiRNA derived from pri-miR-17, a pri-amiRNA derived from pri- miR-20a and at least one other pri-amiRNA. In some embodiments, the plurality of pri-amiRNAs comprises from 5’ to 3’ a pri-amiRNA derived from pri-miR-17, a pri-amiRNA derived from pri-miR-18a, a pri-amiRNA derived from pri-miR-19a, and a pri-amiRNA derived from pri-miR-19b-1. In some embodiments, the plurality of pri-amiRNAs comprises from 5’ to 3’ a pri-amiRNA derived from pri-miR-17, a pri-amiRNA derived from pri-miR-18a, a pri-amiRNA derived from pri-miR-19a, a pri- amiRNA derived from pri-miR-19b-1 and a pri-amiRNA derived from pri-miR-92a-1. The plurality of pri-amiRNAs may comprise a pri-amiRNA derived from pri-miR-19b-1. In some embodiments, the plurality of pri-amiRNAs comprises from 5’ to 3’, a pri-amiRNA not derived from the pri-miR-17-92 cluster and a pri-amiRNA derived from pri-miR-17. The pri-amiRNA not derived from the pri-miR-17-92 cluster may be derived from a pri-miRNA cluster which is not the pri-miR-17-92 cluster. In other embodiments, the plurality of pri- amiRNAs comprises from 5’ to 3’, a pri-amiRNA derived from pri-miR-17 and a pri-amiRNA not derived from the pri-miR-17-92 cluster. The plurality of pri-amiRNAs may comprise, from 5’ to 3’, a pri-amiRNA derived from pri- miR-17 and a pri-amiRNA derived from pri-miR-19b. In some embodiments the plurality of pri-amiRNAs comprises, from 5’ to 3’, a pri-amiRNA derived from pri-miR-17, a pri-amiRNA derived from pri-miR-19b and a pri-amiRNA derived from pri-miR-20a. In some embodiments the plurality of pri-amiRNAs comprises, from 5’ to 3’, a pri-amiRNA derived from pri-miR-19b and a pri-amiRNA derived from pri-miR-20a. In some embodiments the plurality of pri-amiRNAs comprises, from 5’ to 3’, a pri-amiRNA derived from pri-miR-17 and a pri-amiRNA derived from pri-miR-20a. The plurality of pri-amiRNAs may comprise from 5’ to 3’, a pri-amiRNA derived from pri- miR-17, a pri-amiRNA derived from pri-miR-19b and at least one pri-amiRNA not derived from the pri-miR-17-92 cluster. In some embodiments the plurality of pri-amiRNAs comprises, from 5’ to 3’, a pri-amiRNA derived from pri-miR-17, a pri-amiRNA derived from pri-miR-19b, a pri-amiRNA derived from pri-miR-20a and at least one pri-amiRNA not derived from the pri-miR-17-92 cluster. In some embodiments the plurality of pri-amiRNAs comprises, from 5’ to 3’, a pri-amiRNA derived from pri-miR-19b, a pri-amiRNA derived from pri-miR-20a and at least one pri-amiRNA not derived from the pri-miR-17-92 cluster. In some embodiments the plurality of pri-amiRNAs comprises, from 5’ to 3’, a pri-amiRNA derived from pri-miR-17, a pri-amiRNA derived from pri-miR-20a and at least one pri- amiRNA not derived from the pri-miR-17-92 cluster. The plurality of pri-amiRNAs may comprise from 5’ to 3’, at least one pri-amiRNA not derived from the pri-miR-17-92 cluster, a pri-amiRNA derived from pri-miR-17 and a pri- amiRNA derived from pri-miR-19b. In some embodiments the plurality of pri-amiRNAs comprises, from 5’ to 3’, at least one pri-amiRNA not derived from the pri-miR-17-92 cluster, a pri-amiRNA derived from pri-miR-17, a pri-amiRNA derived from pri-miR-19b and a pri-amiRNA derived from pri-miR-20a. In some embodiments the plurality of pri-amiRNAs comprises, from 5’ to 3’, at least one pri-amiRNA not derived from the pri-miR-17-92 cluster, a pri-amiRNA derived from pri-miR-19b and a pri-amiRNA derived from pri-miR- 20a. In some embodiments the plurality of pri-amiRNAs comprises, from 5’ to 3’, at least one pri-amiRNA not derived from the pri-miR-17-92 cluster, a pri-amiRNA derived from pri- miR-17 and a pri-amiRNA derived from pri-miR-20a. The plurality of pri-amiRNAs may be derived from one or more of pri-miR-106a, pri-miR- 18b, pri-miR-20b, pri-miR-19b-2, pri-miR-92a-2 and pri-miR-363. For example, the plurality of pri-amiRNAs may be derived from pri-miR-106a, pri-miR-18b, pri-miR-20b, pri-miR-19b- 2, pri-miR-92a-2 and pri-miR-363. In some embodiments, the plurality of pri-amiRNAs is derived from pri-miR-106a, pri-miR-18b, pri-miR-20b, pri-miR-19b-2 and pri-miR-92a-2. In some embodiments, the plurality of pri-amiRNAs is derived from pri-miR-106a and at least one other pri-miRNA. The plurality of pri-amiRNAs may be derived from pri-miR-106a and at least one other pri-miRNA selected from pri-miR-18b, pri-miR-20b, pri-miR-19b-2, pri-miR-92a-2 and pri-miR-363. The at least one other pri-miRNA may be from a different pri-miRNA cluster (i.e. a cluster which is not the miR-106a-363 cluster). In some embodiments, the plurality of pri-amiRNAs comprises from 5’ to 3’ a pri-amiRNA derived from pri-miR-106a, a pri-amiRNA derived from pri-miR-18b, a pri-amiRNA derived from pri-miR-20b, a pri-amiRNA derived from pri-miR-19b-2 and a pri-amiRNA derived from pri-miR-92a-2. In some embodiments, the plurality of pri-amiRNAs comprises from 5’ to 3’ a pri-amiRNA derived from pri-miR-106a, a pri-amiRNA derived from pri-miR-18b, a pri- amiRNA derived from pri-miR-20b, a pri-amiRNA derived from pri-miR-19b-2, a pri-amiRNA derived from pri-miR-92a-2 and a pri-amiRNA derived from pri-miR-363. For example, the plurality of pri-amiRNAs may be derived from two or more of pri-miR-155, pri-miR-17, pri-miR-18a, pri-miR-19a, pri-miR-19b, pri-miR-20a and pri-miR-92a-1. In some embodiments, the plurality of pri-amiRNAs is derived from two or more of pri-miR-17, pri-miR-18a, pri-miR-19a, pri-miR-19b, pri-miR-20a and pri-miR-92a-1. In some embodiments, the plurality of pri-amiRNAs is derived from pri-miR-17, pri-miR-18a, pri- miR-19a, pri-miR-19b, pri-miR-20a and pri-miR-92a-1. In some embodiments, the plurality of pri-amiRNAs is derived from pri-miR-19b and at least one other pri-miRNA. The plurality of pri-amiRNAs may be derived from pri-miR-19b and at least one other pri-miRNA selected from pri-miR-17, pri-miR-18a, pri-miR-19a, pri-miR-20a and pri-miR-92a-1. In other embodiments, the plurality of pri-amiRNAs is derived from two or more of pri-miR-106a, pri-miR-18b, pri-miR-20b, pri-miR-19b-2, pri-miR-92a-2 and pri-miR-363. In some embodiments, the plurality of pri-amiRNAs comprises from 5’ to 3’, a pri-amiRNA not derived from the pri-miR-106a-363 cluster and a pri-amiRNA derived from pri-miR- 106a. In other embodiments, the plurality of pri-amiRNAs comprises from 5’ to 3’, a pri- amiRNA derived from pri-miR-106a and a pri-amiRNA not derived from the pri-miR-106a- 363 cluster. The plurality of pri-amiRNAs may be derived from one or more of the pri-miRNAs pri-miR- 30a, pri-miR-30b, pri-miR-30c-1, pri-miR-30c-2, pri-miR-30d and pri-miR-30e. For example, the plurality of pri-amiRNAs may be derived from pri-miR-30a, pri-miR-30b, pri- miR-30c-1, pri-miR-30c-2, pri-miR-30d and pri-miR-30e. Thus, the plurality of polynucleotides may each encode a pri-amiRNA derived from one or more of the pri-miRNAs pri-miR-30a, pri-miR-30b, pri-miR-30c-1, pri-miR-30c-2, pri-miR-30d and pri-miR-30e. In some embodiments, the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-30a and at least one other pri-miRNA. The plurality of pri-amiRNAs may be derived from pri-miR-30a and at least one other pri-miRNA selected from pri-miR-30b, pri-miR-30c- 1, pri-miR-30c-2, pri-miR-30d and pri-miR-30e. In some embodiments, the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-30a and and a pri-amiRNA derived from pri-miR-30c-2. In some embodiments the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-30a, a pri-amiRNA derived from pri-miR-30c-2 and a pri-amiRNA derived from pri-miR-30d. In some embodiments the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR- 30c-2 and a pri-amiRNA derived from pri-miR-30d. In some embodiments the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-30a and a pri-amiRNA derived from pri-miR-30d. In some embodiments the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-30a and at least one other pri-amiRNA. The at least one other pri-amiRNA may be derived from a pri-miRNA cluster. The at least one other pri-amiRNA may be derived from the pri-miR-106a-363 cluster or the pri-miR-17-92 cluster. In some embodiments the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-30a, a pri-amiRNA derived from pri-miR-30c-2 and at least one other pri-amiRNA. In some embodiments the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-30a, a pri-amiRNA derived from pri-miR-30-c-2, a pri-amiRNA derived from pri-miR-30d and at least one other pri-amiRNA. In some embodiments the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-30-c-2, a pri-amiRNA derived from pri-miR-30d and at least one other pri-amiRNA. In some embodiments the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-30a, a pri-amiRNA derived from pri-miR-30d and at least one other pri-amiRNA. In some embodiments, the plurality of pri-amiRNAs comprises from 5’ to 3’ a pri-amiRNA derived from pri-miR-30a, a pri-amiRNA derived from pri-miR-30b, a pri-amiRNA derived from pri-miR-30c-1, a pri-amiRNA derived from pri-miR-30c-2 and a pri-amiRNA derived from pri-miR-30d. In some embodiments, the plurality of pri-amiRNAs comprises from 5’ to 3’ a pri-amiRNA derived from pri-miR-30a, a pri-amiRNA derived from pri-miR-30b, a pri- amiRNA derived from pri-miR-30c-1, a pri-amiRNA derived from pri-miR-30c-2, a pri- amiRNA derived from pri-miR-30d and a pri-amiRNA derived from pri-miR-30e. In some embodiments, the plurality of pri-amiRNAs comprises from 5’ to 3’, a pri-amiRNA derived from a pri-miRNA which is not pri-miR30a and a pri-amiRNA derived from pri-miR- 30a. In other embodiments, the plurality of pri-amiRNAs comprises from 5’ to 3’, a pri- amiRNA derived from pri-miR-30a and a pri-amiRNA derived from a pri-miRNA which is not pri-miR30a. In some embodiments, the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-17 and a pri-amiRNA derived from pri-miR-30a. In some embodiments, the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-17, a pri-amiRNA derived from pri-miR-19b and a pri-amiRNA derived from pri-miR-30a. In some embodiments, the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-17, a pri-amiRNA derived from pri-miR-19b, a pri-amiRNA derived from pri-miR-20a and a pri-amiRNA derived from pri-miR-30a. In some embodiments, the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-19b, a pri-amiRNA derived from pri-miR-20a, and a pri- amiRNA derived from pri-miR-30a. In some embodiments the plurality of pri-amiRNAs comprises a pri-amiRNA derived from pri-miR-17, a pri-amiRNA derived from pri-miR-20a and a pri-amiRNA derived from pri-miR-30a. The plurality of pri-amiRNAs may comprise, from 5’ to 3’, a pri-amiRNA derived from pri- miR-30a and a pri-amiRNA derived from pri-miR-17. Alternatively, the plurality of pri- amiRNAs may comprise, from 5’ to 3’, a pri-amiRNA derived from pri-miR-17 and a pri- amiRNA derived from pri-miR-30a. In some embodiments, the plurality of pri-amiRNAs comprises, from 5’ to 3’, a pri-amiRNA derived from pri-miR-30a, a pri-amiRNA derived from pri-miR-17 and a pri-amiRNA derived from pri-miR-19b. In some embodiments, the plurality of pri-amiRNAs comprises, from 5’ to 3’, a pri-amiRNA derived from pri-miR-30a, a pri-amiRNA derived from pri-miR-17, a pri- amiRNA derived from pri-miR-19b and a pri-amiRNA derived from pri-miR-20a. In some embodiments, the plurality of pri-amiRNAs comprises at least six pri-amiRNAs, wherein at least one of the pri-amiRNAs is derived from pri-miR-30a. In some embodiments, the plurality of pri-amiRNAs comprises at least six pri-amiRNAs, wherein at least one of the pri-amiRNAs is derived from pri-miR-17. In some embodiments, the plurality of pri-amiRNAs comprises at least six pri-amiRNAs, wherein at least one of the pri- amiRNAs is derived from pri-miR-19b. In some embodiments, the plurality of pri-amiRNAs comprises at least six pri-amiRNAs, wherein at least one of the pri-amiRNAs is derived from pri-miR-20a. In some embodiments, the plurality of pri-amiRNAs comprises at least six pri-amiRNAs, wherein one of the pri-amiRNAs is derived from pri-miR-30a and another pri-amiRNA is derived from pri-miR-17. In some embodiments, the plurality of pri-amiRNAs comprises at least six pri-amiRNAs, wherein one pri-amiRNA is derived from pri-miR-30a, one pri-amiRNA is derived from pri-miR-17 and one pri-amiRNA is derived from pri-miR-19b. In some embodiments, the plurality of pri-amiRNAs comprises at least six pri-amiRNAs, wherein one pri-amiRNA is derived from pri-miR-30a, one pri-amiRNA is derived from pri-miR-17, one pri-amiRNA is derived from pri-miR-19b and one pri-amiRNA is derived from pri-miR-20a. The plurality of pri-amiRNAs may comprise pri-miRNA loop regions from the pri-miR-17-92 and/or pri-miR-106a-363 clusters. In embodiments comprising a plurality of polynucleotides each encoding a pri-amiRNA, each pri-amiRNA may comprise a pri-miR-17 (SEQ ID NO:72), pri-miR-18a (SEQ ID NO:73), pri- miR-19a (SEQ ID NO:74), pri-miR-19b (SEQ ID NO:75), pri-miR-20a (SEQ ID NO:76) or pri-miR-92a-1 (SEQ ID NO:77) loop region. Alternatively, in embodiments comprising a plurality of polynucleotides each encoding a pri-amiRNA, each pri-amiRNA may comprise a pri-miR-106a (SEQ ID NO:78), pri-miR-18b (SEQ ID NO:79), pri-miR-20b (SEQ ID NO:80), pri-miR-19b-2 (SEQ ID NO:81), pri-miR-92a-2 (SEQ ID NO:82) or pri-miR-363 loop region (SEQ ID NO:83). Each pri-amiRNA may comprise a pri-miR-30a (SEQ ID NO:232), pri-miR- 30b (SEQ ID NO:233), pri-miR-30c-1 (SEQ ID NO:234), pri-miR-30c-2 (SEQ ID NO:235), pri-miR-30d (SEQ ID NO:236) or pri-miR-30e (SEQ ID NO:237) loop region. The plurality of pri-amiRNAs may comprise a pri-amiRNA comprising a pri-miR-19b loop region (SEQ ID NO:75). In some embodiments, the plurality of pri-amiRNAs comprises a pri-amiRNA comprising a pri-miR-17 loop region (SEQ ID NO:72), a pri-amiRNA comprising a pri-miR-18a loop region (SEQ ID NO:73), a pri-amiRNA comprising a pri-miR-19a loop region (SEQ ID NO:74), a pri-amiRNA comprising a pri-miR-19b loop region (SEQ ID NO:75), a pri-amiRNA comprising a pri-miR-20a loop region (SEQ ID NO:76) and a pri- amiRNA comprising a pri-miR-92a-1 loop region (SEQ ID NO:77) . The plurality of pri- amiRNAs may comprise from 5’ to 3’ a pri-amiRNA comprising a pri-miR-17 loop region, a pri-amiRNA comprising a pri-miR-18a loop region, a pri-amiRNA comprising a pri-miR-19a loop region, a pri-amiRNA comprising a pri-miR-19b loop region, a pri-amiRNA comprising a pri-miR-20a loop region and a pri-amiRNA comprising a pri-miR-92 loop region. In some embodiments, the plurality of pri-amiRNAs comprises a pri-amiRNA comprising a pri-miR-106a loop region (SEQ ID NO:78), a pri-amiRNA comprising a pri-miR-18b loop region (SEQ ID NO:79), a pri-amiRNA comprising a pri-miR-20b loop region (SEQ ID NO:80), a pri-amiRNA comprising a pri-miR-19b-2 loop region (SEQ ID NO:81), a pri- amiRNA comprising a pri-miR-92a-2 loop region (SEQ ID NO:82) and a pri-amiRNA comprising a pri-miR-363 loop region (SEQ ID NO:83). In some embodiments, the plurality of pri-amiRNAs comprises from 5’ to 3’ a pri-amiRNA comprising a pri-miR-106a loop region, a pri-amiRNA comprising a pri-miR-18b loop region, a pri-amiRNA comprising a pri- miR-20b loop region, a pri-amiRNA comprising a pri-miR-19b-2 loop region, a pri-amiRNA comprising a pri-miR-92a-2 loop region and a pri-amiRNA comprising a pri-miR-363 loop region. In some embodiments, the plurality of pri-amiRNAs comprises a pri-amiRNA comprising a pri-miR30a loop region (SEQ ID NO:232), a pri-amiRNA comprising a pri-miR-30b loop region (SEQ ID NO:233), a pri-amiRNA comprising a pri-miR-30c-1 loop region (SEQ ID NO:234), a pri-amiRNA comprising a pri-miR-30c-2 loop region (SEQ ID NO:235), a pri- amiRNA comprising a pri-miR-30d loop region (SEQ ID NO:236) and a pri-amiRNA comprising a pri-miR-30e loop region (SEQ ID NO:237). In some embodiments, the plurality of pri-amiRNAs comprises from 5’ to 3’ a pri-amiRNA comprising a pri-miR-30a loop region, a pri-amiRNA comprising a pri-miR-30b loop region, a pri-amiRNA comprising a pri-miR- 30c-1 loop region, a pri-amiRNA comprising a pri-miR-30c-2 loop region, a pri-amiRNA comprising a pri-miR-30d loop region and a pri-amiRNA comprising a pri-miR-30e loop region. In some embodiments, the plurality of pri-amiRNAs comprises a pri-amiRNA comprising a pri-miR-17 loop region and a pri-amiRNA comprising a pri-miR-30a loop region. In some embodiments, the plurality of pri-amiRNAs comprises a pri-amiRNA comprising a pri-miR-17 loop region, a pri-amiRNA comprising a pri-miR-19b loop region and a pri-amiRNA comprising a pri-miR-30a loop region. In some embodiments, the plurality of pri-amiRNAs comprises a pri-amiRNA comprising a pri-miR-17 loop region, a pri-amiRNA comprising a pri-miR-19b loop region, a pri-amiRNA comprising a pri-miR-20a loop region and a pri- amiRNA comprising a pri-miR-30a loop region. In some embodiments, the plurality of pri- amiRNAs comprises a pri-amiRNA comprising a pri-miR-19b loop region, a pri-amiRNA comprising a pri-miR-20a loop region, and a pri-amiRNA comprising a pri-miR-30a loop region. In some embodiments the plurality of pri-amiRNAs comprises a pri-amiRNA comprising a pri-miR-17 loop region, a pri-amiRNA comprising a pri-miR-20a loop region and a pri-amiRNA comprising a pri-miR-30a loop region. The plurality of pri-amiRNAs may comprise, from 5’ to 3’, a pri-amiRNA comprising a pri- miR-30a loop region and a pri-amiRNA comprising a pri-miR-17 loop region. Alternatively, the plurality of pri-amiRNAs may comprise, from 5’ to 3’, a pri-amiRNA comprising a pri- miR-17 loop region and a pri-amiRNA comprising a pri-miR-30a loop region. In some embodiments, the plurality of pri-amiRNAs comprises, from 5’ to 3’, a pri-amiRNA comprising a pri-miR-30a loop region, a pri-amiRNA comprising a pri-miR-17 loop region and a pri-amiRNA comprising a pri-miR-19b loop region. In some embodiments, the plurality of pri-amiRNAs comprises, from 5’ to 3’, a pri-amiRNA comprising a pri-miR-30a loop region, a pri-amiRNA comprising a pri-miR-17 loop region, a pri-amiRNA comprising a pri-miR-19b loop region and a pri-amiRNA comprising a pri-miR-20a loop region. The amiRNA of each pri-amiRNA may be specific for a different target mRNA, as defined above. In some embodiments the amiRNA coding region comprises a polynucleotide encoding a plurality of pri-amiRNAs having at least 40%, 50% or 60% sequence identity to SEQ ID NO:42 (miR-17-92 pri-miRNA cluster) or SEQ ID NO:43 (miR106a-363 pri-miRNA cluster). The amiRNA coding region may comprise a polynucleotide encoding a plurality of pri- amiRNAs having at least about 75% or at least about 80% sequence identity to SEQ ID NO:42 (miR-17-92 pri-miRNA cluster) or SEQ ID NO:43 (miR106a-363 pri-miRNA cluster). In some embodiments the amiRNA coding region comprises a polynucleotide encoding a plurality of pri-amiRNAs comprising flanking sequences of SEQ ID NO:42 (miR-17-92 pri- miRNA cluster) or SEQ ID NO:43 (miR106a-363 pri-miRNA cluster). In some embodiments the amiRNA coding region comprises a polynucleotide encoding a plurality of pri-amiRNAs comprising one or more loop sequences of SEQ ID NO:42 (miR-17-92 pri-miRNA cluster) or SEQ ID NO:43 (miR106a-363 pri-miRNA cluster). In some embodiments the amiRNA coding region comprises a polynucleotide encoding a plurality of pri-amiRNAs comprising flanking sequences of SEQ ID NO:226 (pri-miR-30a). In some embodiments the amiRNA coding region comprises a polynucleotide encoding a plurality of pri-amiRNAs comprising one or more loop sequences of SEQ ID NO:232 (pri- miR-30a). In some embodiments, the amiRNA coding region comprises a polynucleotide encoding a plurality of pri-amiRNAs comprising the flanking sequences of SEQ ID NO:42 (miR-17-92 pri-miRNA cluster), SEQ ID NO:43 (miR106a-363 pri-miRNA cluster) or SEQ ID NO:226 (pri-miR30a) and one or more loop sequences of SEQ ID NO:42 (miR-17-92 pri-miRNA cluster), SEQ ID NO:43 (miR106a-363 pri-miRNA cluster) or SEQ ID NO: 232 (pri-miR30a). In some embodiments, the amiRNA coding region comprises a polynucleotide encoding a plurality of pri-amiRNAs having at least 40%, 50% or 60% sequence identity to SEQ ID NO:42 (miR-17-92 pri-miRNA cluster), SEQ ID NO:43 (miR106a-363 pri-miRNA cluster) or SEQ ID NO:226 (pri-miR30a) and comprising the flanking sequences of SEQ ID NO:42 (miR-17-92 pri-miRNA cluster), SEQ ID NO:43 (miR106a-363 pri-miRNA cluster) or SEQ ID NO:226 (pri-miR30a) and one or more loop sequences of SEQ ID NO:42 (miR-17-92 pri- miRNA cluster), SEQ ID NO:43 (miR106a-363 pri-miRNA cluster) or SEQ ID NO:232 (pri- miR30a). In some embodiments, the amiRNA coding region comprises a polynucleotide encoding a plurality of pri-amiRNAs comprising the flanking sequences of SEQ ID NO:42 (miR-17-92 pri-miRNA cluster) or SEQ ID NO:43 (miR106a-363 pri-miRNA cluster) and one or more loop sequences of SEQ ID NO:42 (miR-17-92 pri-miRNA cluster) or SEQ ID NO:43 (miR106a-363 pri-miRNA cluster). In some embodiments, the amiRNA coding region comprises a polynucleotide encoding a plurality of pri-amiRNAs having at least 40%, 50% or 60% sequence identity to SEQ ID NO:42 (miR-17-92 pri-miRNA cluster) or SEQ ID NO:43 (miR106a-363 pri-miRNA cluster) and comprising the flanking sequences of SEQ ID NO:42 (miR-17-92 pri-miRNA cluster) or SEQ ID NO:43 (miR106a-363 pri-miRNA cluster) and one or more loop sequences of SEQ ID NO:42 (miR-17-92 pri-miRNA cluster) or SEQ ID NO:43 (miR106a-363 pri-miRNA cluster). Target mRNA In some embodiments, the amiRNA is specific for a target mRNA selected from a tumour microenvironment (TME) mRNA, an endogenous TCR mRNA, an endogenous HLA mRNA, a CD3 subunit mRNA, a pro-apoptotic mRNA or any combination thereof. As the skilled person will appreciate, the target mRNA is generated from transcription of the target gene. Thus, “TME mRNA” refers to mRNA generated from transcription of a tumour microenvironment associated gene, while “TCR mRNA” and “HLA mRNA” refer to mRNA generated from transcription of a TCR or HLA gene, respectively. The term “pro-apoptotic mRNA” refers to an mRNA associated with the induction of apoptosis in a cell. An “endogenous” mRNA will be understood to refer to an mRNA endogenous to the host cell to which the construct is introduced (i.e. a mRNA detectable in the wild-type host cell). Various methods are known in the art to detect RNA including, for example, reverse transcription-polymerase chain reaction (RT-PCR), Northern Blots, nuclease protection assays (NPA) and RNA-seq methods. RNA-seq methods function by mapping the number of RNA reads aligned to each gene under each biological condition, to obtain a read count. The reads can then be normalised to provide a normalised read count. Various RT-PCR, Northern Blot, NPA and RNA-seq methods are commercially available and known to those skilled in the art. Such methods can be used to determine if a host cell expresses the target mRNA. Likewise, such methods can also be used to confirm that the amiRNA reduces (i.e. suppresses) the expression of the target mRNA. The amiRNA may reduce expression and/or the detectable amount of the target mRNA by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99%. In some embodiments, the amiRNA reduces expression and/or the detectable amount of the target mRNA by at least about 50%. Preferably, the amiRNA reduces expression and/or the detectable amount of the target mRNA by at least about 70%, more preferably at least about 80%, most preferably at least about 90%. The amiRNA may reduce expression and/or the detectable amount of the target mRNA by at least about one fold, at least about two fold, at least about three fold, at least about four fold, at least about five fold, at least about six fold, at least about seven fold, at least about eight fold, at least about nine fold, at least about 10 fold, at least about 12 fold, at least about 15 fold, at least about 20 fold, at least about 50 fold, at least about 100 fold, at least about 500 fold, at least about 1000 fold, at least about 2000 fold, at least about 5000 fold or at least about 10000 fold. The reduction in expression and/or the detectable amount of the target mRNA may be determined by measuring the expression and/or detectable amount of the protein which the target mRNA encodes. Various methods for measuring protein expression are commercially available and known to those skilled in the art, including, but not limited to flow cytometry, fluorescence microscopy and western blots. Thus, in some embodiments, the amiRNA may reduce expression and/or the detectable amount of a protein encoded by the target mRNA by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99%. In some embodiments, the amiRNA reduces expression and/or the detectable amount of a protein encoded by target mRNA by at least about 50%. Preferably, the amiRNA reduces expression and/or the detectable amount of a protein encoded by the target mRNA by at least about 70%, more preferably at least about 80%, most preferably at least about 90%. The amiRNA may reduce expression and/or the detectable amount of a protein encoded by the target mRNA by at least about one fold, at least about two fold, at least about three fold, at least about four fold, at least about five fold, at least about six fold, at least about seven fold, at least about eight fold, at least about nine fold, at least about 10 fold, at least about 12 fold, at least about 15 fold, at least about 20 fold, at least about 50 fold, at least about 100 fold, at least about 500 fold, at least about 1000 fold, at least about 2000 fold, at least about 5000 fold or at least about 10000 fold. In some embodiments, the amiRNA is specific for a TME mRNA. “Tumour microenvironment (TME)” is a known term of the art, which is used to describe the environment adjacent to a typically in vivo cancerous tumour. The TME may comprise the surrounding blood vessels, immune cells, fibroblasts, signalling molecules and/or the extracellular matrix. Generally, the TME is immunosuppressive. Solid tumour microenvironments (TMEs), in particular, are known to be immunosuppressive. In the context of the present invention, a TME mRNA is typically an mRNA which increases in expression in a tumour microenvironment, for example an mRNA in the host cell. Thus, by being specific for a TME mRNA, the amiRNA can reduce or suppress expression of the TME mRNA in the host cell once the host cell is adjacent to or within the TME. This may further improve efficacy of the host cell in a TME. For example, the TME mRNA may be a PD-1 mRNA. When a host cell which comprises the polynucleotide construct of the invention enters the TME, PD-1 may be upregulated in the host cell. However, when the amiRNA is specific for PD-1 mRNA, the upregulation of PD-1 is suppressed in the host cell, which may advantageously improve efficiency of the host cell in a TME. Various TME mRNAs are known to the skilled person. For example, the TME mRNA may comprise a PD-1 mRNA, a TIM-3 mRNA, a LAG-3 mRNA, a CTLA-4 mRNA, an Adenosine 2A receptor (A2AR) mRNA, a CD39 mRNA, a CD73 mRNA, a THEMIS mRNA, a TGFβ mRNA, a TGFBR2 mRNA, a Protein Tyrosine Phosphatase 1B (PTP1B) mRNA, a p38 mitogen-activated protein kinase (p38 MAPK) mRNA, a PR domain zinc finger protein 1 (PRDM1) (also known as Blimp-1) mRNA, a Thymocyte Selection Associated High Mobility Group Box (TOX) mRNA, a TOX2 mRNA, a NR4A family mRNA, a TIGIT mRNA, a STAT5 mRNA and/or an IL- 10 receptor mRNA. In some embodiments, the TME mRNA is selected from a PD-1 mRNA, a TIM-3 mRNA, a LAG-3 mRNA, a CTLA-4 mRNA, an Adenosine 2A receptor (A2AR) mRNA, a CD39 mRNA, a CD73 mRNA, a THEMIS mRNA, a TGFβ mRNA, a TGFBR2 mRNA, a TIGIT mRNA, a Protein Tyrosine Phosphatase 1B (PTP1B) mRNA, a p38 mitogen-activated protein kinase (p38 MAPK) mRNA, a PR domain zinc finger protein 1 (PRDM1) (also known as Blimp-1) mRNA, a TOX mRNA, a TOX2 mRNA, a NR4A family mRNA, a TIGIT mRNA, a STAT5 mRNA and an IL-10 receptor mRNA. In some embodiments, the TME mRNA comprises a PD-1 mRNA, a TIM-3 mRNA and/or a LAG-3 mRNA. In some embodiments the amiRNA is specific for a pro-apoptotic mRNA. Various pro- apoptotic mRNAs are known to those skilled in the art. For example, the pro-apoptotic mRNA may be selected from or comprise Fas mRNA or TNFr mRNA. In some embodiments, the amiRNA is specific for an endogenous TCR mRNA. In some embodiments the amiRNA is specific for an endogenous HLA mRNA. This enables the amiRNA to suppress the expression of endogenous TCR or HLAs in the host cell to which it is introduced. The suppression of expression respectively reduces the risk of the development of graft versus host disease (GVHD) or the allogeneic rejection of the host cell. Advantageously, this allows allogeneic cells to be universally used with any patient, regardless of the patient’s TCR/HLA background. As such, a single batch of host cells can be generated which can be used to treat multiple patients, regardless of whether they are allogeneic or autologous. This reduces the cost per patient, increases throughput and minimises the biological variability between host cell products. The endogenous TCR mRNA may be an αβ TCR mRNA. Alternatively, the endogenous TCR mRNA may be a γδ TCR mRNA. In some embodiments, the endogenous TCR mRNA is an TCRα mRNA. In some embodiments, the endogenous TCR mRNA is an TCRβ mRNA. In some embodiments, the amiRNA is specific for a CD3 subunit mRNA. For example, the amiRNA may be specific for a CD247 mRNA. The CD3 subunit mRNA may be an endogenous CD3 subunit mRNA. As the skilled person will appreciate, upon expression, CD3 forms a complex with the TCR in T cells. As such, in embodiments where the amiRNA is specific for a CD3 subunit mRNA, specificity for the CD3 subunit advantageously suppresses formation of a CD3-TCR complex. The target endogenous HLA mRNA may comprise an HLA Class I mRNA. Optionally, the HLA Class I mRNA comprises a Beta-2 microglobulin (β2M) mRNA. The target endogenous HLA mRNA may comprise an HLA Class II mRNA. The HLA Class II mRNA may comprise a Class II transactivator (CIITA) mRNA. In some embodiments, the amiRNA comprises a 5’ or 3’ shRNA stem sequence specific for a target mRNA selected from a tumour microenvironment (TME) mRNA, an endogenous TCR mRNA, an endogenous HLA mRNA, a CD3 subunit mRNA, a pro-apoptotic mRNA or any combination thereof, as defined above. In some embodiments, the amiRNA comprises a 5’ or 3’ CRISPR guide RNA stem sequence specific for a target mRNA selected from a tumour microenvironment (TME) mRNA, an endogenous TCR mRNA, an endogenous HLA mRNA, a CD3 subunit mRNA, a pro-apoptotic mRNA or any combination thereof, as defined above. In some embodiments, the amiRNA comprises a 5’ or 3’ shRNA stem sequence specific for a TCRα mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:84. SEQ ID NO:84 is an amiRNA derived from miR-155 which comprises a GIPZ TCRα-specific shRNA stem sequence. Alternatively, the amiRNA may comprise or consist of SEQ ID NO:85. SEQ ID NO:85 is an amiRNA derived from miR-155 which comprises a TCRα-specific shRNA stem sequence. In some embodiments, the amiRNA is derived from miR-17 and is specific for a TCRα mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:86. In some embodiments, the amiRNA is derived from miR-19a and is specific for a TCRα mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:87. In some embodiments, the amiRNA is derived from miR-19b and is specific for a TCRα mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:88. In some embodiments, the amiRNA is derived from miR-18a and is specific for a TCRα mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:89. In some embodiments, the amiRNA is derived from miR-20a and is specific for a TCRα mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:90. In some embodiments, the amiRNA is derived from miR-92a-1 and is specific for a TCRα mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:91. In some embodiments, the amiRNA is specific for a TCRβ mRNA. In some embodiments, the amiRNA is derived from miR-18a and is specific for a TCRβ mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:92. In some embodiments, the amiRNA is derived from miR-20a and is specific for a TCRβ mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:93. In some embodiments, the amiRNA is derived from miR-92a-1 and is specific for a TCRβ mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:94. In some embodiments, the amiRNA comprises a shRNA stem sequence specific for a CD247 mRNA. SEQ ID NOs 95, 96 and 97 are each amiRNAs derived from miR-155 which comprise a CD247-specific shRNA stem sequence. Thus, in some embodiments, the amiRNA comprises or consists of any one of SEQ ID NOs 95, 96 and 97. In some embodiments, the amiRNA is derived from miR-30a and comprises a shRNA stem sequence specific for a CD247 mRNA. For example, the amiRNA may comprise or consist of any one of SEQ ID NOs 238 to 242. In some embodiments, the amiRNA is derived from miR-17 and comprises a shRNA stem sequence specific for a CD247 mRNA. For example, the amiRNA may comprise or consist of any one of SEQ ID NOs 243 to 245. In some embodiments, the amiRNA is derived from miR-20a and comprises a shRNA stem sequence specific for a CD247 mRNA. For example, the amiRNA may comprise or consist of any one of SEQ ID NOs 243 to 246. In some embodiments the amiRNA is specific for a CIITA mRNA. In some embodiments the amiRNA is derived from miR-17 and is specific for a CIITA mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:98. In some embodiments the amiRNA is derived from miR-20a and is specific for a CIITA mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:99. In some embodiments the amiRNA is derived from miR-106a and is specific for a CIITA mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:100. In some embodiments the amiRNA is derived from miR-19b-2 and is specific for a CIITA mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:101. In some embodiments the amiRNA is derived from miR-363 and is specific for a CIITA mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:102. In some embodiments the amiRNA is derived from miR-92a-2 and is specific for a CIITA mRNA. In some embodiments the amiRNA comprises or consists of SEQ ID NO:106. In some embodiments the amiRNA is specific for a β2M mRNA. In some embodiments the amiRNA is derived from miR-18a and is specific for a β2M mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:103. In some embodiments the amiRNA is derived from miR-19a and is specific for a β2M mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:104. In some embodiments the amiRNA is derived from miR-19b and is specific for a β2M mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:105. In some embodiments the amiRNA is derived from miR-92a-2 and is specific for a β2M mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:106. In some embodiments the amiRNA is derived from miR-18b and is specific for a β2M mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:107. In some embodiments the amiRNA is derived from miR-20b and is specific for a β2M mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:108. In some embodiments the amiRNA is derived from miR-92a-2 and is specific for a β2M mRNA. For example, the amiRNA may comprise or consist of SEQ ID NO:109. The amiRNA may comprise or consist of any one of SEQ ID NOs 84-109 or SEQ ID NOs 238 to 246. The amiRNA may comprise or consist of any one of SEQ ID NOs 84-109. In some embodiments, the amiRNA is selected from any one of SEQ ID NOs 86-94 and 98-109. In embodiments comprising a plurality of amiRNAs, the plurality of amiRNAs may be selected from SEQ ID NOs 84-109 or SEQ ID NOs 238 to 246. In embodiments comprising a plurality of amiRNAs, the plurality of amiRNAs may be selected from SEQ ID NOs 84-109. In embodiments comprising a plurality of amiRNAs, the plurality of amiRNAs may be selected from SEQ ID NOs 86-94 and 98-109. In some embodiments, the pri-amiRNA comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a TCRα mRNA. For example, the pri-amiRNA may be derived from pri-miR-155 and comprise a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a TCRα mRNA. An exemplary pri-amiRNA may comprise or consist of SEQ ID NO:110. SEQ ID NO:110 is a pri-amiRNA derived from pri-miR-155 which comprises a GIPZ TCRα-specific shRNA stem sequence. Preferably, the pri-amiRNA further comprises 5’ and/or 3’ flanking sequences. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:111. SEQ ID NO:111 comprises SEQ ID NO:110 with 5’ and 3’ flanking sequences. In some embodiments, the pri-amiRNA comprises SEQ ID NO:112. SEQ ID NO:112 is a pri- amiRNA derived from pri-miR-155 which comprises a TCRα-specific shRNA stem sequence. Preferably, the pri-amiRNA further comprises flanking sequences. Such a pri-amiRNA may comprise or consist of SEQ ID NO:113, which comprises SEQ ID NO:112 with 5’ and 3’ flanking sequences. Pri-amiRNAs which do not comprise flanking sequences may otherwise be referred to throughout as pre-amiRNAs. In some embodiments, the pri-amiRNA is derived from pri-miR-17 and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a TCRα mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:114. In some embodiments, the pri-amiRNA is derived from pri-miR-19a and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a TCRα mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:115. In some embodiments, the pri-amiRNA is derived from pri-miR-19b and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a TCRα mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:116. In some embodiments, the pri-amiRNA is derived from pri-miR-18a and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a TCRα mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:117. In some embodiments, the pri-amiRNA is derived from pri-miR-20a and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a TCRα mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:118. Alternatively, the pri-amiRNA may comprise or consist of SEQ ID NO:214. In some embodiments, the pri-amiRNA is derived from pri-miR-92a-1 and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a TCRα mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:119. Alternatively, the pri-amiRNA may comprise or consist of SEQ ID NO:215. In some embodiments, the pri-amiRNA is derived from pri-miR-30a and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a TCRα mRNA. The pri-amiRNA may comprise a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a TCRβ mRNA. In some embodiments the pri-amiRNA is derived from pri-miR- 18a and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a TCRβ mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:120. In some embodiments the pri-amiRNA is derived from pri-miR-20a and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a TCRβ mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:121. Alternatively, the pri-amiRNA may comprise or consist of SEQ ID NO:216. In some embodiments the pri-amiRNA is derived from pri-miR-92a-1 and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a TCRβ mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:122. Alternatively, the pri-amiRNA may comprise or consist of SEQ ID NO:217. In some embodiments the pri-amiRNA is derived from miR-92a-1 and is specific for a CIITA mRNA. In some embodiments the pri-amiRNA comprises SEQ ID NO:137. In some embodiments the pri-amiRNA is derived from pri-miR-30a and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a TCRβ mRNA. In some embodiments the pri-amiRNA comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a CD247 mRNA. In some embodiments the pri-amiRNA is derived from pri-miR-155 and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a CD247 mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:123, SEQ ID NO:124 or SEQ ID NO:125. Preferably, the pri-amiRNA further comprises 5’ and/or 3’ flanking sequences. Such a pri-amiRNA may comprise or consist of SEQ ID NO:126, SEQ ID NO:127 or SEQ ID NO:128, all of which additionally comprise 5’ and 3’ flanking sequences. In some embodiments, the pri-amiRNA is derived from pri-miR-30a and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a CD247 mRNA. In some embodiments, the pri-amiRNA is derived from pri-miR-30a and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a CD247 mRNA. For example, the amiRNA may comprise or consist of any one of SEQ ID NOs 247 to 256. In some embodiments, the pri-amiRNA is derived from pri-miR-17 and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a CD247 mRNA. In some embodiments, the pri-amiRNA is derived from pri-miR-17 and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a CD247 mRNA. For example, the amiRNA may comprise or consist of any one of SEQ ID NOs 257 to 264. In some embodiments, the pri-amiRNA is derived from pri-miR-20a and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a CD247 mRNA. In some embodiments, the pri-amiRNA is derived from pri-miR-20a and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a CD247 mRNA. For example, the amiRNA may comprise or consist of any one of SEQ ID NOs 265 to 270. In some embodiments the pri-amiRNA comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a CIITA mRNA. In some embodiments the pri-amiRNA is derived from pri-miR-17 and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a CIITA mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:129. In some embodiments the pri-amiRNA is derived from pri-miR-20a and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a CIITA mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:130. Alternatively, the pri-amiRNA may comprise or consist of SEQ ID NO: 218. In some embodiments the pri-amiRNA is derived from pri-miR-92a-1 and is specific for a CIITA mRNA. In some embodiments the amiRNA comprises or consists of SEQ ID NO: 137. In some embodiments the pri-amiRNA is derived from pri-miR-106a and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a CIITA mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:131. In some embodiments the pri-amiRNA is derived from pri-miR-19b-2 and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a CIITA mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:132. In some embodiments the pri-amiRNA is derived from pri-miR-363 and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a CIITA mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:133. The pri-amiRNA may comprise a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a β2M mRNA. In some embodiments, the pri-amiRNA is derived from pri-miR- 18a and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a β2M mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:134. In some embodiments, the pri-amiRNA is derived from pri-miR-19a and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a β2M mRNA. For example, the pri- amiRNA may comprise or consist of SEQ ID NO:135. Alternatively, the pri-amiRNA may comprise or consist of SEQ ID NO:219. In some embodiments, the pri-amiRNA is derived from pri-miR-19b and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a β2M mRNA. For example, the pri- amiRNA may comprise or consist of SEQ ID NO:136. In some embodiments, the pri-amiRNA is derived from pri-miR-92a-2 and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a β2M mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:137. In some embodiments, the pri-amiRNA is derived from pri-miR-18b and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a β2M mRNA. For example, the pri- amiRNA may comprise or consist of SEQ ID NO:138. In some embodiments, the pri-amiRNA is derived from pri-miR-20b and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a β2M mRNA. For example, the pri- amiRNA may comprise or consist of SEQ ID NO:139. In some embodiments, the pri-amiRNA is derived from pri-miR-92a-2 and comprises a 5’ or 3’ shRNA or CRISPR guide RNA stem sequence specific for a β2M mRNA. For example, the pri-amiRNA may comprise or consist of SEQ ID NO:140. The pri-amiRNA may be selected from any one of SEQ ID NOs 110-140, SEQ ID NOs 214- 219 or SEQ ID NOs 247-270. The pri-amiRNA may be selected from any one of SEQ ID NOs 110-140. In some embodiments, the pri-amiRNA is selected from any one of SEQ ID NOs 114-122 and 129-140. In embodiments comprising a plurality of pri-amiRNAs, the plurality of pri-amiRNAs may be selected from SEQ ID NOs 110-140, SEQ ID NOs 214-219 or SEQ ID NOs 247-270. In embodiments comprising a plurality of pri-amiRNAs, the plurality of pri-amiRNAs may be selected from SEQ ID NOs 110-140. In embodiments comprising a plurality of pri-amiRNAs, the plurality of pri-amiRNAs may be selected from SEQ ID NOs 114-122 and 129-140. In embodiments comprising a plurality of pri-amiRNAs derived from a miRNA cluster, the miRNA cluster may be a pri-miR17-92 cluster and the plurality of pri-amiRNAs may comprise 5’ or 3’ shRNA or CRISPR guide RNA stem sequences specific for TCRα and/or TCRβ mRNA. For example, the plurality of pri-amiRNAs may comprise or consist of SEQ ID NO:141. SEQ ID NO:141 is a plurality of pri-amiRNAs derived from the pri-miR17-92 cluster which comprises amiRNAs (comprising shRNA stem sequences) specific for TCRα and TCRβ mRNA. In some embodiments, the plurality of pri-amiRNAs may comprise or consist of SEQ ID NO: 271. SEQ ID NO:271 is a plurality of pri-amiRNAs derived from the pri-miR17-92 cluster which comprises amiRNAs (comprising shRNA stem sequences) specific for TCRα mRNA. In embodiments comprising a plurality of pri-amiRNAs derived from a miRNA cluster, the miRNA cluster may be a pri-miR17-92 cluster and the plurality of pri-amiRNAs may comprise 5’ or 3’ shRNA or CRISPR guide RNA stem sequences specific for β2M and/or CIITA mRNA. For example, the plurality of pri-amiRNAs may comprise or consist of SEQ ID NO:272. SEQ ID NO:272 is a plurality of pri-amiRNAs derived from the pri-miR17-92 cluster which comprises amiRNAs (comprising shRNA stem sequences) specific for β2M and CIITA mRNA. In embodiments comprising a plurality of pri-amiRNAs derived from a miRNA cluster, the miRNA cluster may be a pri-106a-363 cluster and the plurality of pri-amiRNAs may comprise 5’ or 3’ shRNA or CRISPR guide RNA stem sequences specific for β2M and/or CIITA mRNA. For example, the plurality of pri-amiRNAs may comprise or consist of SEQ ID NO:273. SEQ ID NO:273 is a plurality of pri-amiRNAs derived from the pri-miR-106a-363 cluster which comprises amiRNAs (comprising shRNA stem sequences) specific for β2M and CIITA mRNA. In embodiments comprising a plurality of pri-amiRNAs, the plurality of pri-amiRNAs may comprise a pri-amiRNA derived from pri-miR30a and the plurality of pri-amiRNAs may comprise 5’ or 3’ shRNA or CRISPR guide RNA stem sequences specific for TCRα and/or TCRβ mRNA. Alternatively, the plurality of pri-amiRNAs may comprise a pri-amiRNA derived from pri-miR30a and at least one pri-amiRNA derived from the pri-miR17-92 cluster, and the plurality of pri-amiRNAs may comprise 5’ or 3’ shRNA or CRISPR guide RNA stem sequences specific for TCRα and/or TCRβ mRNA. Polynucleotide construct The polynucleotide construct may further comprise a polynucleotide encoding a reporter gene. Suitable reporter genes include, but are not necessarily limited to HNIS, hNET and HSVtK. As used herein, the term polynucleotide refers to a polymer comprising two or more nucleotides. Preferably, the polynucleotide comprises at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides or at least 100 nucleotides. The nucleotides can be naturally occurring or artificial. A nucleotide typically contains a nucleobase, a sugar and at least one linking group, such as a phosphate, 2’O-methyl, 2’ methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate group. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C) The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar and the nucleobase together form a nucleoside. Preferred nucleosides include, but are not limited to, adenosine, guanosine, 5-methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. The nucleosides may be adenosine, guanosine, uridine and cytidine. The nucleotides are typically ribonucleotides or deoxyribonucleotides. The nucleotides may be deoxyribonucleotides. The nucleotides typically contain a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5’ or 3’ side of a nucleotide. Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5- hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5- hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP), 5-methyl-2’-deoxycytidine monophosphate, 5-methyl-2’-deoxycytidine diphosphate, 5-methyl-2’-deoxycytidine triphosphate, 5-hydroxymethyl-2’-deoxycytidine monophosphate, 5-hydroxymethyl-2’-deoxycytidine diphosphate and 5-hydroxymethyl-2’- deoxycytidine triphosphate. The nucleotides may be selected from AMP, UMP, GMP, CMP, dAMP, dTMP, dGMP or dCMP. In some embodiments, the nucleotides are selected from dAMP, dTMP, dGMP or dCMP. The nucleotides may contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2’amino pyrimidines (such as 2’-amino cytidine and 2’-amino uridine), 2’-hyrdroxyl purines (such as , 2’-fluoro pyrimidines (such as 2’- fluorocytidine and 2’fluoro uridine), hydroxyl pyrimidines (such as 5’-α-P-borano uridine), 2’-O-methyl nucleotides (such as 2’-O-methyl adenosine, 2’-O-methyl guanosine, 2’-O- methyl cytidine and 2’-O-methyl uridine), 4’-thio pyrimidines (such as 4’-thio uridine and 4’- thio cytidine) and nucleotides have modifications of the nucleobase (such as 5-pentynyl-2’- deoxy uridine, 5-(3-aminopropyl)-uridine and 1,6-diaminohexyl-N-5-carbamoylmethyl uridine). One or more nucleotides in the polynucleotide may be modified, for instance with a label or a tag. The label may be any suitable label which allows the nucleotides to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. ¹²⁵I, ³⁵S, enzymes, antibodies, antigens, other polynucleotides and ligands such as biotin. The nucleotides in the polynucleotide construct may be attached to each other in any manner. The nucleotides may be linked by phosphate, 2’O-methyl, 2’ methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate linkages. The nucleotides are typically attached by their sugar and phosphate groups. The nucleotides may be connected via their nucleobases as in pyrimidine dimers. The polynucleotide construct may comprise a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). Preferably, the polynucleotide construct comprises DNA. The polynucleotide construct may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid or other synthetic polymers with nucleotide side chains. Substitutions may be used for the practices of codon optimisation and codon wobble, both of which are known to those skilled in the art. Thus, it will be appreciated that codon- optimised and codon-wobbled isolated polynucleotides are also envisaged. In some embodiments, the polynucleotide construct is codon-optimised for human expression. The polynucleotide construct can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence. Direct chemical synthesis of polynucleotides can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68:109; the diethylphosphoramidite method of Beaucage et al., 1981, Tetra. Lett., 22:1859; and the solid support method of U.S. Pat. No. 4,458,066. Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press, NY, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif, 1990; Mattila et al., 1991, Nucleic Acids Res. 19:967; and Eckert et al., 1991, PCR Methods and Applications 1:17. In some embodiments, the amiRNA coding region is downstream of an ori and upstream of the protein-coding region in the polynucleotide construct. In some embodiments, the amiRNA coding region is downstream of the protein-coding region in the polynucleotide construct. In some embodiments, the amiRNA coding region is upstream of an ori and downstream of the protein-coding region in the polynucleotide construct. As the skilled person will appreciate, the term “ori” refers to a nucleotide sequence which is recognised by DNA replication machinery to initiate transcription. The polynucleotide construct may comprise a plurality of oris. The polynucleotide construct may further comprise a promoter nucleotide sequence. The promoter nucleotide sequence may be downstream of an ori and upstream of the amiRNA coding region. In some embodiments the promoter is derived from a mammalian promoter or derived from a mammalian virus. Suitable promoters may be constitutive, cell type- specific, stage-specific, and/or modulatable or regulatable. Useful promoters include, but are not limited to, a viral long-terminal repeat (LTR), the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPS V promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, the EF1 alpha promoter, the phosphoglycerate kinase (PGK) promoter and promoter-enhancer combinations known in the art. Preferably, the promoter is an RNA polymerase II-dependent promoter. In some embodiments the construct does not comprise more than one promoter nucleotide sequence. In some embodiments the promoter comprises a portion or all of a viral long-terminal repeat (LTR). In some embodiments, the construct further comprises a second amiRNA coding region comprising a polynucleotide encoding an amiRNA. The construct may comprise, from 5’ to 3’ a first amiRNA coding region, a protein-coding region and a second amiRNA coding region. Alternatively, the construct may comprise from 5’ to 3’ the second amiRNA coding region, a protein-coding region and the first amiRNA coding region. In embodiments comprising a plurality of amiRNA coding regions, each amiRNA coding region preferably differs from the other amiRNA coding regions. For example, each amiRNA coding region may each encode a different plurality of amiRNAs, or each encode a plurality of amiRNAs each derived from different miRNA clusters. This may advantageously help to avoid sequence similarity in separate areas of the construct, which in turn may assist in avoiding recombination when using transduction. The second amiRNA coding region may be as defined above in relation to the first coding region. For example, the second amiRNA coding region may comprise a plurality of polynucleotides each encoding an amiRNA. Thus, the plurality of polynucleotides may encode a plurality of amiRNAs, for example at least six amiRNAs. The second amiRNA coding region may comprise at least two, at least three, at least four, at least five or at least six polynucleotides each encoding an amiRNA. The second amiRNA coding region may comprise less than 10, less than nine, less than eight or less than seven polynucleotides each encoding an amiRNA. The plurality of amiRNAs may be derived from a miRNA cluster, for example an miR-17-92 or miR-106a-363 cluster. In some embodiments, the plurality of amiRNAs encoded by the plurality of polynucleotides in the second amiRNA coding region may be derived from a miR-106a-363 cluster. In some embodiments, the plurality of amiRNAs encoded by the plurality of polynucleotides in the first amiRNA coding region may be derived from a miR-17-92 cluster. In some embodiments, the plurality of amiRNAs encoded by the plurality of polynucleotides in the first amiRNA coding region may comprise a amiRNA derived from miR30a. In some embodiments, the plurality of amiRNAs encoded by the plurality of polynucleotides in the first amiRNA coding region may be derived from a miR-17-92 cluster and miR30a, as defined above. The plurality of amiRNAs encoded by a plurality of polynucleotides in the second amiRNA coding region may be as defined above. The plurality of amiRNAs encoded by a plurality of polynucleotides in the second amiRNA coding region may be derived from one or more of the miRNAs miR-30a, miR-155, miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR- 92a-1. The plurality of amiRNAs encoded by a plurality of polynucleotides in the second amiRNA coding region may be derived from one or more of the miRNAs miR-155, miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1. For example, the plurality of amiRNAs may be derived from miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR- 92a-1. In some embodiments, the plurality of amiRNAs comprises from 5’ to 3’ an amiRNA derived from miR-17, an amiRNA derived from miR-18a, an amiRNA derived from miR-19a, and an amiRNA derived from miR-19b-1. In some embodiments, the plurality of amiRNAs comprises from 5’ to 3’ an amiRNA derived from miR-17, an amiRNA derived from miR-18a, an amiRNA derived from miR-19a, an amiRNA derived from miR-19b-1 and an amiRNA derived from miR-92a-1. The plurality of amiRNAs may comprise an amiRNA derived from miR-19b-1. In some embodiments the plurality of amiRNAs comprises from 5’ to 3’ an amiRNA derived from miR-30a and an amiRNA derived from miR-17. In some embodiments the plurality of amiRNAs comprises from 5 to 3’ an amiRNA derived from miR-30a, an amiRNA derived from miR-17 and an amiRNA derived from miR-19a. In some embodiments the plurality of amiRNAs comprises from 5 to 3’ an amiRNA derived from miR-30a, an amiRNA derived from miR-17, an amiRNA derived from miR-19a and an amiRNA derived from miR-20a. The plurality of amiRNAs encoded by a plurality of polynucleotides in the second amiRNA coding region may be derived from one or more of the miRNAs miR-106a, miR-18b, miR- 20b, miR-19b-2, miR-92a-2 and miR-363. For example, the plurality of amiRNAs may be derived from miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2 and miR-363. In some embodiments, the plurality of amiRNAs comprises from 5’ to 3’ an amiRNA derived from miR-106a, an amiRNA derived from miR-18b, an amiRNA derived from miR-20b, an amiRNA derived from miR-19b-2, an amiRNA derived from miR-92a-2 and an amiRNA derived from miR-363. In some embodiments, the second amiRNA coding region is downstream of the protein- coding region. In some embodiments, the first amiRNA coding region is downstream of the protein-coding region. In some embodiments the second amiRNA coding region is adjacent to the first amiRNA coding region. For example, the construct may comprise from 5’ to 3’ (i) the first amiRNA coding region, (ii) the second amiRNA coding region and (iii) the protein coding region. In some embodiments, the construct may comprise from 5’ to 3’ (i) the protein coding region, (ii) the first amiRNA coding region and (iii) the second amiRNA coding region. In some embodiments, the construct comprises from 5’ to 3’ (i) the first amiRNA coding region, (ii) the protein-coding region and (iii) the second amiRNA coding region. In some embodiments, the construct comprises from 5’ to 3’ (i) an RNA polymerase II- dependent promoter, (ii) the first amiRNA coding region, (iii) the protein-coding region and (iv) the second amiRNA coding region. Optionally, the construct further comprises two LTRs. In some embodiments, the two LTRs flank the amiRNA and protein-coding regions. In some embodiments, the construct comprises from 5’ to 3’ (i) a 5’ LTR, (ii) the amiRNA coding region, (iii) the protein-coding region, and (iv) a 3’ LTR. In some embodiments, the construct comprises from 5’ to 3’ (i) a 5’ LTR, (ii) the first amiRNA coding region, (iii) the protein-coding region, (iv) the second amiRNA coding region and (v) a 3’ LTR. The construct may comprise from 5’ to 3’, (i) the 5’ LTR, (ii) a splice donor, (iii) the amiRNA coding region, (iv) a splice acceptor, (v) the protein-coding region, and (vi) the 3’ LTR. Alternatively, the construct may comprise, from 5’ to 3’ (i) the 5’ LTR, (ii) a splice donor, (iii) the first amiRNA coding region, (iv) a splice acceptor, (v) the protein-coding region, (vi) the second amiRNA coding region and (vii) the 3’ LTR. Chimeric Antigen Receptor Various CARs are suitable for use and may be encoded by the polynucleotide construct. In particular, the CAR may comprise or consist of a first, second, third, or fourth generation CAR. First-generation CARs comprise or consist of a binding domain that is capable of specifically binding to an epitope on a target antigen, a transmembrane domain, and one or more intracellular signalling domains. Typically, first-generation CARs comprise or consist of a binding domain that is capable of specifically binding to an epitope on a target antigen, a transmembrane domain, and one intracellular signalling domain. The extracellular binding domain may comprise a single‐chain variable fragment (scFv) from a monoclonal antibody. A first-generation CAR typically comprises a CD3ζ chain domain or a variant thereof as the intracellular signalling domain, which is the primary transmitter of signals. In addition to the components specified for first-generation CARs, second-generation CARs also contain a co‐stimulatory domain, such as CD28 and/or 4‐1BB. Typically, second generation CARs contain one co-stimulatory domain, such as CD28 or 4-1BB. The inclusion of an intracellular co-stimulatory domain improves T-cell proliferation, cytokine secretion, resistance to apoptosis, and in vivo persistence. The co-stimulatory domain of a second- generation CAR is typically in cis with and upstream of the one or more intracellular signalling domains. Generally, the co-stimulatory domain of a second-generation CAR is typically in cis with and upstream of the one intracellular signalling domains. Third‐generation CARs combine multiple co-stimulatory domains in cis with one or more intracellular signalling domains, to augment T-cell activity. Typically, third generation CARs combine two co-stimulatory domains in cis with an intracellular signalling domain. For example, a third-generation CAR may comprise co-stimulatory domains derived from CD28 and 41BB, together with an intracellular signalling domain derived from CD3 zeta. Other third-generation CARs may comprise co-stimulatory domains derived from CD28 and OX40, together with an intracellular signalling domain derived from CD3 zeta. Fourth‐generation CARs (also known as TRUCKs or armoured CARs), combine the features of a second‐generation CAR with further factors to enhance anti-tumour activity (e.g., cytokines, co‐stimulatory ligands, chemokines receptors or further chimeric receptors of immune regulatory or cytokine receptors). The factors may be in trans or in cis with the CAR, typically in trans with the CAR. In some embodiments, the CAR is specific for a cancer antigen. The cancer antigen may be a solid tumour cancer antigen. By “specific”, in the context of the CAR, this will be understood to refer to being capable of specifically binding to a target antigen. Cancer antigens include, but are not necessarily limited to ErbB1, ErbB3, ErbB4, ErbB2, mucins, PSMA, carcinoembryonic antigen (CEA), mesothelin, GD2, MUC1, folate receptor, NKG2D ligands, ligands bound by other NK receptors such as NKp30, NKp44 or NKp46, GPC3, CAIX, FAP, NY-ESO-1, gp100, PSCA, ROR1, PD-L1, PD-L2, EpCAM, EGFRvIII, CD19, CD20, GD3, CLL-1, ductal epithelial mucin, CA-125, GP36, TAG-72, glycosphingolipids, glioma-associated antigen, beta-hCG, AFP (alpha-fetoprotein) and lectin-reactive AFP, thyroglobulin, receptor for advanced glycation end products (RAGE), TERT, telomerase, carboxylesterase, M-CSF, M-CSF receptor, PSA, tyrosinase, survivin, PCTA-1, melanoma- associated antigen (MAGE), for example MAGE A1, MAGE A2, MAGE A4, MAGE A8, CD22, IGF-1, IGF-2, IGF-1 receptor, MHC-associated tumour peptide, 5T4, tumour stroma- associated antigens, WT1, MLANA, CA 19-9, epithelial tumour antigen (ETA), BCMA, cancer testis antigens such as CTA New York (o)esophageal squamous cell carcinoma (NYESO) and glycoprotein 100 (GP100), preferentially expressed antigen in melanoma (PRAME), collagen type IV alpha 3 chain (COL6A3), MR1, CD1c, human epidermal growth factor receptor 2 (HER2), solute carrier family 3 member 2 (SLC3A2) and avb6 integrin. In some embodiments, the cancer antigen is selected from Axl, ErbB1, ErbB2, ErbB3, ErbB4, mucins, PSMA, carcinoembryonic antigen (CEA), mesothelin, GD2, MUC1, folate receptor, NKG2D ligands, ligands bound by other NK receptors such as NKp30, NKp44 or NKp46, GPC3, CAIX, FAP, NY-ESO-1, gp100, PSCA, ROR1, PD-L1, PD-L2, EpCAM, EGFRvIII, CD19, CD20, CD22, GD3, CLL-1, ductal epithelial mucin, CA-125, GP36, TAG-72, glycosphingolipids, glioma-associated antigen, beta-hCG, AFP (alpha-fetoprotein) and lectin- reactive AFP, thyroglobulin, receptor for advanced glycation end products (RAGE), TERT, carboxylesterase, M-CSF, M-CSF receptor, PSA, tyrosinase, survivin, PCTA-1, melanoma- associated antigen (MAGE), for example MAGE A1, MAGE A2, MAGE A4, MAGE A8, IGF-1, IGF-2, IGF-1 receptor, MHC-associated tumour peptide, 5T4, tumour stroma-associated antigens, WT1, MLANA, CA 19-9, epithelial tumour antigen (ETA), BCMA, cancer testis antigens such as CTA New York (o)esophageal squamous cell carcinoma (NYESO) and glycoprotein 100 (GP100), preferentially expressed antigen in melanoma (PRAME), collagen type IV alpha 3 chain (COL6A3), MR1, CD1c, solute carrier family 3 member 2 (SLC3A2) and avb6 integrin. In some embodiments, the cancer antigen is selected from NYESO, GP100, PRAME, COL6A3, MR1, CD1c, HER2, SLCA2, CD19, PSMA, AFP, CEA, CA-125, MUC1, ETA, tyrosinase and MAGE. In some embodiments, the CAR is an anti-CD19, anti-SLC3A2 or anti-PSMA CAR. In some embodiments, the CAR is an anti-CD19 or anti-PSMA CAR. MAGE may be selected from MAGE A1, MAGE A2, MAGE A4 or MAGE A8. In some embodiments, the CAR is not specific for MAGE-A4. The CAR may be linked to a reporter protein, for example GFP, MYC epitope flag or a FLAG epitope tag. Other suitable reporter proteins will be known to those skilled in the art. In some embodiments, the CAR comprises a second-generation CAR. Suitable CAR intracellular signalling domains may include any suitable signalling domain, including any region comprising an Immune-receptor-Tyrosine-based-Activation-Motif (ITAM), as reviewed for example by Love et al. Cold Spring Harbor Perspect. Biol 2010 2(6)l a002485. In some embodiments, the signalling domain comprises DAP12, which is discussed in further detail below. In some embodiments, the signalling domain comprises the intracellular domain of human CD3 [zeta] chain as described for example in US Patent No 7,446,190, or a variant thereof. In particular, this comprises the domain which spans amino acid residues 52-163 of the full- length human CD3 zeta chain. It has a number of polymorphic forms (e.g. Sequence ID: gb|AAF34793.1 (SEQ ID NO:142) and gb|AAA60394.1 (SEQ ID NO: 143)). Various T-cell co-stimulatory domains are known from previous work to engineer CAR T- cells. The CAR may comprise one or more of these domains. Suitable co-stimulatory domains include, but are not necessarily limited to members of the B7/CD28 family such as B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-1, PD-L2 or PDCD6; or ILT/CD85 family proteins such as LILRA3, LILRA4, LILRB1, LILRB2, LILRB3 or LILRB4; or tumour necrosis factor (TNF) superfamily members such as 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, Lymphotoxin-alpha, OX40, RELT, TACI, TL1A, TNF-alpha or TNF RII; or members of the SLAM family such as 2B4, BLAME, CD2, CD2F-10, CD48, CD58, CD84, CD229, CRACC, NTB-A or SLAM; or members of the TIM family such as TIM-1, TIM-3 or TIM-4; or other co- stimulatory molecules such as CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP12, Dectin- 1, DPPIV, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3 or TSLP R. In some embodiments, the CAR comprises a co-stimulatory domain selected from B7/CD28 family such as B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, DAP10, PD-1, PD-L2 or PDCD6; or ILT/CD85 family proteins such as LILRA3, LILRA4, LILRB1, LILRB2, LILRB3 or LILRB4; or tumour necrosis factor (TNF) superfamily members such as 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, Lymphotoxin-alpha, OX40, RELT, TACI, TL1A, TNF-alpha or TNF RII; or members of the SLAM family such as 2B4, BLAME, CD2, CD2F-10, CD48, CD58, CD84, CD229, CRACC, NTB-A or SLAM; or members of the TIM family such as TIM-1, TIM-3 or TIM-4; or other co- stimulatory molecules such as CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP12, Dectin- 1, DPPIV, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3 or TSLP R. The CAR co-stimulatory domain may be selected from CD28, CD27, ICOS, 4-1BB, OX40, CD30, GITR, HVEM, DR3, DAP10 or CD40. In some embodiments, the CAR co-stimulatory domain comprises CD28, 4-1BB, OX40, CD40, DAP10 or CD27. The CAR co-stimulatory domain may be selected from CD28, CD27, ICOS, 4-1BB, OX40, CD30, GITR, HVEM, DR3 or CD40. In some embodiments, the CAR co-stimulatory domain comprises CD28, 4-1BB, OX40, CD40 or CD27. In some embodiments, the CAR co- stimulatory domain comprises CD28, 4-1BB or OX40. In some embodiments, the CAR co- stimulatory domain comprises 4-1BB or OX40. The CAR costimulatory domain may comprise CD28. The transmembrane domain may comprise a CD8 α, CD28, CD4 or CD3 zeta transmembrane domain. In some embodiments, the transmembrane domain is a CD28 transmembrane domain. The CAR may comprise an NKG2D protein or variant thereof. In the context of the present invention, the term “NKG2D protein” refers to one or more NKG2D domains, or variants thereof. By NKG2D domain, this may refer to a specified domain, for example an intracellular, extracellular and/or transmembrane region. Alternatively, the term NKG2D domain may be used to refer to a portion of a full NKG2D protein. Thus, the term “NKG2D protein” encompasses full NKG2D proteins, fragments of NKG2D proteins and one or more domains of NKG2D proteins. In some embodiments, the CAR comprises an extracellular NKG2D domain or variant thereof. In such embodiments, it will be appreciated that the binding domain of the CAR will comprise this extracellular NKG2D domain or variant thereof. For the purposes of the present invention, the terms protein and polypeptide are used interchangeably. As used herein, the term “variant” in the context of a protein or nucleotide sequence encompasses a sequence which is a naturally occurring polymorphic form of the basic sequence as well as synthetic variants, in which one or more nucleotides or amino acids within the sequence are inserted, removed or replaced. The variant may otherwise be referred to as a functional variant, in that while one or more of the nucleotides/amino acids within the chain are inserted, removed, or replaced, relative to the basic sequence, the protein encoded by/of the variant substantially retains the functional activity of the protein encoded by/of the basic sequence. “Substantially retains” will be understood to refer to a functional activity of at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% of the protein encoded by/of the basic sequence. A variant of the present invention may have a functional activity equivalent or improved to the basic sequence. Functional variants also encompass truncated versions of the protein or nucleotide sequence. Truncated versions are shortened versions of the basic nucleotide or peptide sequence which produces a biological effect in the translated protein which is equivalent to or improved relative to the protein encoded by the basic sequence. In some embodiments, the NKG2D protein is a human NKG2D protein. Wild-type human NKG2D is encoded by the amino acid sequence having UniProt accession no: P26718 (SEQ ID NO:144). Thus, in some embodiments, the NKG2D protein comprises or consists of SEQ ID NO:144. In other embodiments, the NKG2D protein is a murine NKG2D protein. As the skilled person will appreciate, the term murine refers to rat and mouse. Thus, the NKG2D protein may be a mouse NKG2D protein. Wild-type mouse NKG2D is encoded by the amino acid sequence having UniProt accession no: O54709 (SEQ ID NO:145). The first 66 amino acids are considered to be the intracellular domain, amino acids 67-89 the transmembrane domain, and amino acids 90-232 the extracellular domain. Thus, in some embodiments, the NKG2D protein comprises or consists of SEQ ID NO:145. In some embodiments, the NKG2D protein is a rat NKG2D protein. Wild-type rat NKG2D is encoded by the amino acid sequence having UniProt accession no: O70215 (SEQ ID NO:146). The first 51 amino acids are considered to be the intracellular domain, amino acids 52-74 the transmembrane domain, and amino acids 75-215 the extracellular domain. Thus, in some embodiments, the NKG2D protein comprises or consists of SEQ ID NO:146. In some embodiments, the NKG2D protein comprises a murine NKG2D transmembrane domain or a variant thereof. In some embodiments, the NKG2D protein comprises a human NKG2D extracellular domain or a variant thereof. The NKG2D protein may be a chimeric NKG2D protein. In the context of the present invention, the term “chimeric NKG2D protein” refers to a NKG2D protein which is formed of NGK2D domains, or variants thereof, from two or more different organisms. This may comprise NKG2D domains from at least human and murine sources. In some embodiments, the chimeric NKG2D protein is a human-murine chimeric protein. Murine may be selected from rat, mouse, and combinations thereof. Thus, the chimeric NKG2D protein may be human-mouse. Alternatively, the chimeric NKG2D protein may be human-rat, or human, rat and mouse. The chimeric NKG2D protein may comprise a murine NKG2D transmembrane domain and a human NKG2D extracellular domain, or variants thereof. In some embodiments, the chimeric NKG2D protein or a variant thereof comprises N- terminal to C-terminal a murine NKG2D transmembrane domain or a variant thereof and a human NKG2D extracellular domain or a variant thereof. In some embodiments, the murine NKG2D transmembrane domain is a mouse NKG2D transmembrane domain. An exemplary mouse NKG2D transmembrane domain sequence is SEQ ID NO:147. SEQ ID NO:147 may otherwise be identified as amino acids 67-89 of UniProt accession no: O54709. In some embodiments, the mouse NKG2D transmembrane domain comprises or consists of SEQ ID NO:147. Other mouse NKG2D transmembrane domains are envisaged. For example, the mouse NKG2D transmembrane domain (such as, for example, SEQ ID NO:147) may further comprise a portion of a mouse NKG2D extracellular domain, and optionally a portion of a mouse NKG2D intracellular domain. The portion of the mouse NKG2D extracellular domain may be at the N-terminus of the mouse NKG2D transmembrane domain. The portion of the mouse NKG2D intracellular domain may be at the C-terminus of the mouse NKG2D transmembrane domain. Alternatively, the portion of the mouse NKG2D extracellular domain may be at the C-terminus of the mouse NKG2D transmembrane domain. The portion of the mouse NKG2D intracellular domain may be at the N-terminus of the mouse NKG2D transmembrane domain. By “portion”, this may be 1, 2, 3, 4, 6, 7, 8, 9 or 10 amino acids. Each portion may be at least 5, and less than 11 amino acids. In an embodiment, the portion of the mouse NKG2D extracellular domain may be 6 amino acids. In an embodiment, the portion of the mouse NKG2D intracellular domain may be 10 amino acids. An exemplary mouse NKG2D transmembrane domain, which comprises SEQ ID NO:147, a portion of a mouse NKG2D extracellular domain and a portion of a mouse NKG2D intracellular domain is SEQ ID NO:148. SEQ ID NO:148 represents amino acids 61-97 of UniProt accession no: O54709. In some embodiments, the mouse NKG2D transmembrane domain comprises or consists of SEQ ID NO:148. Rat NKG2D transmembrane domains are also suitable transmembrane domains for the present invention. Thus, in some embodiments, the murine NKG2D transmembrane domain is a rat NKG2D transmembrane domain. One example of a rat NKG2D transmembrane domain is SEQ ID NO:149, which corresponds to amino acids 52-74 of UniProt accession no: O70215. The rat NKG2D transmembrane domain may comprise or consist of SEQ ID NO:149. A variant of the murine NKG2D transmembrane domain may have at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the murine NKG2D transmembrane domain, for example to a mouse NKG2D transmembrane domain (such as SEQ ID NO:147 or SEQ ID NO:148) or to a rat NKG2D transmembrane domain (such as SEQ ID NO:149). A variant may have at least 90%, optionally at least 95% sequence identity to a mouse NKG2D transmembrane domain or a rat NKG2D transmembrane domain. Alternatively, a variant murine NKG2D transmembrane domain may comprise a polypeptide comprising one or more point (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations that add, delete or substitute any of the amino acids compared to SEQ ID NO:147, SEQ ID NO:148 or SEQ ID NO:149. The functional activity of the variant can be measured using functional assays, such as MTT and measuring cytokine secretion by ELISA. Exemplary human NKG2D extracellular domains include, but are not necessarily limited to, SEQ ID NO:150 and SEQ ID NO:151. SEQ ID NO:151 comprises SEQ ID NO:150, with the additional 9 amino acid sequence IWSAVFLNS (SEQ ID NO:152) at the N-terminus. Another exemplary human NKG2D extracellular domain is SEQ ID NO:153. SEQ ID NO:153 corresponds to SEQ ID NO:150, except that the eight most N-terminal amino acids have been removed in SEQ ID NO:153, as compared to SEQ ID NO:150. Thus, in some embodiments, the human NKG2D extracellular domain comprises or consists of SEQ ID NO:153. Alternatively, the human NKG2D extracellular domain may comprise or consist of SEQ ID NO:150. In some embodiments, the human NKG2D extracellular domain comprises or consists of SEQ ID NO:151. A variant of the human NKG2D extracellular domain may have at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity to the human NKG2D extracellular domain, for example SEQ ID NO:150, SEQ ID NO:151 or SEQ ID NO:152. The variant may have at least 90% or at least 95% sequence identity to the human NKG2D extracellular domain. In some embodiments, the variant comprises a polypeptide comprising one or more point (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations that add, delete or substitute any of the amino acids compared to SEQ ID NO:150, SEQ ID NO:151 or SEQ ID NO:152. The one or more point mutations may be one or more point (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) deletions of any of the amino acids compared to SEQ ID NO:150. Alternatively, the one or more point mutations may be one or more point (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) deletions of any of the amino acids compared to SEQ ID NO:151. Optionally, the one or more point mutations are one or more point (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) deletions of any of the amino acids compared to SEQ ID NO:150 and SEQ ID NO:151. In some embodiments, the one or more point deletions are at the N-terminus of SEQ ID NO:150. Alternatively, the one or more point deletions are at the N-terminus of SEQ ID NO:151. Such deletions at the N-terminus of the human NKG2D extracellular domain result in a variant which is a truncated human NKG2D extracellular domain. Variant functional activity may be measured as described above. In some embodiments, the NKG2D protein comprises or consists of a human NKG2D transmembrane domain and a human NKG2D extracellular domain, or variants thereof. For example, the NKG2D protein may comprise or consist of SEQ ID NO:154 (which specifies the human NKG2D transmembrane domain and a human NKG2D extracellular domain), or a variant thereof. A variant may have at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity to SEQ ID NO:154. In some embodiments, a variant is a polypeptide comprising one or more point (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations that add, delete or substitute any of the amino acids compared to SEQ ID NO:154. In some embodiments, the NKG2D protein further comprises an intracellular NKG2D domain or a variant thereof. Optionally, the intracellular NKG2D domain or a variant thereof is at the N-terminus of a NKG2D transmembrane domain, optionally a murine NKG2D transmembrane domain, or variant thereof. In some embodiments, the intracellular NKG2D domain or a variant thereof is N-terminal to a NKG2D transmembrane domain, optionally to a murine NKG2D transmembrane domain, or a variant thereof. In some embodiments the intracellular NKG2D domain or a variant thereof is located at the N-terminus of the NKG2D protein or variant thereof. The intracellular NKG2D domain may be a human NKG2D intracellular domain. An exemplary human NKG2D intracellular domain is SEQ ID NO:155. Another exemplary human NKG2D intracellular domain is SEQ ID NO:156. SEQ ID NO:156 corresponds to SEQ ID NO:155, except that the last amino acid at the C-terminus has been removed. The human NKG2D intracellular domain may comprise or consist of SEQ ID NO:155. In some embodiments, the human NKG2D intracellular domain comprises or consists of SEQ ID NO:156. Alternatively, the intracellular NKG2D domain may be a murine NKG2D intracellular domain. The murine NKG2D intracellular domain comprises or consists of a short isoform murine NKG2D intracellular domain. The murine NKG2D intracellular domain may be a mouse NKG2D intracellular domain. Exemplary short isoform mouse NKG2D intracellular domains include, but are not necessarily limited to, SEQ ID NO:157 and SEQ ID NO:158. In some embodiments, the intracellular NKG2D domain comprises or consists of SEQ ID NO:157. In other embodiments, the intracellular NKG2D domain comprises or consists of SEQ ID NO:158. Alternatively, the murine NKG2D intracellular domain may be a rat NKG2D intracellular domain. For example, the rat NKG2D intracellular domain may comprise or consist of SEQ ID NO:159. SEQ ID NO:159 corresponds to amino acids 1-51 of UniProt accession number: O70215 (SEQ ID NO:146). An intracellular NKG2D domain variant may have at least at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity to the intracellular NKG2D domain, for example a human (such as SEQ ID NO:155 or SEQ ID NO:156), mouse (such as SEQ ID NO:157 or SEQ ID NO:158) or rat (such as SEQ ID NO:159) intracellular NKG2D domain. An intracellular NKG2D domain variant may have at least 90% or at least 95% sequence identity to the intracellular NKG2D domain. In some embodiments, a variant is a polypeptide comprising one or more point (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations that add, delete or substitute any of the amino acids compared to any of SEQ ID NOs:155-159. In some embodiments, the NKG2D protein does not comprise an NKG2D intracellular domain. In some embodiments, the NKG2D protein does not comprise a mouse NKG2D intracellular domain. The NKG2D protein may not comprise a murine NKG2D intracellular domain. Optionally, the NKG2D protein does not comprise an NKG2D intracellular domain. In some embodiments the NKG2D protein does not comprise a human NKG2D intracellular domain. The protein coding region may further comprise a polynucleotide encoding a DNAX- activating protein 10 (DAP10) and/or a DNAX-activating protein 12 (DAP12) polypeptide, or variants thereof. For example, the protein coding region may further comprise a polynucleotide encoding a DAP12 polypeptide or a variant thereof. The DAP12 polypeptide may be mammalian, for example, murine (such as mouse or rat) or human. In some embodiments, the DAP12 polypeptide is human. Wild-type human DAP12 has the amino acid sequence having UniProt accession no: O43914 (SEQ ID NO:160). The first 21 amino acids are considered to be a signal/leader sequence, amino acids 22-40 the extracellular domain, amino acids 41-61 the transmembrane domain, and amino acids 62- 113 the cytoplasmic/intracellular domain. The DAP12 polypeptide may comprise or consist of SEQ ID NO:160. The DAP12 polypeptide may comprise a truncated DAP12 polypeptide. For example, the DAP12 polypeptide may comprise or consist of a truncated DAP12 comprising only amino acids 62-113 of SEQ ID NO:160 (i.e. the intracellular domain). Such a sequence is referred to as SEQ ID NO:161. Other truncated DAP12 polypeptides may comprise amino acids 41-61 of SEQ ID NO:160, such a sequence comprising merely the transmembrane domain of human DAP12, and referred to here as SEQ ID NO:162. Thus, in some embodiments, the DAP12 polypeptide comprises or consists of SEQ ID NO:162. Another truncated DAP12 polypeptide may comprise amino acids 22-61 of SEQ ID NO:160 (i.e. the extracellular and transmembrane domains), referred to as SEQ ID NO:163 herein. In some embodiments, the DAP12 polypeptide may comprise or consist of SEQ ID NO:163. Another truncated DAP12 polypeptide is SEQ ID NO:164. SEQ ID NO:164 comprises only amino acids 22-113 of SEQ ID NO:160 (i.e. lacking amino acids 1-21, the signal/leader sequence). In some embodiments, the DAP12 polypeptide comprises or consists of SEQ ID NO:164. In some embodiments, the DAP12 polypeptide comprises SEQ ID NO:165. SEQ ID NO:165 comprises a human DAP12 transmembrane domain and a human DAP12 intracellular domain (amino acids 41-113 of UniProt accession no: 043914). The DAP12 polypeptide, such as SEQ ID NO:165, may further comprise an extracellular domain peptide sequence. For example, the DAP12 polypeptide, such as SEQ ID NO:165, may further comprise a human DAP12 extracellular domain peptide sequence. The DAP12 polypeptide may be a murine polypeptide, optionally a mouse polypeptide. Wild- type mouse DAP12 has the amino acid sequence having UniProt accession no: O54885 (SEQ ID NO:166). The first 21 amino acids are considered to be a signal/leader sequence, amino acids 22-42 the extracellular domain, amino acids 43-63 the transmembrane domain, and amino acids 64-114 the cytoplasmic/intracellular domain. The DAP12 polypeptide may comprise or consist of SEQ ID NO:166. The murine DAP12 polypeptide may comprise a truncated murine DAP12 polypeptide. In some embodiments, the murine DAP12 polypeptide comprises or consists of a truncated murine DAP12 polypeptide only amino acids 64-114 of SEQ ID NO:166 (i.e. the intracellular domain). Such a sequence is referred to as SEQ ID NO:167. In other embodiments, the DAP12 polypeptide may comprise or consist of amino acids 43- 63 of SEQ ID NO:166. Such a sequence is referred to as SEQ ID NO:168. Another truncated DAP12 polypeptide of the invention is SEQ ID NO:169. SEQ ID NO:169 comprises the murine extracellular (aa 22-42) and murine transmembrane DAP12 (aa 43- 63) regions. Thus, in some embodiments the DAP12 polypeptide comprises or consists of SEQ ID NO:169. A further exemplary truncated murine DAP12 polypeptide is amino acids 22-114 of SEQ ID NO:166. Such a sequence comprises the murine extracellular, transmembrane and intracellular DAP12 domains, and is referred to herein as SEQ ID NO:170. Thus, in an embodiment, the DAP12 polypeptide comprises or consists of SEQ ID NO:170. A DAP12 variant may have at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity to the DAP12 polypeptide, for example to a human DAP12 polypeptide (such as any of SEQ ID NO:160, 161, 162, 163, 164 and 165) or to a mouse DAP12 polypeptide (such as any of SEQ ID NO:166, 167, 168, 169 and 170). The DAP12 variant may comprise a peptide comprising one or more (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) point mutations that add, delete or substitute any of the amino acids of the amino acids of DAP12 (such as that of wild-type human DAP12 (e.g. SEQ ID NO:160, 161, 162, 163, 164 and 165) or wild-type mouse DAP12 (e.g. SEQ ID NO:166, 167, 168, 169 and 170). In some embodiments, the protein coding region further comprises a polynucleotide encoding a DAP10 polypeptide or a variant thereof. The DAP10 polypeptide may be mammalian, for example, murine (such as mouse or rat) or human. In some embodiments, the DAP10 polypeptide is human. Wild-type human DAP10 has the amino acid sequence having UniProt accession no: Q9UBK5 (SEQ ID NO:171). This is a 93aa polypeptide. The first 18aa are considered to be a signal/leader sequence, amino acids 19-48 the extracellular domain, amino acids 49-69 the transmembrane domain, and amino acids 70-93 the cytoplasmic/intracellular domain. Thus, in some embodiments, the DAP10 polypeptide may comprise or consist of SEQ ID NO:171. The DAP10 polypeptide may comprise a truncated DAP10 polypeptide. For example, the DAP10 polypeptide may comprise or consist of a truncated DAP10 polypeptide comprising amino acids 19-93 of SEQ ID NO:171 (i.e. lacking amino acids 1-18, the signal/leader sequence). Such a sequence is referred to as SEQ ID NO:173 herein. Thus, the DAP10 polypeptide may comprise or consist of SEQ ID NO:173. Another truncated DAP10 polypeptide may comprise amino acids 70-93 of SEQ ID NO:171 (i.e. the intracellular domain), referred to as SEQ ID NO:172 herein. The DAP10 polypeptide may comprise or consist of SEQ ID NO:172. Other truncated DAP10 polypeptides may comprise or consist of amino acids 19-69 of SEQ ID NO:171, such a sequence comprising merely the extracellular and transmembrane domains of DAP10, and referred to herein as SEQ ID NO:174. A further truncated DAP10 polypeptide may comprise or consist of amino acids 1-71 of SEQ ID NO:171 (i.e. the signal/leader sequence, extracellular domain, transmembrane domain and 2 amino acids from the cytoplasmic/intracellular domain), referred to as SEQ ID NO:175 herein. A further truncated DAP10 polypeptide may comprise or consist of amino acids 19-71 of SEQ ID NO:171 (i.e. the extracellular domain, transmembrane domain and 2 amino acids from the cytoplasmic/intracellular domain), referred to as SEQ ID NO:176 herein. A yet further truncated DAP10 polypeptide may comprise or consist of amino acids 49-93 of SEQ ID NO:171 (i.e. the transmembrane and cytoplasmic/intracellular domains), referred to as SEQ ID NO:177 herein. A yet further truncated DAP10 polypeptide may comprise or consist of amino acids 49-69 of SEQ ID NO:171 (i.e. the transmembrane domain), referred to as SEQ ID NO:178 herein. In other embodiments the DAP10 polypeptide or variant thereof is murine, optionally mouse. Wild-type mouse DAP10 has the amino acid sequence having UniProt accession no: Q9QUJ0 (SEQ ID NO:180). This is a 79aa polypeptide. The first 17aa are considered to be a signal/leader sequence, amino acids 18-35 the extracellular domain, amino acids 36-56 the transmembrane domain, and amino acids 57-79 the cytoplasmic/intracellular domain. Thus, in some embodiments, the DAP10 polypeptide may comprise or consist of SEQ ID NO:180. In some embodiments, the DAP10 polypeptide comprises or consists of a truncated mouse DAP10 polypeptide. A truncated mouse DAP10 polypeptide comprising only amino acids 18- 79 of SEQ ID NO:180 (i.e. lacking amino acids 1-18, the signal/leader sequence) may be used as the DAP10 polypeptide of the invention. Such a sequence is referred to as SEQ ID NO:181 herein. The DAP10 polypeptide may comprise or consist of SEQ ID NO:181. Another truncated mouse DAP10 polypeptide is SEQ ID NO:179. SEQ ID NO:179 comprises only amino acids 57-79 (intracellular region) of SEQ ID NO:179. Thus, in some embodiments, the DAP10 polypeptide comprises or consists of SEQ ID NO:179. A DAP10 polypeptide variant may have at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity to a human DAP10 polypeptide (such as any of SEQ ID NO:171, 172, 173, 174, 175, 176, 177 or 178) or a murine (optionally mouse) DAP10 polypeptide (such as SEQ ID NO:179, 180 or 181). The variant may have at least 90% or at least 95% sequence identity to a human DAP10 polypeptide or a murine, optionally mouse DAP10 polypeptide. In some embodiments, the functional activity of the variant is measured by assessment of tyrosine phosphorylation of DAP10 and/or recruitment and activation of the p85 subunit of phosphatidylinositol 3-kinase and the downstream anti-apoptotic kinase, AKT. In some embodiments, the DAP10 polypeptide variant is a peptide comprising one or more point (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations that add, delete or substitute any of the amino acids compared to any of SEQ ID NOs 171-181. Optionally, the DAP10 polypeptide variant is a peptide comprising one or more point (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations that add, delete or substitute any of the amino acids compared to any of SEQ ID NOs 171-178. Alternatively, the DAP10 polypeptide variant is a peptide comprising one or more point (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations that add, delete or substitute any of the amino acids compared to any of SEQ ID NOs 179-181. In some embodiments the protein coding region further comprises a polynucleotide encoding a DAP10 polypeptide and a polynucleotide encoding a DAP12 polypeptide, or variants thereof. The DAP10 and DAP12 polypeptides, or variants thereof, may be as defined above. In some embodiments, the protein coding region comprises a polynucleotide encoding a DAP10 polypeptide, or variant thereof, fused to a DAP12 polypeptide, or variant thereof. This may otherwise be referred to as a DAP10/12 fusion protein. Any of the DAP10 and DAP12 polypeptides, or variants thereof as defined above may be suitable for such a fusion protein. For example, the DAP12 polypeptide or variant thereof may be fused to a human DAP10 extracellular domain peptide sequence (such as SEQ ID NO:182). The DAP12 polypeptide may comprise a human DAP12 transmembrane domain and a human DAP12 intracellular domain (amino acids 41-113 of UniProt accession no: 043914), for example SEQ ID NO:165. Fusion may be direct or by a linker. In some embodiments, the construct further comprises a polynucleotide encoding a CXCR2 polypeptide. The CXCR2 polypeptide may be mammalian. In some embodiments, the CXCR2 polypeptide is a human CXCR2 polypeptide. The CXCR2 polypeptide may comprise SEQ ID NO:274. The protein coding region may comprise a polynucleotide encoding a DAP10 polypeptide, or variant thereof, fused to an NKG2D protein, or variant thereof. The NKG2D protein or variant thereof may be as defined above. In such embodiments, when expressed, the NKG2D protein or variant thereof will further comprise the DAP10 polypeptide or variant thereof. In some embodiments, fusion is to the N-terminus of the NKG2D protein or variant thereof. Fusion of the DAP10 polypeptide, or variant thereof, may be to the murine NKG2D transmembrane domain or a variant thereof in the NKG2D protein or variant thereof. Fusion may be direct or may be by a linker. Suitable exemplary linkers are described in more detail below. In other embodiments, the DAP12 polypeptide, or variant thereof, is fused to the NKG2D protein or variant thereof. Optionally, fusion is to the N-terminus of the NKG2D protein. In some embodiments, a DAP12 intracellular domain, as described herein, is fused to the NKG2D protein or variant thereof. Fusion of the DAP12 polypeptide, or variant thereof, may be to the murine NKG2D transmembrane domain or a variant thereof in the NKG2D protein or variant thereof. Optionally, the DAP12 intracellular domain used in such a fusion construct is human. For example, the DAP12 intracellular domain used in such a fusion construct may comprise or consist of SEQ ID NO:161. Fusion is optionally using a linker which comprises a cleavage site. In such embodiments, the linker can be cleaved to separate the DAP12 polypeptide or variant thereof and the NKG2D protein or variant thereof. As such, the protein coding region may comprise a polynucleotide encoding a DAP12 polypeptide or variant thereof and the NKG2D protein or variant thereof. In some embodiments, the CAR comprises an NKG2D protein or variant thereof fused to a DAP10 polypeptide or variant thereof. Preferably, the DAP10 polypeptide or variant thereof is fused to a DAP12 polypeptide or variant thereof. The NKG2D protein, DAP10 polypeptide, DAP12 polypeptide and variants thereof may be as defined above. The protein coding region may further comprise a polynucleotide encoding an immune signalling receptor polypeptide comprising an immunoreceptor tyrosine-based activation motif (ITAM). An ITAM is a conserved sequence of four amino acids that is repeated twice in the cytoplasmic tails of non-catalytic tyrosine phosphorylated receptors. The protein coding region may comprise a polynucleotide encoding a DAP10 and/or DAP12 polypeptide, or variants thereof, fused to an ITAM. The DAP12 and/or DAP10 polypeptide, or variant(s) thereof, and the ITAM may be directly fused together. Alternatively, they may be joined by a linker. The ITAM may be fused to the N- or the C-terminus of the DAP12 and/or DAP10 polypeptide or variant(s) thereof. Optionally, the ITAM is fused to the C-terminus of the DAP12 and/or the DAP10 polypeptide or variant(s) thereof. The zeta chain of a T-cell receptor, the eta chain of a T-cell receptor, the delta chain of a T- cell receptor, the gamma chain of a T-cell receptor, or the epsilon chain of a T-cell receptor (i.e. CD3 chains) or the gamma subunit of the FcR1 receptor may comprise the ITAM. Thus the protein coding region may comprise a polynucleotide encoding the zeta chain of a T-cell receptor, the eta chain of a T-cell receptor, the delta chain of a T-cell receptor, the gamma chain of a T-cell receptor, or the epsilon chain of a T-cell receptor (i.e. CD3 chains) or the gamma subunit of the FcR1 receptor. The protein coding region may comprise a polynucleotide encoding a CD3-zeta chain or gamma subunit of the FcR1 receptor. Various sequences may be attached to the N- or C-terminus of the CAR and/or to the DAP10 and/or DAP12 polypeptides (or variants thereof) disclosed herein. Such sequences may be encoded by a polynucleotide in the protein coding region. These sequences may be functional, such as signal peptides, purification tags/sequences, or half-life extension moieties, or may simply comprise spacer sequences. Alternatively, they may comprise a function such as a T-cell stimulatory function. Any of the polypeptides described herein may further comprise a signal peptide (otherwise referred to as a leader sequence). In particular, the DAP10 polypeptide or variant thereof and/or the DAP12 polypeptide or variant thereof, may further comprise a signal peptide. The signal peptide may optionally be fused to the N-terminus of the polypeptide. Various peptides are suitable as signal peptides. One suitable signal peptide is the CD8 α signal peptide sequence (amino acids 1-21 of UniProt: P01732 or a shortened derivative comprising amino acids 1-18). This is a commonly used T-cell sequence and is referred to as SEQ ID NO:183 herein. Thus, in some embodiments, the signal peptide is derived from a CD8α signal peptide. The signal peptide may comprise or consist of SEQ ID NO:183. In some embodiments, a signal peptide is fused to the N-terminus of the DAP10 polypeptide or variant thereof. The signal peptide may comprise SEQ ID NO:184 (aa 1-17 of SEQ ID NO:180). In some embodiments, a signal peptide is fused to the N-terminus of the DAP12 polypeptide or variant thereof. In some embodiments, the signal peptide comprises or consists of SEQ ID NO:185 (aa 1-21 of SEQ ID NO:166) or SEQ ID NO:186 (aa 1-21 of SEQ ID NO:160). In some embodiments, the DAP10, DAP12 and/or CAR polypeptide, or variants thereof, may further comprise a purification tag. The purification tag may be at the N or the C terminus of the polypeptide. Advantageously, purification tags may assist with purification. Examples of purification tags include, but are not necessarily limited to, a His-tag, a FLAG-tag, Arg-tag, T7-tag, Strep-tag, S-tag, aptamer-tag, V5 tag, AviTag^TM or myc epitope tag. In some embodiments the purification tag is a His-tag (usually comprising 5-10 histidine residues), for example a 6 His tag (i.e. HHHHHH) (SEQ ID NO:187). In other embodiments the purification tag is a FLAG tag (i.e. DYKDDDDK) (SEQ ID NO:188). In other embodiments, the purification tag is an AviTag^TM (i.e. GLNDIFEAQKIEWHE) (SEQ ID NO:189). In other embodiments, the purification tag is a V5 tag (GKPIPNPLLGLDST) (SEQ ID NO:190) or (IPNPLLGLD) (SEQ ID NO:191). In other embodiments, the purification tag is a myc epitope tag recognised by the 9e10 antibody (EQKLISEEDL) (SEQ ID NO:192). Various other tags are well known in the art. In some embodiments, the DAP10, DAP12 and/or CAR polypeptide, or variants thereof, may comprise a combination of purification tags, for example one or more tags at the N- terminus, one or more tags at the C-terminus, or one or more tags at each of the N- terminus and the C-terminus. Examples of such combinations include a His tag (H) combined with an AviTag (A), or a His tag (H) combined with both an AviTag (A) and a FLAG tag (F). The tags may be in either orientation, thus the AviTag/His tag may have the orientation N-AH-C or N-HA-C, while the Avi/His/FLAG tag may have the orientation N-AHF- C, N-FHA-C, etc. In some embodiments, a DAP10 or DAP12 polypeptide, or variant thereof, comprises a FLAG tag (i.e. DYKDDDDK) (SEQ ID NO:188). In particular, the DAP12 polypeptide or variant thereof may comprise a FLAG tag (SEQ ID NO:188). The FLAG tag may be positioned at or towards the N-terminus of the polypeptide, for example a DAP12 polypeptide or variant thereof. As described above, the DAP10 and/or DAP12 polypeptide, or variant thereof, may be fused to the CAR. Fusion may be direct or with a linker The linker may be a peptide linker. Peptide linkers are commonly used in fusion polypeptides and methods for selecting or designing linkers are well-known (see, e.g., Chen X et al., 2013, Adv. Drug Deliv. Rev. 65(10):135701369 and Wriggers W et al., 2005, Biopolymers 80:736-746.). Peptide linkers generally are categorized as i) flexible linkers, ii) helix forming linkers, and iii) cleavable linkers, and examples of each type are known in the art. Flexible linkers may contain a majority of amino acids that are sterically unhindered, such as glycine and alanine. The hydrophilic amino acid Ser is also conventionally used in flexible linkers. Examples of flexible linkers include, without limitation: polyglycines (e.g., (Gly)4 and (Gly)5), polyalanines poly(Gly-Ala), and poly(Gly-Ser) (e.g., (Glyn-Sern)n or (Sern-Glyn)n, wherein each n is independently an integer equal to or greater than 1). The peptide linker sequence may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or more amino acid residues in length. For example, a peptide linker can be from about 5 to about 50 amino acids in length; from about 10 to about 40 amino acids in length; from about 15 to about 30 amino acids in length; or from about 15 to about 20 amino acids in length. Variation in peptide linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. The peptide linker sequence may be comprised of naturally or non-naturally occurring amino acids, or a mixture of both naturally and non-naturally occurring amino acids. In some embodiments, the linker comprises the amino acid methionine, optionally at the C- terminus of the linker. In some embodiments, the linker comprises or consists of the amino acids glycine and serine. More specifically, the linker sequence may be SGSG (SEQ ID NO:193). In other embodiments, the linker sequence is GSGGG (SEQ ID NO:194). The linker sequence may be GSGG (SEQ ID NO:195). In other embodiments, a linker may contain glycine (G), serine (S) and proline (P) in a random or repeated patter. In a particular example, n is 1 and the linker is GPPGS (SEQ ID NO:196). In general, the linker is not immunogenic when administered to a subject, such as a human. Thus, linkers may be chosen such that they have low immunogenicity or are thought to have low immunogenicity. The linkers described herein are exemplary, and the linker can include other amino acids, such as Glu and Lys, if desired. Peptide linkers may also include cleavable linkers. The linkers may comprise further domains and/or features, such as a furin cleavage site (such as RRKR)(SEQ ID NO:197), a P2A ribosomal skip peptide (ATNFSLLKQAGDVEENPGP)(SEQ ID NO:198) and/or a T2A ribosomal skip peptide (EGRGSLLTCGDVEENPGP)(SEQ ID NO: 199). Examples of linkers comprising these domains include SGSG + a P2A ribosomal skip peptide (SGSGATNFSLLKQAGDVEENPGP)(SEQ ID NO:200), SGSG + a T2A ribosomal skip peptide (SGSGEGRGSLLTCGDVEENPGP)(SEQ ID NO:201), and versions also including a furin cleavage site, i.e. furin cleavage site + SGSG + a P2A ribosomal skip peptide (RRKRSGSGATNFSLLKQAGDVEENPGP) (SEQ ID NO:202) and furin cleavage site + SGSG + a T2A ribosomal skip peptide (RRKRSGSGEGRGSLLTCGDVEENPGP) (SEQ ID NO:203). Alternative ribosomal skip peptides that may be used in the invention include F2A (VKQTLNFDLLKLAGDVESNPGP) (SEQ ID NO:204) and E2A (QCTNYALLKLAGDVESNPGP) (SEQ ID NO:205). The furin cleavage site, P2A ribosomal skip peptide or T2A ribosomal skip peptide may comprise an additional methionine at the C-terminus. An exemplary linker comprising an additional methionine is SEQ ID NO:206, which includes SGSG + a P2A ribosomal skip peptide (SGSGATNFSLLKQAGDVEENPGP)(SEQ ID NO:200) + a methionine (M). In some embodiments the protein coding region further comprises a polynucleotide encoding a chimeric costimulatory receptor (CCR). In the context of the present invention, CCRs comprise a binding domain which specifically interacts with an epitope on a target antigen, a transmembrane domain and a co- stimulatory signalling domain. In some embodiments, the protein coding region comprises a polynucleotide encoding a second-generation CAR and a polynucleotide encoding a CCR. The combination of the second-generation CAR and the CCR may otherwise be referred to as “parallel chimeric activating receptors” or “pCAR”. CCRs typically do not comprise an intracellular activation domain. Thus, engagement of the CCR alone is not able to activate the host cell in which it is expressed. However, co- expression of a CCR with a CAR enables the CCR to bind to a second epitope to the CAR. This provides additional intracellular signalling to support activation of the host cell by the CAR. The CCR binding domain may specifically interact with a different epitope to the CAR binding domain. In other embodiments, the CCR binding domain specifically interacts with the same epitope as the CAR binding domain. In some embodiments, the CCR binding domain specifically interacts with a different epitope on the same target antigen to the CAR binding domain. The CCR transmembrane domain is typically positioned between the CCR binding domain and the CCR co-stimulatory signalling domain. Suitable CCR co-stimulatory signalling domains may include, but not necessarily be limited to B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-1, PD-L2 or PDCD6; or ILT/CD85 family proteins such as LILRA3, LILRA4, LILRB1, LILRB2, LILRB3 or LILRB4; or tumour necrosis factor (TNF) superfamily members such as 4- 1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, Lymphotoxin-alpha, OX40, RELT, TACI, TL1A, TNF-alpha or TNF RII; or members of the SLAM family such as 2B4, BLAME, CD2, CD2F-10, CD48, CD58, CD84, CD229, CRACC, NTB-A or SLAM; or members of the TIM family such as TIM-1, TIM-3 or TIM-4; or other co-stimulatory molecules such as CD7, CD81, CD96, CD160, CD200, CD300a, CRTAM, DAP12, Dectin-1, DPPIV, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3 or TSLP R. In some embodiments, the CCR co-stimulatory signalling domain is selected from B7-1, B7- 2, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, DAP10, PD-1, PD-L2 or PDCD6; or ILT/CD85 family proteins such as LILRA3, LILRA4, LILRB1, LILRB2, LILRB3 or LILRB4; or tumour necrosis factor (TNF) superfamily members such as 4- 1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, Lymphotoxin-alpha, OX40, RELT, TACI, TL1A, TNF-alpha or TNF RII; or members of the SLAM family such as 2B4, BLAME, CD2, CD2F-10, CD48, CD58, CD84, CD229, CRACC, NTB-A or SLAM; or members of the TIM family such as TIM-1, TIM-3 or TIM-4; or other co-stimulatory molecules such as CD7, CD81, CD96, CD160, CD200, CD300a, CRTAM, DAP12, Dectin-1, DPPIV, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3 or TSLP R. In some embodiments, the CCR co-stimulatory signalling domain is selected from CD28, CD27, ICOS, 4-1BB, OX40, CD30, GITR, HVEM, DR3, CD81 and CD40, or variants thereof. In some embodiments, the CCR co-stimulatory signalling domain is selected from 4-1BB, OX40, CD40 and CD27, or variants thereof. In some embodiments, the CCR co-stimulatory signalling domain comprises 4-1BB, OX40 or CD27. The CCR transmembrane domain may be different to the CAR transmembrane domain. This may assist with separation of the CAR and CCR on the surface of a host cell. In some embodiments the CCR transmembrane domain is selected from CD8 α, CD28, CD4 and CD3 zeta transmembrane domains. In some embodiments, the CCR transmembrane domain comprises a CD28 transmembrane domain. The full length CD28 protein is a 220 amino acid protein of SEQ ID NO:207. Thus, in some embodiments the CCR transmembrane domain comprises SEQ ID NO:207 or a variant thereof. In some embodiments, the CCR transmembrane domain comprises SEQ ID NO:208 or a variant thereof, wherein SEQ ID NO:208 is the CD28 transmembrane domain. In some embodiments the CAR co-stimulatory signalling domain comprises or consists of SEQ ID NO:209. Optionally, the protein coding region further comprises a polynucleotide encoding a chimeric cytokine receptor. The chimeric cytokine receptor may be a 4αβ chimeric cytokine receptor. In the 4 αβ chimeric cytokine receptor, the ectodomain of the IL-4 receptor- α chain is joined to the transmembrane and endodomains of IL-2/15 receptor-β. This allows the selective expansion and enrichment of host cells engineered to comprise the construct ex vivo by the culture of these cells in a suitable support medium, which, in the case of 4 αβ, would comprise IL-4 as the sole cytokine support. Alternatively, the protein coding region may comprise a polynucleotide encoding a chimeric cytokine receptor in which the ectodomain of the IL-4 receptor- α chain is joined to the transmembrane and endodomains of another receptor that is naturally bound by a cytokine that also binds to the common ^ chain. In some embodiments, the protein coding region further comprises a polynucleotide encoding a cytokine. The cytokine may comprise IL-2, IL-7, IL-9, IL-15, IL-17 and/or IL-21. In some embodiments, the protein coding region further comprises a polynucleotide encoding one or more of the following: HLA-E, HLA-G, HLA-E single chain trimer, CD80, 4- 1BB ligand, hyaluronidase, and neuraminidase. In some embodiments, the amiRNA coding region comprises a polynucleotide encoding an amiRNA specific for an endogenous TCR mRNA, wherein the amiRNA is derived from miR- 155. In some embodiments, the amiRNA coding region comprises a polynucleotide encoding an amiRNA specific for an endogenous TCR mRNA, wherein the amiRNA is derived from miR- 155 and the protein-coding region comprises a polynucleotide encoding a CAR comprising an NKG2D protein or variant thereof fused to a DAP10/12 fusion polypeptide or variant thereof. In some embodiments, the amiRNA coding region comprises a polynucleotide encoding an amiRNA specific for a CD3 mRNA, wherein the amiRNA is derived from miR-155 and the protein-coding region comprises a polynucleotide encoding a CAR comprising an NKG2D protein or variant thereof associated with a DAP10/12 fusion polypeptide or variant thereof. In some embodiments, the amiRNA coding region comprises six polynucleotides encoding amiRNAs derived from miR-17, miR-18, miR-19a, miR-19b, miR-20 and miR-92a-1, wherein each amiRNA is specific for an endogenous TCR mRNA. In some embodiments, the amiRNA coding region comprises six polynucleotides encoding amiRNAs derived from miR-17, miR-18, miR-19a, miR-19b, miR-20 and miR-92a-1, wherein each amiRNA is specific for an endogenous TCR mRNA and the protein-coding region comprises a polynucleotide encoding a CAR comprising an NKG2D protein or variant thereof fused to a DAP10/12 fusion polypeptide or variant thereof. In some embodiments, the amiRNA coding region comprises six polynucleotides encoding amiRNAs derived from miR-17, miR-18, miR-19a, miR-19b, miR-20 and miR-92a-1, wherein the amiRNAs are specific for a β2M or CIITA mRNA and the protein-coding region comprises a polynucleotide encoding a CAR comprising an NKG2D protein or variant thereof fused to a DAP10/12 fusion polypeptide or variant thereof. In some embodiments, the amiRNA coding region comprises six polynucleotides encoding amiRNAs derived from miR-17, miR-18, miR-19a, miR-19b, miR-20 and miR-92a-1, wherein each amiRNA is specific for an endogenous TCR mRNA and the protein-coding region comprises a polynucleotide encoding a CAR comprising an NKG2D protein or variant thereof and a polynucleotide encoding a DAP10/12 fusion polypeptide or variant thereof. In some embodiments, the amiRNA coding region comprises six polynucleotides encoding amiRNAs derived from miR-17, miR-18, miR-19a, miR-19b, miR-20 and miR-92a-1, wherein the amiRNAs are specific for a β2M or CIITA mRNA and the protein-coding region comprises a polynucleotide encoding a CAR comprising an NKG2D protein or variant thereof and a polynucleotide encoding a DAP10/12 fusion polypeptide or variant thereof. Vector Preferably, the polynucleotide construct is in an expression vector. In some embodiments, the construct is in a retroviral or lentiviral vector. Optionally, the construct is in an SFG retroviral vector. Also provided is a vector comprising the construct as defined above. It will be appreciated that the vector may be as defined above. For example, the vector is preferably an expression vector. Generally, the vector is one vector (i.e. a single vector). The inventors have demonstrated that a one-vector approach is highly efficient at expressing both the amiRNA and the CAR in host cells. This is unexpected, given that the construct of the vector encodes both the amiRNA and the CAR. By utilising one vector, the inventors have also found that host cells can be engineered to express both the amiRNA and the CAR with only one, rather than two, engineering steps. This reduces the cost and complexity of the manufacturing process. Such an approach also reduces the time engineered host cells spend ex-vivo, thus improving their viability and persistence when used in vivo. Various viral and non-viral vectors are known to those skilled in the art. Non-viral vectors include plasmids, episomal vectors, and human artificial chromosomes (see, e.g., Harrington et al., 1997, Nat Genet. 15:345). For example, non-viral vectors useful for expression of the amiRNA and the CAR of the invention in mammalian (e.g., human) cells include pThioHis A, B and C, pcDNA3.1/His, pEBVHis A, B and C, (Invitrogen, San Diego, Calif.), MPS V vectors, and numerous other vectors known in the art for expressing other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, 1995, Annu. Rev. Microbiol. 49:807; and Rosenfeld et al., 1992, Cell 68: 143. In particular, retroviral, lentiviral, adenoviral or adeno-associated viral vectors are commonly used for expression in immune cells such as T-cells. Examples of such vectors include the SFG retroviral expression vector (see Riviere et al., 1995, Proc. Natl. Acad. Sci. (USA) 92:6733-6737). In some embodiments, the vector is a retroviral or lentiviral vector. Optionally, the vector is an SFG retroviral vector. In some embodiments the vector is a lentiviral vector. Lentiviral vectors include self-inactivating lentiviral vectors (so-called SIN vectors). The choice of expression vector depends on the intended host cells in which the vector is to be expressed. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen, et al., 1986, Immunol. Rev. 89:49-68), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPS V promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, the EF1 alpha promoter, the phosphoglycerate kinase (PGK) promoter and promoter-enhancer combinations known in the art. Cultures of transformed organisms can be expanded under non-inducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells. In addition to promoters, other regulatory elements may also be required or desired for efficient expression of the amiRNA and CAR of the invention. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. In addition, the efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf et al., 1994, Results Probl. Cell Differ. 20:125; and Bittner et al., 1987, Meth. Enzymol., 153:516). For example, the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells. In some embodiments, a non-coding region of the vector comprises the construct. For example, a non-coding region of a retroviral vector may comprise the construct. The genetic engineering of host cells can be carried out according to standard cloning and expression techniques, which are known in the art (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). The vector of the invention may be introduced into a host cell using such techniques. Thus, also provided is a host cell comprising the construct or the vector as defined above. The construct or vector may be transfected or transduced into the host cell using standard techniques. There is also provided a population of host cells comprising the construct or the vector as defined above. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Host cells It is possible to express the amiRNA and the CAR of the invention in either prokaryotic or eukaryotic host cells. Representative host cells include many E. coli strains, mammalian cell lines, such as CHO, CHO-K1, and HEK293; insect cells, such as Sf9 cells; and yeast cells, such as S. cerevisiae and P. pastoris. In some embodiments, the host cell is a mammalian host cell. For example, the host cell may be a mouse, rat, human, dog, cat, horse, cow, primate, goat or sheep host cell. In some embodiments, the host cell is a human or mouse host cell, preferably a human host cell. Cell lines which may be used include the NK cell line NK-92. Mammalian host cells encompassed by the invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220 used with a DH FR selectable marker, e.g., as described in R.J. Kaufman and P.A. Sharp, 1982, Mol. Biol. 159:601-621) NSO myeloma cells, COS cells and SP2 cells. In some embodiments the host cells are CHO K1PD cells. In some embodiments the host cells are NSO1 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system shown in WO 87/04462, WO 89/01036 and EP 338,841. In some embodiments, the host cell is an immuno-responsive cell. In the context of the present invention, an immuno-responsive cell is an immune cell. Typically, an immuno- responsive cell can be involved in an immune response, preferably an inflammatory immune response, for example to respond to cancer. Preferably, the immuno-responsive cell is selected from the group consisting of a Natural Killer (NK) cell, a T-cell, a B-cell, a Natural Killer T-(NKT) cell, or any combination thereof. The immuno-responsive cell may be selected from the group consisting of a T-cell, a B-cell or an NK cell. In some embodiments, the immuno-responsive cell is a T-cell. The T-cell may be an αβ T- cell. In other embodiments, the T-cell is a γδ T-cell. In some embodiments the T-cell is a CD4⁺ T-cell. Alternatively, the T-cell is a CD8⁺ T-cell. In some embodiments, the T-cell is an αβ CD4⁺ T-cell. In some embodiments, the T-cell is a γδ CD8⁺ T-cell. The T-cell may be a γδ CD4- CD8- T-cell Preferably, the immuno-responsive cell is a primary cell. More preferably, the immuno- responsive cell is a human primary cell. By “primary cell” this will be understood to refer to a cell that has been obtained from a subject. Primary cells are not immortalised cells from a cell line. The immuno-responsive cell may comprise or consist of a primary T-cell. Optionally, the primary cell is a primary human T-cell. The primary cell may be autologous. Alternatively, the primary cell may be allogeneic. In embodiments comprising a population of primary cells, the population may comprise a mixture of autologous and allogenic cells. As the skilled person will appreciate, autologous cells are cells from the same subject, i.e. cells which have been obtained from a subject which will be administered back to the same subject. Allogeneic cells are cells obtained from a different subject to the subject to which the cells will be administered. The different subjects are typically from the same species. Allogenic cells are thus genetically different to the subject to which they are administered. Alternatively, the immuno-responsive cell may comprise or consist of an immortalised immuno-responsive cell from a cell line. Pharmaceutical composition The invention also provides a pharmaceutical composition comprising the construct, the vector and/or the host cell as defined above. Preferably, the pharmaceutical composition further comprises a pharmaceutically or physiologically acceptable diluent and/or carrier. The carrier and/or diluent is generally selected to be suitable for the intended mode of administration and can include agents for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, colour, isotonicity, odour, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Typically, these carriers and/or diluents include aqueous or alcoholic/aqueous solutions, emulsions, or suspensions, including saline and/or buffered media. Suitable further agents for inclusion in the pharmaceutical compositions include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulphite, or sodium hydrogen- sulphite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as free serum albumin, gelatin, or immunoglobulins), colouring, flavouring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as Polysorbate 20 or Polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides, such as sodium or potassium chloride, or mannitol sorbitol), delivery vehicles, excipients and/or pharmaceutical adjuvants. The carrier and/or diluent may be a parenteral, optionally intravenous vehicle. Suitable parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates may be included. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. In some cases, one might include agents to adjust tonicity of the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in a pharmaceutical composition. For example, in many cases it is desirable that the composition is substantially isotonic. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, may also be present. The precise formulation will depend on the route of administration. Additional relevant principle, methods and components for pharmaceutical formulations are well known (see, e.g., Allen, Loyd V. Ed, (2012) Remington's Pharmaceutical Sciences, 22^nd Edition). A pharmaceutical composition of the present invention can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled person, the route and/or mode of administration will vary depending upon the desired results. Routes of administration for pharmaceutical compositions of the invention include intravenous, intramuscular, intradermal, intraperitoneal, intrapleural, subcutaneous, intratumoural, spinal, or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intratumoural, intrapleural and intra-sternal injection and infusion. In some embodiments, the pharmaceutical composition is administered intratumourally. In other embodiments, administration is intrapleural or intraperitoneal. When parenteral administration is contemplated, the pharmaceutical compositions are usually in the form of a sterile, pyrogen-free, parenterally acceptable composition. A particularly suitable vehicle for parenteral injection is a sterile, isotonic solution, properly preserved. The pharmaceutical composition can be in the form of a lyophilizate, such as a lyophilized cake. Alternatively, the pharmaceutical composition of the invention can be administered by a non-parenteral route, such as a topical, epidermal, or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically. In some embodiments, the pharmaceutical composition is for subcutaneous administration. Typically, the pharmaceutical compositions for subcutaneous administration contain suitable stabilizers (e.g., amino acids, such as methionine, and or saccharides such as sucrose), buffering agents and tonicifying agents. Alternatively, the pharmaceutical composition may be for intravenous administration. Kit The invention also provides a kit comprising the comprising the construct, vector, host cell and/or the pharmaceutical composition as defined above. The kit may further comprise instructions for use. In some embodiments, the construct, vector, host cell and/or the pharmaceutical composition is provided in an aqueous solution, optionally buffered solution and/or at a temperature of at least -20°C. Method of preparing a host cell Also provided is a method of preparing a host cell as defined above, the method comprising the steps of: (i) introducing the construct or the vector as defined above into a host cell, and (ii) culturing the host cell such that the CAR is expressed. Advantageously, expression of the CAR also acts as a marker for expression of the amiRNA. In some embodiments step (i) comprises introducing one vector as defined above into a host cell. The host cell is preferably an immuno-responsive cell. Preferably, the host cell is isolated. Method of treating or preventing a disease in a subject Also provided is a method of treating or preventing a disease in a subject, wherein the method comprises administering to the subject the construct, vector, host cell and/or pharmaceutical composition of the invention. Preferably, the host cell is an immuno-responsive cell. More preferably, the host cell is a T- cell. In some embodiments, the host cell comprises a population of host cells. In some embodiments, the host cell comprises a population of immuno-responsive cells. Preferably, the host cell comprises a population of T cells. The method typically comprises administering a therapeutically effective amount or a prophylactically effective amount of the construct, vector, host cell and/or pharmaceutical composition of the invention. A therapeutically effective amount is an amount which ameliorates one or more symptoms, such as all the symptoms, of the disease and/or abolishes one or more symptoms, such as all the symptoms, of the disease. The therapeutically effective amount preferably cures the disease. A prophylactically effective amount is an amount which prevents the onset of the disease and/or prevents the onset of one or more symptoms, such as all the symptoms, of the disease. The prophylactically effective amount preferably prevents the subject from developing the disease. Suitable amounts are discussed in more detail below. The construct, vector, host cell and/or pharmaceutical composition of the invention may be administered to a subject that displays symptoms of disease. The construct, vector, host cell and/or pharmaceutical composition of the invention may be administered to a subject that is asymptomatic, i.e. does not display symptoms of disease. The construct, vector, host cell and/or pharmaceutical composition of the invention may be administered when the subject’s disease status is unknown or the patient is expected not to have a disease. The construct, vector, host cell and/or pharmaceutical composition of the invention may be administered to a subject that is predisposed, such as genetically predisposed, to developing the disease. The subject may be a mammal. Optionally, the subject is a human, horse, dog or cat. In some embodiments, the subject is human. Alternatively, the subject may be a horse. Various diseases are suitable for treatment or prophylaxis by administration of the construct, vector, host cell and/or pharmaceutical composition of the invention. Any disease which can be treated or prevented using immunotherapy is envisaged. In some embodiments, the disease comprises cancer, allogeneic rejection and/or graft versus host disease. In some embodiments, the disease is cancer, allogeneic rejection and graft versus host disease. In this way, the severity or development of graft versus host disease or allogeneic rejection can be reduced or avoided. Optionally, the disease is cancer and the host cell is an immunoresponsive cell. Preferably, the immunoresponsive cell is a population of T-cells. The cancer may include, but not necessarily be limited to, a solid tumour cancer, a soft tissue tumour, a metastatic lesion, and a haematological cancer. For example, the cancer can be liver cancer, lung cancer, breast cancer, prostate cancer, lymphoid cancer, colon cancer, renal cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, such as squamous cell carcinoma of the head and neck (SCCHN), cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the oesophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukaemias including acute myeloid leukaemia, chronic myeloid leukaemia, acute lymphoblastic leukaemia, chronic lymphocytic leukaemia, solid tumours of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumour angiogenesis, spinal axis tumour, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, myelodysplastic syndrome (MDS), chronic myelogenous leukaemia-chronic phase (CMLCP), diffuse large B-cell lymphoma (DLBCL), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), hepatocellular carcinoma (HCC), gastrointestinal stromal tumours (GIST), non-small cell lung carcinoma (NSCLC), cutaneous melanoma, mucosal melanoma, cutaneous squamous cell carcinoma (CSCC), small-cell lung cancer, squamous cell carcinoma of the lung, Merkle cell carcinoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers. In some embodiments, the cancer is selected from the above group. Preferably, the cancer is a solid tumour cancer. In some embodiments, the cancer is selected from the group consisting of cancer of the head and/or neck, ovarian cancer, malignant mesothelioma, breast cancer, pancreatic cancer, colorectal cancer, lung cancer, gastric cancer, bladder cancer, prostate cancer, oesophageal cancer, endometrial cancer, hepatobiliary cancer, duodenal carcinoma, thyroid carcinoma, cancer of the central nervous system or renal cell carcinoma. In some embodiments, the cancer is selected from ovarian cancer, breast cancer, optionally triple-negative breast cancer, pancreatic cancer, malignant mesothelioma, and combinations of said cancers. The subject may have been pre-treated with a chemotherapeutic agent. The administration of the construct, vector, host cell and/or pharmaceutical composition of the invention to the subject may result in a decrease in tumour size of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100%, when compared to an untreated tumour. In embodiments administering a population of host cells, preferably a population of immuno-responsive cells, the number of host cells administered to the subject should take into account the route of administration, the cancer being treated, the weight of the subject and/or the age of the subject. In general, from about 1 x 10⁶ to about 1 x 10¹¹ host cells are administered to the subject. In some embodiments, from about 1 x 10⁷ to about 1 x 10¹⁰ host cells, or from about 1 x 10⁸ to about 1 x 10⁹ host cells are administered to the subject. The invention also provides the construct, vector, host cell and/or pharmaceutical composition of the invention for use in any of the therapeutic methods described above. Thus, also provided is the construct, vector, host cell and/or pharmaceutical composition of the invention for use in the treatment or prevention of a disease. This may otherwise be referred to for use in therapy. In particular, the invention provides the construct, vector, host cell and/or pharmaceutical composition of the invention for use in the treatment or prevention of cancer. Also provided is the use of the construct, vector, host cell and/or pharmaceutical composition of the invention for the manufacture of a medicament for the treatment or prevention of a disease. Optionally, the disease is cancer. Further provided is use of the construct, vector, host cell and/or pharmaceutical composition of the invention for therapy. Also provided is use of the construct, vector, host cell and/or pharmaceutical composition of the invention for the treatment or prevention of cancer. Further provided is a method for generating an immune response to a target cell in a subject in need thereof, wherein the method comprises administering to the subject the construct, vector, host cell and/or pharmaceutical composition of the invention as defined above. Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, mean “including but not limited to”, and do not exclude other components, integers or steps. Moreover, the singular encompasses the plural unless the context otherwise requires in particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application, it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and/or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and/or features of any embodiment can be combined in any way and/or combination, unless such features are incompatible. BRIEF DESCRIPTION OF THE DRAWINGS One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which: Figure 1 is a schematic of the TCRα-specific amiRNA generated. The endogenous 5’ stem and 3’ stem sequences from miR-155 were replaced with a previously validated shRNA or CRISPR guide RNA (anti-sense strand) and its complementary sequence (sense strand), respectively (dashed lines). Figure 2 is a schematic of the miR-155-based pri-amiRNA and CAR polynucleotide sequence embedded within the non-coding region of the SFG retroviral vector. SD = splice donor, SA = splice acceptor. Figure 3 is a schematic depicting the four original amiRNA constructs. Two different TCRα- specific sequences (GIPZ and CH) were used, with the resulting amiRNA cloned into either the BsrI or BglII sites. These were expressed in the non-coding region of SFG, upstream of a γδ CAR, designated γδ CAR 2. It will be appreciated that the constructs shown contain pri- amiRNA. Figure 4 is a schematic demonstrating the switch in position of the 3’ stem sequence from its original position after the loop region (left) to preceding the loop region (right). Figure 5 (A) The expression of a γδ control TCR, γδ CAR 2 and the endogenous αβ TCR were assessed by flow cytometry, with the median fluorescence intensity (MFI) of the γδ TCR detailed in Figure (D). GD control TCR = γδ TCR, UT = untransduced. The MFI of the endogenous αβ TCR was quantified within the γδ TCR⁺ (B) and γδ TCR- (C) fractions of T-cells transduced to express the γδ CAR 2 alone, the γδ TCR (control GD TCR), or the γδ CAR 2 co- expressed with a TCR α-specific amiRNA (CH or GIPZ). UT = untransduced T-cells. Figure 6 CAR T-cells were stained for CD4 and CD8 and the expression of both the CAR and the endogenous αβ TCR was assessed in T-cells transduced with a CAR alone (N1012), a control CAR (NKG2D), or the N1012 CAR with a TCR α-specific amiRNA (N1012_GIPZ). Expression of the N1012 CAR and the αβ TCR was determined by flow cytometry (A). This flow cytometry was used to determine the percentage of CAR T-cells that lacked detectable levels of the endogenous αβ TCR (B) and the overall expression level of the endogenous αβ TCR within the total transduced T-cell population, as assessed by MFI (C). Figure 7 The MFI of the endogenous αβ TCR was assessed by flow cytometry on the indicated days in T-cells transduced with a CAR alone (N1012), a control CAR (NKG2D), or the vector containing the CAR and the integrated amiRNA (N1012_GIPZ). Figure 8 Eleven days post-transduction, the expression level of both the CAR (NKG2D) and CD3 were assessed by flow cytometry in CD4 and CD8 subpopulations in T-cells expressing the CAR alone (N1012) or the CAR co-expressed with one of three different CD247-specific amiRNA (A). The percentage change in MFI of CD3 in relation to that observed in untransduced (UT) T-cells was calculated for both CD4⁺ and CD8⁺ as follows (MFI CD3 in transduced population/MFI CD3 in UT T-cells)*100 (panel B). Figure 9 is a schematic detailing the structure of the clustered single-vector construct, in which an amiRNA cluster is inserted between the SD and SA sites, upstream of the CAR gene. The cluster consists of six individual amiRNA embedded within the endogenous flanking sequences (miR-17-92 pri-miRNA). Figure 10 The expression of the γδ TCR (control GD TCR), γδ CAR 2 and the αβ TCR were assessed 17 days post-transduction in T-cells transduced with γδ CAR 2 alone, a γδ TCR (control GD TCR), or γδ CAR 2 combined with the integrated TCRx6 amiRNA clusters. Expression of the endogenous αβ TCR was assessed in the CAR⁺ (A) and CAR- fractions (B) from each cell population. Expression of the CAR from the different constructs was also assessed by flow cytometry (C). Figure 11 Expression of the endogenous αβ TCR was assessed by flow cytometry on Day 5 and again on Day 17 post-transduction. The expression level of the endogenous αβ TCR (A) and the number of γδ TCR⁺ αβ TCR⁺ T-cells (B) was assessed in T-cells transduced with the γδ CAR 2 alone, a γδ TCR (control GD TCR), or γδ CAR 2 co-expressed with the TCRx6 amiRNA cluster. Figure 12 The expression level of the endogenous αβ TCR within the γδ TCR⁺ population was assessed by flow cytometry at the indicated timepoints in T-cells transduced with γδ CAR 2, γδ CAR 2 co-expressed with a single amiRNA (GIPZ-Bsr), or γδ CAR 2 co-expressing the amiRNA cluster (TCRx6_Bsr or TCRx6_Bgl). Figure 13 The expression of both the CAR (NKG2D) and the endogenous αβ TCR were assessed in the CD4⁺ subpopulation of T-cells transduced with the CAR alone (N1012), the CAR co-expressed with the TRACx6 amiRNA cluster (N1012_TRACx6) or in untransduced T- cells 17 days post-transduction (A). Expression of the endogenous αβ TCR was quantified in two individual donors on Day 10 and Day 17 post-transduction (B). n.d. = not detectable. Figure 14 T-cells expressing the CAR alone (N1012), the N1012 CAR co-expressed with the TRACx6 amiRNA cluster (N1012_TRACx6), a control CAR (NKG2D) or untransduced T-cells (UT) were co-cultured with two mesothelioma cells lines (Ren and Ju77) or a pancreatic ductal adenocarcinoma cell line (BxPC3) at a 1:1 CAR T-cell: tumour cell ratio. Tumour cell viability was assessed after 72 hours by MTT assay and expressed as a percentage of tumour cell viability when grown in the absence of T-cells (A). To assess the ability of the CAR T-cells to undergo multiple rounds of stimulation, T-cells were seeded at a 1:1 ratio with Ren cells. After 72 hours, the T-cells were removed and immediately cultured with fresh Ren tumour cells. This ‘re-stimulation assay’ was repeated until tumour cell viability exceeded 60% of that observed in the absence of T-cells. The number of rounds of successful re-stimulation were calculated (B). Figure 15 The expression of both HLA Class I and HLA Class II in untransduced (UT) αβ T- cells, or in those transduced to express either the CAR alone (N1012) or the CAR co- expressed with an amiRNA cluster targeting both β2M and CIITA was assessed by flow cytometry (A). The expression of HLA Class I (panel B) and HLA Class II (panel C) within the CAR+ T-cells was quantified by flow cytometry as median fluorescence intensity. Figure 16 The expression of both HLA Class I and HLA Class II in untransduced (UT) γδ T- cells, or in those transduced to express either the CAR alone (N1012) or the CAR co- expressed with an amiRNA cluster targeting both β2M and CIITA was assessed by flow cytometry (A). The expression of HLA Class I (panel B) and HLA Class II (panel C) within the CAR+ T-cells was quantified by flow cytometry as median fluorescence intensity. Figure 17 Five different pri-amiRNA sequences (derived from pri-miR-30a), targeting CD247, were cloned into the SFG γ-retroviral vector, alongside puromycin (PuroR) and green fluorescent protein (GFP) marker genes (A). The surface expression of CD3ε in Jurkat cells transduced with the constructs individually was assessed by flow cytometry (B) and the median fluorescent intensity (MFI) of CD3ε, which quantifies the relative level of surface expression was calculated (C). Figure 18 To assess the relative efficiency of silencing achieved from each individual pri- amiRNA within the miR17-92 cluster, six variants were generated in which the amiRNA 5’ strand and 3’ strand sequences targeting the TCRα chain were substituted for amiRNA 5’ strand and 3’ strand sequences targeting the red fluorescent protein, tdTomato (A). When transduced into Jurkat cells stably expressing tdTomato, the relative expression of tdTomato (as determined by calculating the median fluorescent intensity, MFI) was determined by flow cytometry (B). The impact upon the surface expression of the αβTCR (due to the replacement of a TCRα-specific amiRNA with one targeting tdTomato) was also assessed by flow cytometry and calculation of the MFI of the αβTCR (C). Figure 19 To identify whether silencing efficiency can be improved by co-expressing two pri- amiRNA, four constructs were generated in which the CD247-specific pri-amiRNA (derived from pri-miRNA-30a) was chained to a second pri-amiRNA targeting CD247 and derived from pri-miRNA-20a (A). Surface expression of CD3ε was assessed by flow cytometry in Jurkat cells transduced to stably express these constructs (B) and the median fluorescent intensity (MFI) of CD3ε, which quantifies the relative level of surface expression was calculated (C). EXAMPLES Introduction MicroRNA (miRNA) are small, non-coding, RNA that inhibit gene expression by binding to the 3’ untranslated region of target mRNA, thereby promoting degradation or translational repression. MicroRNA are present within longer primary transcripts (pri-miRNA) that are processed into smaller precursor RNA (pre-miRNA) by the enzyme Drosha. These pre- miRNA are then further processed into mature miRNA by the enzyme Dicer, before being loaded onto the RISC complex and mediating RNA interference. The specificity of a miRNA can be altered to target a gene of interest by replacing the sequences 5’ and 3’ to the loop region of its pri- or pre-miRNA with sequences from a previously validated shRNA or CRISPR guide RNA. The resulting modified miRNA will be referred to herein as “amiRNA”. The resulting pri-amiRNA preferably retain the flanking sequences of the endogenous pri-miRNA, meaning that expression is driven by standard RNA polymerase II-dependent promoters, removing the need for alternative promoters. Multiple pri-miRNAs are often found together in a miRNA cluster. This provides potential to target more than one gene. Here, we describe the generation of a novel polynucleotide construct in which we have combined CAR expression with amiRNA-mediated target gene modulation. We have demonstrated the feasibility and flexibility of this approach against a variety of different target genes. Materials and Methods Method of amiRNA design miRNA genomic sequences were obtained from UCSC genome browser, including 30bp upstream and 30bp downstream of the annotated miRNA sequence. Mature 5’ and 3’ miRNA sequence were identified from miRBase annotation. To identify which of the 5’ and 3’ strand is the major strand that ends up entering the RNAi machinery, read count data was analysed. Typically, one sequence will be present in high levels (major strand) and the other will have very low read counts (minor strand). The remaining sequence of the miRNA was defined as either the loop, 5’ stem sequence or 3’ stem sequence. The miRNA sequence ((5’Stem)-(Major/Minor Strand)-(Loop)-(Minor/Major Strand)- (3’Stem)) was run through Mfold web tool to identify endogenous miRNA secondary structure. Following this, a sequence targeting the gene of interest (GOI) was inserted into the endogenous major strand or the endogenous major strand was replaced with this sequence targeting the GOI. If the endogenous major strand was longer than 21nt, which is the default output for the shRNA design tools, then the location of the targeting sequence in the GOI mRNA was identified and the targeting sequence extended 5’ to obtain the necessary number of bases to have a targeting sequence that was the same length as the endogenous major strand. The endogenous minor strand was then replaced with the reverse complement sequence of the targeting sequence, or the reverse complement sequence was inserted into the endogenous minor strand. Then (using miRBase as a guide) the new minor strand sequence was modified (by deletions and/or base modifications) to achieve a corresponding or similar secondary structure to the endogenous miRNA. For example, endogenous mir17 encodes a 22nt 5’ mature sequence and a 21nt 3’ mature sequence, where the 5’ mature sequence is the major one incorporated into RNAi. The artificial miRNA based on this may comprise the structure: (30nt 5’ mi17 Genomic Flank)-(mir17 5’ Stem)-(22nt anti-sense targeting sequence)-(mir17 Loop)-(21nt sense targeting sequence modified to mimic mir17 secondary structure)-(mir17 3’ stem)-(30nt 3’ mir17 Genomic Flank) Generation of virus by triple transfection In order to generate virus, 1.65x10⁶ HEK293T cells were seeded in a 10cm² tissue culture dish in 10mL of IMDM media containing 10% FBS and 2mM L-glutamine (I10 media) and incubated for 24 hours at 37 ºC at 5% CO₂ in order to adhere. The following day, the following transfection mix was generated (the volumes stated are per plate):

Table 2: Transfection Mixes Upon completion of the 15-minute incubation, Mix B was added dropwise to the HEK293T cells, gently swirled and the cells placed back into the incubator. Forty-eight hours after transfection, the supernatant was harvested into labelled, pre-chilled, 50mL Falcons and stored on ice whilst supernatants from all plates were harvested. The HEK293T cells were fed with 10mL fresh I10 media and placed back into the incubator. The harvested supernatant was placed into the fridge overnight. After an additional 24 hours, the supernatant was harvested once again from the HEK283T cells and combined with the supernatant harvested 48 hours after transfection. The combined supernatant was aliquoted into pre-labelled tubes, snap frozen and subsequently stored at -80 ^C until required. Isolation, activation and transduction of T-cells Peripheral blood mononuclear cells (PBMCs) were isolated using standard Ficoll Paque- mediated density centrifugation. Once re-suspended at a concentration of 3x10⁶ cells/mL in RPMI + 5% normal human AB serum and 2mM L-glutamine (‘R5’ media), αβ T-cells were activated using paramagnetic beads coated with anti-human CD3 and anti-human CD28 antibodies (1:2 cell:bead ratio), or phytohaemagglutinin (PHA) at a concentration of 5ug/mL. Forty-eight hours after activation, 1x10⁶ T-cells were plated onto RetroNectin- coated non-tissue culture treated plates and mixed with 3mL viral supernatant harvested from transiently transfected HEK 293T cells. T-cells were fed with fresh R5 media supplemented with 100IU/mL IL-2, thrice weekly. For the activation of gamma-delta T-cells, PBMCs were re-suspended at a concentration of 3x10⁶/mL in R5 media supplemented with 100IU/mL IL-2 and 5ng/mL recombinant TGF- β and subsequently plated onto immobilised pan anti-γδ TCR antibody (800ng/mL). Seventy- two hours after activation, 1x10⁶ PBMCs were plated onto RetroNectin-coated non-tissue culture treated plates and mixed with 3mL viral supernatant harvested from transiently transfected HEK 293T cells. T-cells were enumerated every 48 hours by trypan blue exclusion. If T-cell density exceeded 1x10⁶/mL, the T-cells were fed with 100% volume of fresh media supplemented with 100IU/mL IL-2 and 5ng/mL TGF- β. If the T-cell density was below 1x10⁶/mL, the T-cells were fed solely with 100IU/mL IL-2 and 5ng/mL TGF- β. Assessment of gene-transfer efficiency and target gene knockdown using flow cytometry To determine expression of the N1012 CAR, T-cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-human CD4, phycoerythrin (PE)-conjugated anti- human NKG2D and allophycocyanin (APC)-conjugated anti-human CD8 α antibodies on ice for 30 minutes. Expression of the γδ CAR2 and the γδ control TCR was assessed using a FITC-conjugated pan anti- γδ TCR antibody. Expression of the endogenous αβ TCR within the transduced and untransduced fractions was achieved by co-staining with an allophycocyanin cyanine7 (APC/Cy7)-conjugated anti-human αβ TCR antibody. After washing in 2mL ice-cold PBS, the cells were re-suspended in 0.5mL ice-cold PBS and assessed by flow cytometry. Due to endogenous expression of NKG2D in CD8⁺ T-cells, gene transfer efficiency of N1012⁺ T-cells was calculated within the CD4⁺ T-cells and compared to that seen in untransduced (UT) T-cells. Assessment of HLA Class I and HLA Class II expression in N1012⁺ T-cells was assessed by staining with AlexaFluor488-conjugated anti-human HLA_A/B/C and phycoerythrin cyanine7 (PE/Cy7)-conjugated anti-human HLA_DP/DQ/DR, along with APC- conjugated anti-human CD4 and PE-conjugated anti-human NKG2D. Quantification of tumour cell viability using MTT assay (cytotoxicity assay) Tumour cells were co-cultured with CAR T-cells at an effector:target ratio of 1:1. After 72 hours, the T-cells were removed and 500µL MTT solution (500µg/mL) added per well. The plates were incubated at 37°C and 5% CO₂ for approximately 1 hour. Following removal of the MTT solution, the resulting formazan crystals were solubilised in DMSO (500µL/well) and the absorbance measured at 560nm. Tumour cell viability was calculated as follows: (Absorbance of monolayer with T-cells/absorbance of monolayer without T-cells)*100. To assess the ability of the CAR T-cells to undergo multiple rounds of target cell lysis (‘re- stimulation experiments’), the CAR T-cells were co-cultured with tumour cells at a 1:1 effector:target ratio. After 72 hours, the T-cells were gently removed and each well gently washed with 1mL PBS. Following removal of the PBS, 500 μL MTT solution was added per well and the plates incubated at 37°C and 5% CO2 for approximately 1 hour. Following removal of the MTT solution, the resulting formazan crystals were solubilised in DMSO (500 ^L/well) and the absorbance measured at 560nm. Tumour cell viability was calculated as follows: (Absorbance of monolayer with T-cells/absorbance of monolayer without T- cells)*100. This assay was undertaken without exogenous IL-2 supplementation and repeated twice weekly until the T-cells failed to mediate greater than 40% target cell lysis. Example 1: Generation of an amiRNA + CAR co-expressing construct The inventors initially sought to develop a single vector comprising the polynucleotide construct of the invention. Figure 1 is a schematic demonstrating the endogenous structure of the pri-miR-155 microRNA (left), in which the stem and loop structure is preceded by and precedes 5’ and 3’ flanking sequences, respectively. The horizontal lines between the two stem regions indicate Watson-Crick base pairing. For the generation of an artificial pri-microRNA (pri-amiRNA, encoding an amiRNA), parts of the stem regions were replaced by sequences specific for the gene of interest (dashed line, right hand of the figure). All other sequences remained identical to the endogenous pri-miR-155 sequences to ensure efficient RNA processing. The sequences 5’ and 3’ to the loop region of one shRNA and one CRISPR guide RNA targeting the T-cell receptor (TCR) α chain were embedded within the sequence of the miR- 155 pri-miRNA to form pri-amiRNA. These TCR α-specific sequences replaced the endogenous pri-miR-155 5’ stem sequence and 3’ stem sequence. The shRNA anti-sense strand was placed 5’ to the pri-miR-155 loop region to become the 5’ stem sequence (Figure 1, right). This was because the 5’ stem sequence of the naturally occurring miR-155 is typically more likely to be incorporated into RISC. Therefore, positioning of the shRNA anti- sense strand here provides the greatest chance of RISC incorporation. DNA encoding each pri-amiRNA was subsequently cloned into the truncated pol region that sits between the splice donor (SD) and splice acceptor (SA) sites of an SFG ^-retroviral vector to allow efficient processing of the amiRNA from the spliced mRNA. The vector also comprised a polynucleotide encoding a CAR (Figure 2) to allow co-expression of the amiRNA and the CAR from the SFG γ-retroviral backbone. Four different constructs were generated (Figure 3) for the co-expression of two different TCR α chain-targeting amiRNA (GIPZ and CH) along with γδ CAR 2. DNA encoding both amiRNA were cloned into either the unique BsrGI or BglII sites found within the truncated pol gene in the SFG ^-retroviral vector. The strand involved in gene silencing (the anti-sense strand) is either the 5’ stem sequence or the 3’ stem sequence, depending upon the particular miRNA in question. A schematic of this is shown in Figure 4. In the example given in Figure 1, the anti-sense strand is the 5’ stem sequence. The number of bases replaced within the 5’ stem and 3’ stem sequences differed depending upon the length of the new targeting sequence being introduced. In the event that the new targeting sequence is shorter than the endogenous stem sequence, the original number of base pairs within the endogenous stem sequence will be maintained by preserving the requisite additional bases required to maintain the correct structure. Example 2: Functionality of the amiRNA + CAR co-expressing construct To confirm the function of the embedded TCR α-specific amiRNA, αβ T-cells were transduced to express the γδ CAR 2 alone or in combination with the GIPZ or CH TCR α-specific amiRNA that had been cloned into either the BsrGI or BglII sites within the truncated pol region present within SFG. Control T-cells were either left untransduced (UT) or modified to express a control γδ TCR (GD control TCR). Following eleven days culture ex vivo, expression of the γδ TCR and the endogenous αβ TCR was assessed by flow cytometry using FITC-conjugated anti-human pan- γδ TCR and APC/Cy7-conjugated anti-human pan- αβ TCR antibodies, respectively. Gating was applied using untransduced (UT) T-cells as a control, in which the populations of γδ and endogenous αβ T-cells were clearly evident. A reduction in endogenous αβ TCR expression was evident within the transduced fraction of T-cells expressing either of the γδ CAR 2 or control γδ TCR alone, when compared to the untransduced fraction within the same population (Figure 5A). This likely reflects the natural competition of the γδ CAR 2 or control γδ TCR constructs with the endogenous αβ TCR for the CD3 complex. Co-expression of the γδ CAR 2 construct with either the GIPZ or CH TCR α- specific amiRNA resulted in a further reduction in the expression of the αβ TCR within the CAR⁺ T-cells (Figure 5A-B). Reductions in endogenous αβ TCR surface expression were only observed in those T-cells expressing the γδ CAR 2, with no change observed within the untransduced ( γδ CAR 2-) fraction of each population (Figure 5C). This demonstrates the stringency of this ‘single vector’ approach, in which gene silencing occurs exclusively within the transduced T-cell population (Figure 5B). To confirm the efficacy of the embedded amiRNA when co-expressed with a different CAR construct, surface expression of the endogenous αβ TCR was assessed in primary human T- cells transduced to express an NKG2D-based CAR (N1012). The N1012 CAR is an adaptor CAR in which NKG2D is co-expressed with a DAP10/12 fusion protein as described in PCT/EP2020/076566 filed on September 23, 2020, incorporated by reference in its entirety herein. The CAR comprises a complex comprising a human NKG2D homodimer, each monomeric unit of which associates with a fused DAP10/12 homodimer. The human NKG2D protein has a human extracellular, transmembrane, and intracellular NKG2D domain. Each monomer of the fusion DAP10/12 homodimer comprises a human DAP10 extracellular, transmembrane and intracellular domain and a human DAP12 intracellular domain. N1012 consists of the peptide sequence SEQ ID NO:210 and is encoded by the nucleotide sequence SEQ ID NO:211. Following eleven days of culture ex vivo, CAR expression and endogenous αβ TCR expression was assessed by flow cytometry using PE-conjugated anti-human NKG2D and APC/Cy7-conjugated anti-human αβ TCR antibodies, respectively. Co-staining with FITC- conjugated anti-human CD4 and APC-conjugated anti-human CD8 allowed assessment of CAR expression and αβ TCR silencing in the two individual populations. T-cells expressing either N1012 alone, or a control construct (NKG2D) alone showed no difference in endogenous αβ TCR surface expression compared to untransduced (UT) T-cells. In contrast, co-expression of N1012 with the GIPZ TCR α-specific miR155-based amiRNA (N1012_GIPZ) resulted in a substantial reduction in endogenous αβ TCR expression in over 45% of T-cells (Figure 6A). The number of T-cells lacking surface expression of the endogenous αβ TCR (Figure 6B) and the overall reduction in surface endogenous αβ TCR expression within the transduced population (Figure 6C) was similar in both CD4⁺ and CD8⁺ T-cells. To investigate the kinetics of amiRNA function, the surface expression of the endogenous αβ TCR was assessed by flow cytometry on day 4, day 7 and day 11 post-transduction within T-cells expressing the CAR alone (N1012) or co-expressing the CAR and the GIPZ TCR α- specific miR155-based amiRNA (N1012_GIPZ). Control T-cells were transduced to express NKG2D alone. The level of endogenous αβ TCR was similar in T-cells transduced with either N1012 or NKG2D alone and remained relatively consistent across the timepoints investigated (Figure 7). Contrastingly, the surface expression of the αβ TCR was already lower in N1012_GIPZ T-cells than control cells by day 4 post-transduction and continued to decrease over time. These data confirm the function of the amiRNA and demonstrate that the efficiency of gene silencing increases over time. To demonstrate the flexibility of the single construct approach, three different amiRNA targeting CD3 ^ (CD247) were generated and cloned into the SFG vector encoding the N1012 CAR either into the unique BglII site found within the truncated pol gene (N1012_247_B and N1012_247_C), or the unique XhoI site found between the end of the protein coding region and the 3’ LTR (N1012_247_AX). CD4⁺ and CD8⁺ cells transduced with amiRNA constructs demonstrated a lower surface expression of CD3 than untransduced T-cells and a construct encoding the N1012 CAR alone, as assessed by flow cytometry. Of the three constructs, the N1012_247_B construct was the most effective at gene silencing (Figure 8). Example 3: A plurality of amiRNAs and a CAR can be co-expressed from a single construct The endogenous miR17-92 microRNA cluster contains six individual microRNA (miR-17, miR-18a, miR-19, miR20a, miR-19b and miR-92a-1). To determine whether gene silencing could be further enhanced by simultaneous targeting with multiple amiRNA, the endogenous stem sequences of miR-17, miR-19 and miR-19b were replaced with sequences targeting TCR α mRNA, whilst those of miR-18, miR-20a and miR-92-1 were replaced with sequences targeting the TCR β chain mRNA (collectively termed the ‘TCRx6 cluster’). To ensure optimal processing of each amiRNA, base pair mismatches and bulges found within the endogenous stem regions were replicated within the new targeted sequences. The cluster was cloned into the unique BsrGI or BglII sites present within the truncated pol gene within the SFG vector, thus giving the TCRx6_Bsr and TCRx6_Bgl constructs, respectively (Figure 9). To confirm efficacy of the TCRx6 cluster, primary human αβ T-cells were transduced to express either the γδ CAR 2 alone, the control γδ TCR, or the γδ CAR 2 co-expressed with the TCRx6 cluster. Surface expression of the CAR and the endogenous αβ TCR was assessed by flow cytometry 17 days post-transduction. Within the transduced fraction, the surface expression of the endogenous αβ TCR was substantially reduced in those co-expressing the TCRx6 cluster alongside the γδ CAR 2, compared to T-cells expressing either the γδ CAR 2 alone or in control T-cells (Figure 10A). This disparity in αβ TCR expression was not observed within the untransduced fraction of each population (Figure 10B). Furthermore, the reduction in endogenous αβ TCR expression coincided with an increase in the expression of the γδ CAR 2 (Figure 10C). This may have been as a result of the reduced competition for the CD3 complex. These data demonstrate that multiple amiRNA can be expressed simultaneously and work synergistically to enhance gene silencing. To investigate the kinetics of gene silencing achieved by the TCRx6 cluster, surface expression of the endogenous αβ TCR was assessed by flow cytometry at day 5 and day 17 post-transduction. A reduction in surface αβ TCR expression in T-cells co-expressing the γδ CAR 2 and the TCRx6 cluster was already evident five days after transduction when compared to the level expressed in T-cells transduced with the γδ CAR 2 alone (Figure 11A, left). By day 17, this difference had increased substantially, with almost negligible surface expression of the αβ TCR detected in both TCRx6_Bsr and TCRx6_Bgl constructs (Figure 11A, right). This was also associated with a reduction in the number of transduced T-cells upon which αβ TCR surface expression could be detected (Figure 11B). This confirms that co-expression of multiple amiRNA within the structure of an endogenous cluster can achieve potent gene silencing and that the level of silencing achieved increases over time. To compare the level of silencing achieved by an amiRNA cluster with that observed using a single amiRNA, primary human αβ T-cells were transduced to express the γδ CAR 2 alone, co-expressed with either a TCR α-specific amiRNA (GIPZ_Bsr), or with the TCRx6 cluster (either TCRx6_Bsr or TCRx6_Bgl; Figure 12). Surface expression of the endogenous αβ TCR was monitored by flow cytometry at six defined timepoints during culture. A reduction in endogenous αβ TCR surface expression was observed over time within the gene-modified fraction of the T-cells transduced to express the γδ CAR 2 alone, likely reflecting the competition for the components of the CD3 complex. This reduction in αβ TCR expression was enhanced in the GIPZ_Bsr T-cells and even greater silencing was observed in those T- cells co-expressing the γδ CAR 2 with the TCRx6 cluster. In all cases, a reduction in the relative expression of the αβ TCR was observed over time. These data confirm that the amiRNA cluster achieves greater gene silencing than is mediated by a single amiRNA and that the level of silencing achieved increases over time. To investigate whether even greater silencing of αβ TCR expression could be achieved by targeting all six amiRNA against the same target gene, the TCRx6 cluster was redesigned to exclusively target the TCR α chain (herein called ‘TRACx6’). When co-expressed with the N1012 CAR, dramatic reductions in the surface expression of the endogenous αβ TCR were observed compared to those transduced with the N1012 CAR or a control CAR alone (Figure 13A). Indeed, within seventeen days of transduction, expression of the αβ TCR was undetectable in N1012_TRACx6 T-cells by flow cytometry (Figure 13B). Example 4: Anti-tumour efficacy of T-cells co-expressing amiRNA and the CAR To investigate whether inclusion of the TRACx6 cluster affected the anti-tumour efficacy of αβ T-cells expressing the N1012 CAR, the T-cells were cultured with mesothelioma (Ren and Ju77) and pancreatic ductal adenocarcinoma (BxPC3) cells at a 1:1 effector:target ratio and tumour cell viability assessed by MTT assay after 72 hours. Compared to control T-cells, N1012 and N1012_TRACx6 demonstrated potent and equivalent lysis of the target cells (Figure 14A). N1012 and N1012_TRACx6 T-cells also demonstrated equivalent function in re-stimulation assays (Figure 14B), thus confirming that the inclusion of the amiRNA cluster into the SFG vector doesn’t appear to affect the anti-tumour efficacy of the CAR T-cells. Example 5: A plurality of amiRNAs can target different mRNAs from the same construct To demonstrate the flexibility of using the miR17-92 cluster as a basis for generating amiRNA clusters, the miR17-92 cluster was re-designed to target both beta-2 microglobulin ( β2M) and the class II transactivator (CIITA), which are required for the surface expression of HLA Class I (HLA-A/B/C) and HLA Class II (HLA-DP/DQ/DR), respectively. Here, the endogenous stem sequences of miR-17, miR-20a and miR-92a-1 were modified to include sequences targeted against CIITA, whilst the endogenous stem sequences of miR-18a, miR- 19a and miR-19b were modified to include sequences targeted against β2M. The resulting cluster is referred to as β2M_CIITA herein. The surface expression of both HLA Class I and Class II on primary human αβ T-cells was assessed by flow cytometry 10 days after transduction (Figure 15A). Whilst HLA Class I expression was lower in T-cells expressing the N1012 CAR alone than in untransduced T-cells, co-expression of N1012 and the β2M_CIITA cluster resulted in a substantial further reduction in HLA Class I expression (Figure 15B). T- cells co-expressing N1012 and the β2M_CIITA cluster had substantially lower HLA Class II expression than that observed in untransduced T-cells (Figure 15C). To investigate the function of the β2M_CIITA cluster further, primary human γδ T-cells were transduced to express either the N1012 CAR alone or N1012 co-expressed with the β2M_CIITA cluster (Figure 16A). Gamma-delta T-cells were chosen due to the high endogenous expression of HLA Class II by these cells. As observed with αβ T-cells, whilst N1012 T-cells expressed lower levels of HLA Class I than untransduced T-cells, a substantial further reduction in HLA Class I expression was noted in those cells transduced to co- express N1012 and the β2M_CIITA cluster (Figure 16B). In addition, a clear reduction in HLA Class II expression was noted in T-cells co-expressing the N1012 CAR and the β2M_CIITA cluster when compared to T-cells expressing N1012 alone (Figure 16C). Taken together, these data demonstrate that the targeting profile of artificial miRNA can be flexibly altered against different genes of interest. Example 6: Generation of a further amiRNA cluster Five different sequences specific for CD3ζ (CD247) were designed and placed individually into miR30a (thus giving artificial miR30a, Figure 17A). These CD3ζ-specific amiRNA were embedded just upstream of the 3’ long terminal repeat (LTR) within the SFG γ-retroviral vector. Efficient knockdown of the αβTCR (as detailed by staining for CD3ε) was observed with some of these constructs (Figure 17B). This demonstrates the provision of a further amiRNA cluster. In addition, this data shows that amiRNA clusters can be located 3’ of the coding region in a vector. Example 7: Silencing efficiency of individual miRNA within artificial miRNA cluster To investigate the silencing efficiency of each individual miRNA within the artificial miR17-92 cluster, the TRACx6 cluster (Examples 3 and 4) was re-designed. Six iterations were generated in which the TCRα-specific amiRNA at a different single position was replaced with an amiRNA specific for the red fluorescent protein, tdTomato (Figure 18A). For example, in construct ‘Pos1’ the TCRα-specific stem sequences in the artificial miR17 have been replaced with stem sequences targeting tdTomato, whilst retaining the TCRα-specific sequences in the other amiRNA in the cluster (amiR18a, amiR19a, amiR20a amiR19b and amiR92). When transduced into tdTomato-expressing Jurkat cells, a reduction in the MFI of tdTomato was observed when the tdTomato-specific amiRNA was present at certain, but not all, positions (Figure 18B). This amiRNA-induced reduction in tdTomato expression was mirrored by a matched increase in surface expression of the αβTCR (Figure 18C), by virtue of the replacement of the TCRα-specific amiRNA. These data suggest a hierarchy in the processing efficiency of individual miRNA within the miR17-92 cluster. Example 8: “Chaining” individual amiRNA together to form artificial clusters To further improve the processing of amiRNA within the miR17-92 cluster, the ability to ‘chain’ individual amiRNA together to form artificial clusters was investigated. These ‘chains’ are artificial clusters containing miRNA that would not endogenously be found in a cluster, or in an order different to that found in endogenous clusters. To investigate the feasibility of this approach, four different CD3-specific amiRNAs were developed, based upon the endogenous miR17 backbone. These were then attached directly to the 3’ flanking region of the miR30a-based, CD3ζ-specific amiRNA identified in Example 6 (miRCD3z-3), resulting in an amiRNA cluster containing two, optimised amiRNA (Figure 19A). Compared to the control (PuroR-GFP), a reduction in αβTCR expression (identified by staining for CD3ε) was observed in Jurkat cells transduced with the single CD3ζ-specific amiRNA (PuroR-GFP-CD3z-3, Figure 19B). A further reduction in CD3ε expression was observed in all constructs containing both CD3ζ-specific amiRNA. These data demonstrate the feasibility of chaining together individual amiRNA to generate artificial clusters and demonstrate that this can be used to enhance gene silencing.

LIST OF SEQUENCES SEQ ID NO:1 dd SEQ ID NO:2 dw SEQ ID NO:3 gu SEQ ID NO:4 ua SEQ ID NO:5 ga SEQ ID NO:6 ag SEQ ID NO:7 ddd SEQ ID NO:8 ddw SEQ ID NO:9 dwd SEQ ID NO:10 wdd SEQ ID NO:11 wwd SEQ ID NO:12 dww SEQ ID NO:13 wdw SEQ ID NO:14 www SEQ ID NO:15 agu SEQ ID NO:16 aua SEQ ID NO:17 uga SEQ ID NO:18 gua SEQ ID NO:19 uag SEQ ID NO:20 nddd SEQ ID NO:21 nddw SEQ ID NO:22 ndwd SEQ ID NO:23 nwdd SEQ ID NO:24 nwwd SEQ ID NO:25 ndww SEQ ID NO:26 nwdw SEQ ID NO:27 nwww SEQ ID NO:28 uagu SEQ ID NO:29 gaua SEQ ID NO:30 cuga SEQ ID NO:31 ggua SEQ ID NO:32 uuag SEQ ID NO:33 nnddd SEQ ID NO:34 dnddd SEQ ID NO:35 dndwd SEQ ID NO:36 dnwdd SEQ ID NO:37 dnwwd SEQ ID NO:38 dndww SEQ ID NO:39 dnwdw SEQ ID NO:40 dnwww SEQ ID NO:41 guagu Endogenous miR-17-92 pri-miRNA cluster (SEQ ID NO:42) uaaugucaaagugcuuacagugcagguagugauaugugcaucuacugcagugaaggcacuuguagcauuauggugac agcugccucgggaagccaaguugggcuuuaaagugcagggccugcugauguugagugcuuuuuguucuaaggugcau cuagugcagauagugaaguagauuagcaucuacugcccuaagugcuccuucuggcauaagaaguuauguauucaucca auaauucaagccaagcaaguauauagguguuuuaauaguuuuuguuugcaguccucuguuaguuuugcauaguugc acuacaagaagaauguaguugugcaaaucuaugcaaaacugaugguggccugcuauuuccuucaaaugaaugauuuu uacuaauuuuguguacuuuuauugugucgauguagaaucugccuggucuaucugaugugacagcuucuguagcacua aagugcuuauagugcagguaguguuuaguuaucuacugcauuaugagcacuuaaaguacugcuagcuguagaacucc agcuucggccugucgcccaaucaaacuguccuguuacugaacacuguucuaugguuaguuuugcagguuugcauccag cugugugauauucugcugugcaaauccaugcaaaacugacugugguagugaaaagucuguagaaaaguaagggaaac ucaaaccccuuucuacacagguugggaucgguugcaaugcuguguuucuguaugguauugcacuugucccggccuguu gaguuuggu Endogenous miR106a-363 pri-miRNA cluster (SEQ ID NO:43) ccuuggccauguaaaagugcuuacagugcagguagcuuuuugagaucuacugcaauguaagcacuucuuacauuacca uggugauuuagucaauggcuacugagaacuguaguuugugcauaauuaaguaguugaugcuuuugagcugcuucuu auaaugugucucuuguguuaaggugcaucuagugcaguuagugaagcagcuuagaaucuacugcccuaaaugccccu ucuggcacaggcugccuaauauacagcauuuuaaaaguaugccuugaguaguaauuugaauaggacacauuucagug guuuguuuuuugccuuuuuauuguuuguugggaacagaugguggggacugugcaguguacaguuguguacagagga uaagauuggguccuaguaguaccaaagugcucauagugcagguaguuuuggcaugacucuacuguaguaugggcacu uccaguacucuuggauaacaaaucucuuguugauggagagaauauucaaagacauugcuacuuacaauuaguuuugc agguuugcauuucagcguauauauguauauguggcugugcaaauccaugcaaaacugauugugauaaugugugcuuc cuacgucugugugaacacaccuucaugcguaucuccagcacucaugcccauucaucccuggguggggauuuguugcau uacuuguguucuauauaaaguauugcacuugucccggccuguggaagaaaggaggauuuuuaucgucuucuuauuuu aacuuuuaaaagccguaaguucugauauuuagucauuguaaaaugaucuguuuugcuguugucggguggaucacga ugcaauuuugaugaguaucauaggagaaaaauugcacgguauccaucuguaaacc miR-17 mature miRNA (SEQ ID NO:44) caaagugcuuacagugcagguagu miR-18a mature miRNA (SEQ ID NO:45) uaaggugcaucuagugcagaua miR-19a mature miRNA (SEQ ID NO:46) ugugcaaaucuaugcaaaacuga miR-20a mature miRNA (SEQ ID NO:47) uaaagugcuuauagugcaggua miR-92a-1 mature miRNA (SEQ ID NO:48) uauugcacuugucccggccugu miR-106a mature miRNA (SEQ ID NO:49) aaaagugcuuacagugcagguag miR-18b mature miRNA (SEQ ID NO:50) uaaggugcaucuagugcaguuag miR-20b mature miRNA (SEQ ID NO:51) caaagugcucauagugcagguag miR-19b-2 mature miRNA (SEQ ID NO:52) ugugcaaauccaugcaaaacuga miR-92a-2 mature miRNA (SEQ ID NO:53) uauugcacuugucccggccugu miR-363 mature miRNA (SEQ ID NO:54) aauugcacgguauccaucugua miR-155 mature miRNA SEQ ID NO:55 uuaaugcuaaucgugauagggguu miR-155 5’ flanking region (SEQ ID NO:56) acaaaccaggaaggggaaaucugugguuuaaauucuuuaugccucauccucugagugcugaaggcuugcuguaggcu guaugcug miR-155 3’ flanking region (SEQ ID NO:57) guguaugaugccuguuacuagcauucacauggaacaaauugcugccgugggaggaugacaaagaagcaugagucaccc ugcugg miR-155 pri-miRNA (SEQ ID NO:58) cuguuaaugcuaaucgugauagggguuuuugccuccaacugacuccuacauauuagcauuaaca miR-17 pri-miRNA (SEQ ID NO:59) uaaugucaaagugcuuacagugcagguagugauaugugcaucuacugcagugaaggcacuuguagcauua miR-18a pri-miRNA (SEQ ID NO:60) uguucuaaggugcaucuagugcagauagugaaguagauuagcaucuacugcccuaagugcuccuucuggca miR-19a pri-miRNA (SEQ ID NO:61) guccucuguuaguuuugcauaguugcacuacaagaagaauguaguugugcaaaucuaugcaaaacugaugguggcc miR-19b pri-miRNA (SEQ ID NO:62) acugaacacuguucuaugguuaguuuugcagguuugcauccagcugugugauauucugcugugcaaauccaugcaaa acugacugugguagugaaaagu miR-20a pri-miRNA (SEQ ID NO:63) uaaagugcuuauagugcagguaguguuuaguuaucuacugcauuaugagcacuuaa miR-92a-1 pri-miRNA (SEQ ID NO:64) acagguugggaucgguugcaaugcuguguuucuguaugguauugcacuugucccggccugu miR-106a pri-miRNA (SEQ ID NO:65) ccuuggccauguaaaagugcuuacagugcagguagcuuuuugagaucuacugcaauguaagcacuucuuacauuacca ugg miR-18b pri-miRNA (SEQ ID NO:66) uguguuaaggugcaucuagugcaguuagugaagcagcuuagaaucuacugcccuaaaugccccuucuggca miR-20b pri-miRNA (SEQ ID NO:67) aguaccaaagugcucauagugcagguaguuuuggcaugacucuacuguaguaugggcacuuccaguacu miR-19b-2 pri-miRNA (SEQ ID NO:68) acauugcuacuuacaauuaguuuugcagguuugcauuucagcguauauauguauauguggcugugcaaauccaugca aaacugauugugauaaugu miR-92a-2 pri-miRNA (SEQ ID NO:69) ucaucccuggguggggauuuguugcauuacuuguguucuauauaaaguauugcacuugucccggccuguggaaga miR-363 pri-miRNA (SEQ ID NO:70) uguugucggguggaucacgaugcaauuuugaugaguaucauaggagaaaaauugcacgguauccaucuguaaacc miR-155 loop region (SEQ ID NO:71) uuuugccuccaacuga miR-17 loop region (SEQ ID NO:72) gauaugugc miR-18a loop region (SEQ ID NO:73) gugaaguagauuagcauc miR-19a loop region (SEQ ID NO:74) uacaagaagaauguagu miR-19b loop region (SEQ ID NO:75) gcugugugauauucugc miR-20a loop region (SEQ ID NO:76) guguuuaguuauc miR-92a-1 loop region (SEQ ID NO:77) cuguguuucuguaugg miR-106a loop region (SEQ ID NO:78) cuuuuugagaucua miR-18b loop region (SEQ ID NO:79) ugaagcagcuuagaaucuac miR-20b loop region (SEQ ID NO:80) uuuuggcaugacucu miR-19b-2 loop region (SEQ ID NO:81) gcguauauauguauauguggc miR-92a-2 loop region (SEQ ID NO:82) uuguguucuauauaaag miR-363 loop region (SEQ ID NO:83) ugaugaguaucauaggagaaa miR-155-derived amiRNA (GIPZ TCR α-specific sequence) (SEQ ID NO:84) Uggauuuagagucucucag miR-155-derived amiRNA (CH TCR α-specific sequence) (SEQ ID NO:85) Ucucucagcugguacacggc miR-17-derived amiRNA (containing a TCR α-specific sequence) (SEQ ID NO:86) Uggauuuagagucucucagguagu miR-19a-derived amiRNA (containing a TCR α-specific sequence) (SEQ ID NO:87) Uacacaucagaauccuuaccuga miR-19b-derived amiRNA (containing a TCR α-specific sequence) (SEQ ID NO:88) ugcaaagucagauuuguugcuga miR-18a-derived amiRNA (containing a TCR α-specific sequence) (SEQ ID NO:89) aauaaugcuguuguugaaggca miR-20a-derived amiRNA (containing a TCR α-specific sequence) (SEQ ID NO:90) caugucuagcacaguuuuguua miR-92-1-derived amiRNA (containing a TCR α-specific sequence) (SEQ ID NO:91) uaaauucggguaggauccaguu miR-18a-derived amiRNA (containing a TCR β-specific sequence) (SEQ ID NO:92) aucucauagaggaugguggaua miR-20a-derived amiRNA (containing a TCR β-specific sequence) (SEQ ID NO:93) uccuuucucuugaccaugggua miR-92-1-derived amiRNA (containing a TCR β-specific sequence) (SEQ ID NO:94) Ucuccgagagcccguagaacgu mir-155-derived amiRNA (CD247-specific sequence A) (SEQ ID NO:95) aacuucacucucaggaacaag mir-155-derived amiRNA (CD247-specific sequence B) (SEQ ID NO:96) Uauagagcugguucuggcccu miR-155-derived amiRNA (CD247-specific sequence C) (SEQ ID NO:97) Uucaucccaaucucacuguag miR-17-derived amiRNA (containing a CIITA-specific sequence) (SEQ ID NO:98) uacucucaccgaucacuucguagu miR-20a-derived amiRNA (containing a CIITA-specific sequence) (SEQ ID NO:99) uagaugaauauacuggagagua miR-106a-derived amiRNA (containing a CIITA-specific sequence) (SEQ ID NO:100) gcugaacuggucgcaguugauag miR-19b-2-derived amiRNA (containing a CIITA-specific sequence) (SEQ ID NO:101) ugauauuggcauaagccucccga miR-363-derived amiRNA (containing a CIITA-specific sequence) (SEQ ID NO:102) caucgcuguuaagaagcuccua miR-18a-derived amiRNA (containing a β2M-specific sequence) (SEQ ID NO:103) aaucuuuggaguacgcuggaua miR-19a-derived amiRNA (containing a β2M-specific sequence) (SEQ ID NO:104) acuuaacuaucuugggcugugga miR-19b-derived amiRNA (containing a β2M-specific sequence) (SEQ ID NO:105) aaacccagacacauagcaacuga miR-92-1-derived amiRNA (containing a β2M-specific sequence) (SEQ ID NO:106) uauuguacaagcuuagccuugu miR-18b-derived amiRNA (containing a β2M-specific sequence) (SEQ ID NO:107) aaucuuuggaguacgcugguuag miR-20b-derived amiRNA (containing a β2M-specific sequence) (SEQ ID NO:108) acuuaacuaucuugggcugugag miR-92a-2-derived amiRNA (containing a β2M-specific sequence) (SEQ ID NO:109) uauaaacccagacacauagcaa pri-miR-155-derived pri-amiRNA (containing GIPZ TCR α-specific sequence) (SEQ ID NO:110) uggauuuagagucucucaguuuugccuccaacugacugagagacucuaaaucca pri-miR-155-derived pri-amiRNA (containing GIPZ TCR α-specific sequence) (SEQ ID NO:111) Acaaaccaggaaggggaaaucugugguuuaaauucuuuaugccucauccucugagugcugaaggcuugcuguaggcu guaugcuguggauuuagagucucucaguuuugccuccaacugacugagagacucuaaauccaguguaugaugccugu uacuagcauucacauggaacaaauugcugccgugggaggaugacaaagaagcaugagucacccugcugg pri-mir-155-derived pri-amiRNA (containing CH TCR α-specific sequence) (SEQ ID NO:112) ucucucagcugguacacggcuuuugccuccaacugagccguguaccagcugagaga pri-miR-155-derived pri-amiRNA (containing CH TCR α-specific sequence) (SEQ ID NO:113) Acaaaccaggaaggggaaaucugugguuuaaauucuuuaugccucauccucugagugcugaaggcuugcuguaggcu guaugcugucucucagcugguacacggcuuuugccuccaacugagccguguaccagcugagagaguguaugaugccug uuacuagcauucacauggaacaaauugcugccgugggaggaugacaaagaagcaugagucacccugcugg pri-miR-17-derived pri-amiRNA (containing a TCR α-specific sequence) (SEQ ID NO:114) uaauguuggauuuagagucucucagguagugauaugugcaucuacugagugagucuaaaucguagcauua pri-miR-19a-derived pri-amiRNA (containing a TCR α-specific sequence) (SEQ ID NO:115) guccucuguuagguaaggauucauguguuacaagaagaauguaguuacacaucagaauccuuaccuga pri-miR-19b-derived pri-amiRNA (containing a TCR α-specific sequence) (SEQ ID NO:116) Acugaacacuguucuaugguuagcaacaaaugacuuugaccagcugugugauauucugcugcaaagucagauuuguu gcugacugugguagugaaaagu Pri-miR-18a-derived pri-amiRNA (containing a TCR α-specific sequence) (SEQ ID NO:117) uguucaauaaugcuguuguugaaggcagugaaguagauuagcaucugcuuccacacuagcuuuauc pri-miR-20a-derived pri-amiRNA (containing a TCR α-specific sequence) (SEQ ID NO:118) caugucuagcacaguuuuguuaguguuuaguuaucuacaaaacugugcuagacaag pri-mir-92-1-derived pri-amiRNA (containing a TCR α-specific sequence) (SEQ ID NO:119) acuggauccuaaccguaauuuacuguguuucuguaugguaaauucggguaggauccagu pri-miR-18a-derived pri-amiRNA (containing a TCR β-specific sequence) (SEQ ID NO:120) uguucaucucauagaggaugguggauagugaaguagauuagcaucuaccacaaucagcuaagagac pri-miR-20a-derived pri-amiRNA (containing a TCR β-specific sequence) (SEQ ID NO:121) uccuuucucuugaccauggguaguguuuaguuaucuaccauggucaagagaaagca pri-miR-92-1-derived pri-amiRNA (containing a TCR β-specific sequence) (SEQ ID NO:122) acguucuacgggcaucuucggagacuguguuucuguauggucuccgagagcccguagaacgu pri-mir-155-derived pri-amiRNA (containing CD247-specific sequence A) (SEQ ID NO:123) Aacuucacucucaggaacaaguuuugccuccaacugacuuguuccugagagugaaguu Pri-mir-155-derived pri-amiRNA (containing CD247-specific sequence B) (SEQ ID NO:124) uauagagcugguucuggcccuuuuugccuccaacugaagggccagaaccagcucuaua pri-mir-155-derived pri-miRNA (containing CD247-specific sequence C) (SEQ ID NO:125) uucaucccaaucucacuguaguuuugccuccaacugacuacagugagauugggaugaa pri-miR-155-derived pri-amiRNA (containing CD247-specific sequence A) (SEQ ID NO:126) Acaaaccaggaaggggaaaucugugguuuaaauucuuuaugccucauccucugagugcugaaggcuugcuguaggcu guaugcugaacuucacucucaggaacaaguuuugccuccaacugacuuguuccugagagugaaguuguguaugaugcc uguuacuagcauucacauggaacaaauugcugccgugggaggaugacaaagaagcaugagucacccugcugg pri-miR-155-derived pri-amiRNA (containing CD247-specific sequence B) (SEQ ID NO:127) Acaaaccaggaaggggaaaucugugguuuaaauucuuuaugccucauccucugagugcugaaggcuugcuguaggcu guaugcuguauagagcugguucuggcccuuuuugccuccaacugaagggccagaaccagcucuauaguguaugaugcc uguuacuagcauucacauggaacaaauugcugccgugggaggaugacaaagaagcaugagucacccugcugg pri-mir-155-derived pri-amiRNA (containing CD247-specific sequence C) (SEQ ID NO:128) acaaaccaggaaggggaaaucugugguuuaaauucuuuaugccucauccucugagugcugaaggcuugcuguaggcu guaugcuguucaucccaaucucacuguaguuuugccuccaacugacuacagugagauugggaugaaguguaugaugcc uguuacuagcauucacauggaacaaauugcugccgugggaggaugacaaagaagcaugagucacccugcugg pri-miR-17-derived pri-amiRNA (containing a CIITA-specific sequence) (SEQ ID NO:129) uaauguuacucucaccgaucacuucguagugauaugugcaucuagaagucaugggugagaggcagcauua pri-miR-20a-derived pri-amiRNA (containing a CIITA-specific sequence) (SEQ ID NO:130) uagaugaauauacuggagaguaguguuuaguuaucuaucuccaguauauucaucaa pri-miR-106a-derived pri-amiRNA (containing a CIITA-specific sequence) (SEQ ID NO:131) ccuuggccaugugcugaacuggucgcaguugauagcuuuuugagaucuacaacuucgaccaguucacgcacauuacca ugg pri-miR-19b-2-derived pri-amiRNA (containing a CIITA-specific sequence) (SEQ ID NO:132) acauugcuacuuacaauugggaggcuuugcaauaucuucagcguauauauguauauguggcugauauuggcauaagc cucccgauugugauaaugu pri-miR-363-derived pri-amiRNA (containing a CIITA-specific sequence) (SEQ ID NO:133) uguuguggagcuucugcacuagcgauguugaugaguaucauaggagaaacaucgcuguuaagaagcuccuaaacc pri-miR-18a-derived pri-amiRNA (containing a β2M-specific sequence) (SEQ ID NO:134) uguucaaucuuuggaguacgcuggauagugaaguagauuagcaucuaccagaguaugccauagauc pri-miR-19a-derived pri-amiRNA (containing a β2M-specific sequence) (SEQ ID NO:135) guccucuguucacagcccaagaguuaaguacaagaagaauguaguacuuaacuaucuugggcugugga pri-miR-19b-derived pri-amiRNA (containing a β2M-specific sequence) (SEQ ID NO:136) acugaacacuguucuaugguuaguugcuaugucugggucauugcugugugauauucugcaaacccagacacauagcaa cugacugugguagugaaaagu pri-miR-92-1-derived pri-amiRNA (containing a β2M-specific sequence) (SEQ ID NO:137) acaaggcuaagccuguuacaauacuguguuucuguaugguauuguacaagcuuagccuugu pri-miR-18b-derived pri-amiRNA (containing a β2M-specific sequence) (SEQ ID NO:138) uguguaaucuuuggaguacgcugguuagugaagcagcuuagaaucuacugcccagaguaugccauagauca pri-miR-20b-derived pri-amiRNA (containing a β2M-specific sequence) (SEQ ID NO:139) aguacacuuaacuaucuugggcugugaguuuuggcaugacucuacugcacagcccaagaguuaaguacu pri-miR-92a-derived pri-amiRNA (containing a β2M-specific sequence) (SEQ ID NO:140) ucaucccuuugcuaugucugggucauuuacuuguguucuauauaaaguauaaacccagacacauagcaaggaaga miR17-92 TCR α and TCR β-specific pri-amiRNA cluster (SEQ ID NO:141) uaauguuggauuuagagucucucagguagugauaugugcaucuacugagugagucuaaaucguagcauuauggugac agcugccucgggaagccaaguugggcuuuaaagugcagggccugcugauguugagugcuuuuuguucaucucauaga ggaugguggauagugaaguagauuagcaucuaccacaaucagcuaagagacuaagaaguuauguauucauccaauaa uucaagccaagcaaguauauagguguuuuaauaguuuuuguuugcaguccucuguuagguaaggauucauguguua caagaagaauguaguuacacaucagaauccuuaccugaugcuauuuccuucaaaugaaugauuuuuacuaauuuugu guacuuuuauugugucgauguagaaucugccuggucuaucugaugugacagcuucuguagcacuccuuucucuugac cauggguaguguuuaguuaucuaccauggucaagagaaagcaaguacugcuagcuguagaacuccagcuucggccugu cgcccaaucaaacuguccuguuacugaacacuguucuaugguuagcaacaaaugacuuugaccagcugugugauauuc ugcugcaaagucagauuuguugcugacugugguagugaaaagucuguagaaaaguaagggaaacucaaaccccuuuc uacacguucuacgggcaucuucggagacuguguuucuguauggucuccgagagcccguagaacguugaguuuggu SEQ ID NO:142 CD3 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:143 CD3 RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:144 human NKG2D – UniProt accession no: P26718 MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIA VAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNW YESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLT IIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV SEQ ID NO:145 (mouse NKG2D) MALIRDRKSHHSEMSKCHNYDLKPAKWDTSQEQQKQRLALTTSQPGENGIIRGRYPIEKL KISPMFVVRVLAIALAIRFTLNTLMWLAIFKETFQPVLCNKEVPVSSREGYCGPCPNNWI CHRNNCYQFFNEEKTWNQSQASCLSQNSSLLKIYSKEEQDFLKLVKSYHWMGLVQIPANG SWQWEDGSSLSYNQLTLVEIPKGSCAVYGSSFKAYTEDCANLNTYICMKRAV SEQ ID NO:146 rat NKG2D MSKCHNYDLKPAKWDTSQEHQKQRSALPTSRPGENGIIRRRSSIEELKISPLFVVRVLVA AMTIRFTVITLTWLAVFITLLCNKEVSVSSREGYCGPCPNDWICHRNNCYQFFNENKAWN QSQASCLSQNSSLLKIYSKEEQDFLKLVKSYHWMGLVQSPANGSWQWEDGSSLSPNELTL VKTPSGTCAVYGSSFKAYTEDCSNPNTYICMKRAV SEQ ID NO:147 mouse NKG2D transmembrane domain; UniProt accession no: 054709 VVRVLAIALAIRFTLNTLMWLAI SEQ ID NO:148 mouse NKG2D transmembrane domain KISPMFVVRVLAIALAIRFTLNTLMWLAIFKETFQPV SEQ ID NO:149 rat NKG2D Transmembrane Domain - UniProt accession no: O70215 aa 52- 74 LFVVRVLVAAMTIRFTVITLTWL SEQ ID NO:150 human extracellular NKG2D domain LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLV KSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV SEQ ID NO:151 human extracellular NKG2D domain IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKE DQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYI CMQRTV SEQ ID NO:152 IWSAVFLNS SEQ ID NO:153 PLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMG LVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV SEQ ID NO:154 human NKG2D aa52-216 – transmembrane and extracellular domain PFFFCCFIAV AMGIRFIIMV AIWSAVFLNS LFNQEVQIPL TESYCGPCPK NWICYKNNCY QFFDESKNWY ESQASCMSQN ASLLKVYSKE DQDLLKLVKS YHWMGLVHIP TNGSWQWEDG SILSPNLLTI IEMQKGDCAL YASSFKGYIE NCSTPNTYIC MQRTV SEQ ID NO:155 human NKG2D intracellular MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENAS SEQ ID NO:156 human NKG2D intracellular domain MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENA SEQ ID NO:157 murine intracellular NKG2D domain, short isoform -UniProt accession no: O54709-2 aa13-66 MSKCHNYDLKPAKWDTSQEQQKQRLALTTSQPGENGIIRGRYPIEKLKISPMF SEQ ID NO:158 murine intracellular NKG2D domain MSKCHNYDLKPAKWDTSQEQQKQRLALTTSQPGENGIIRGRYPIEKL SEQ ID NO:159 rat NKG2D Intracellular Domain- UniProt accession no: O70215 aa 1-51 MSKCHNYDLKPAKWDTSQEHQKQRSALPTSRPGENGIIRRRSSIEELKISP SEQ ID NO:160 DAP12 human polypeptide UniProt accession no: O43914 MGGLEPCSRL LLLPLLLAVS GLRPVQAQAQ SDCSCSTVSP GVLAGIVMGD LVLTVLIALA VYFLGRLVPR GRGAAEAATR KQRITETESP YQELQGQRSD VYSDLNTQRP YYK SEQ ID NO:161 human DAP12 polypeptide – cytoplasmic/intracellular domain aa62-113 YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK SEQ ID NO:162 human DAP12 polypeptide – transmembrane domain aa41-61 GVLAGIVMGD LVLTVLIALA V SEQ ID NO:163 human DAP12 polypeptide aa22-61 – extracellular and transmembrane domains LRPVQAQAQS DCSCSTVSPG VLAGIVMGDL VLTVLIALAV SEQ ID NO:164 human DAP12 polypeptide – extracellular, transmembrane, and intracellular LRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPY QELQGQRSDVYSDLNTQRPYYK SEQ ID NO:165 human DAP12 polypeptide – transmembrane and intracellular GVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRP YYK SEQ ID NO:166 murine (mouse) DAP12 extracellular, transmembrane and intracellular domains, with a leader sequence (UniProt accession no: O54885 aa 1-114) MGALEPSWCLLFLPVLLTVGGLSPVQAQSDTFPRCDCSSVSPGVLAGIVLGDLVLTLLIA LAVYSLGRLVSRGQGTAEGTRKQHIAETESPYQELQGQRPEVYSDLNTQRQYYR SEQ ID NO:167 murine (mouse) DAP12 Intracellular Domain (UniProt accession no: O54885 aa 64-114) YSLGRLVSRGQGTAEGTRKQHIAETESPYQELQGQRPEVYSDLNTQRQYYR SEQ ID NO:168 murine (mouse) DAP12 Transmembrane Domain (UniProt accession no: O54885 aa 43-63) GVLAGIVLGDLVLTLLIALAV SEQ ID NO:169 murine (mouse) DAP12 extracellular and transmembrane domains (UniProt accession no: O54885 aa 22-63) LSPVQAQSDTFPRCDCSSVSP GVLAGIVLGDLVLTLLIALAV SEQ ID NO:170 murine (mouse) DAP12 extracellular, intracellular, and transmembrane domains (UniProt accession no: O54885 aa 22-114) LSPVQAQSDTFPRCDCSSVSP GVLAGIVLGDLVLTLLIALAV YSLGRLVSRGQGTAEGTRKQHIAETESPYQELQGQRPEVYSDLNTQRQYYR SEQ ID NO:171 human DAP10 UniProt accession no: Q9UBK5 MIHLGHILFL LLLPVAAAQT TPGERSSLPA FYPGTSGSCS GCGSLSLPLL AGLVAADAVA SLLIVGAVFL CARPRRSPAQ EDGKVYINMP GRG SEQ ID NO:172 human DAP10 intracellular domain LCARPRRSPAQEDGKVYINMPGRG SEQ ID NO:173 human DAP10 QTTPGERSSL PAFYPGTSGS CSGCGSLSLP LLAGLVAADA VASLLIVGAV FLCARPRRSP AQEDGKVYIN MPGRG SEQ ID NO:174 human DAP10 extracellular and transmembrane domains QTTPGERSSL PAFYPGTSGS CSGCGSLSLP LLAGLVAADA VASLLIVGAV F SEQ ID NO:175 amino acids 1-71 of human DAP10 MIHLGHILFL LLLPVAAAQT TPGERSSLPA FYPGTSGSCS GCGSLSLPLL AGLVAADAVA SLLIVGAVFL C SEQ ID NO:176 amino acids 19-71 of human DAP10 QTTPGERSSL PAFYPGTSGS CSGCGSLSLP LLAGLVAADA VASLLIVGAV FLC SEQ ID NO:177 amino acids 49-93 of human DAP10 LLAGLVAADA VASLLIVGAV FLCARPRRSP AQEDGKVYIN MPGRG SEQ ID NO:178 amino acids 49-69 of human DAP10 LLAGLVAADA VASLLIVGAV F SEQ ID NO:179 murine (mouse) DAP10 Intracellular Domain – UniProt accession no: Q9QUJ0 aa 57-79 CMRPHGRPAQEDGRVYINMPGRG SEQ ID NO:180 mouse DAP10- UniProt accession no: Q9QUJ0 MDPPGYLLFLLLLPVAASQTSAGSCSGCGTLSLPLLAGLVAADAVMSLLIVGVVFVCMRP HGRPAQEDGRVYINMPGRG SEQ ID NO:181 mouse DAP10 - UniProt accession no: Q9QUJ0 SQTSAGSCSGCGTLSLPL LAGLVAADAVMSLLIVGVVFV CMRPHGRPAQEDGRVYINMPGRG SEQ ID NO:182 human DAP10 extracellular domain QTTPGERSSL PAFYPGTSGS CSGCGSLSLP SEQ ID NO:183 CD8α signal peptide MALPVTALLLPLALLLHAARP SEQ ID NO:184 murine (mouse) DAP10 signal peptide - UniProt accession no: Q9QUJ0 aa1- 17 MDPPGYLLFLLLLPVAA SEQ ID NO:185 murine (mouse) DAP12 signal peptide MGALEPSWCLLFLPVLLTVGG SEQ ID NO:186 human DAP12 Signal Peptide - UniProt accession no: O43914 aa 1-21 MGGLEPCSRLLLLPLLLAVSG SEQ ID NO:187 His tag HHHHHH SEQ ID NO:188 FLAG tag DYKDDDDK SEQ ID NO:189 Avi tag GLNDIFEAQKIEWHE SEQ ID NO:190 V5 tag GKPIPNPLLGLDST SEQ ID NO:191 V5 tag IPNPLLGLD SEQ ID NO:192 Myc tag EQKLISEEDL SEQ ID NO:193 SGSG linker SGSG SEQ ID NO:194 linker GSGGG SEQ ID NO:195 linker GSGG SEQ ID NO:196 linker GPPGS SEQ ID NO:197 furin cleavage site RRKR SEQ ID NO:198 P2A skip peptide ATNFSLLKQAGDVEENPGP SEQ ID NO:199 T2A skip peptide EGRGSLLTCGDVEENPGP SEQ ID NO:200 SGSG + P2A SGSGATNFSLLKQAGDVEENPGP SEQ ID NO:201 SGSG + T2A SGSGEGRGSLLTCGDVEENPGP SEQ ID NO:202 furin + SGSG + P2A RRKRSGSGATNFSLLKQAGDVEENPGP SEQ ID NO:203 furin + SGSG + T2A RRKRSGSGEGRGSLLTCGDVEENPGP SEQ ID NO:204 F2A skip peptide VKQTLNFDLLKLAGDVESNPGP SEQ ID NO:205 E2A skip peptide QCTNYALLKLAGDVESNPGP SEQ ID NO:206 SGSG linker + P2A ribosomal skip peptide + methionine SGSGATNFSLLKQAGDVEENPGPM SEQ ID NO:207 CD28 MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYG NYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHL CPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRS SEQ ID NO:208 FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO:209 IEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVR SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO:210 N1012 polypeptide MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCA RPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQ RPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQ KQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICY KNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGS ILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV SEQ ID NO:211 N1012 DNA ATGATCCACCTGGGCCACATCCTGTTCCTGCTGCTGCTGCCCGTGGCCGCTGCCCAGACCACCCCTG GCGAGCGGAGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGC CTGAGCCTGCCCCTGCTGGCCGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGG CGCCGTGTTCCTGTGCGCCAGGCCCAGGCGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAA CATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTGGTGCCCAGGGGCAGGGGCGCTGCCGAGGCT GCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAGCTGCAGGGCCAGCGGAG CGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAGAGGCGGAAAAGGTCTGGGAGTG GGGCTACCAATTTCTCTCTCCTCAAGCAAGCCGGAGACGTTGAGGAAAACCCTGGaCCcATGGGCTG GATCCGGGGACGGAGGAGCCGGCACAGCTGGGAGATGAGCGAGTTCCACAACTACAACCTGGACC TGAAGAAGAGCGACTTCAGCACCCGGTGGCAGAAGCAGCGGTGCCCCGTGGTGAAGAGCAAGTGC CGGGAGAACGCCAGCCCCTTCTTCTTCTGCTGCTTCATCGCCGTGGCtATGGGCATCCGGTTCATCA TCATGGTGGCCATCTGGAGCGCCGTGTTCCTGAACAGCCTGTTCAACCAGGAGGTGCAGATCCCCC TGACCGAGAGCTACTGCGGCCCCTGCCCCAAGAACTGGATCTGCTACAAGAACAACTGCTACCAGTT CTTCGACGAGAGCAAGAACTGGTACGAGAGCCAGGCCAGCTGCATGAGCCAGAACGCCAGCCTGCT GAAGGTGTACAGCAAGGAGGACCAGGACCTGCTGAAGCTGGTGAAGAGCTACCACTGGATGGGCC TGGTGCACATCCCCACCAACGGCAGCTGGCAGTGGGAGGACGGCAGCATCCTGAGCCCCAACCTGC TGACCATCATCGAGATGCAGAAGGGCGACTGCGCCCTGTACGCCAGCAGCTTCAAGGGCTACATCG AGAACTGCAGCACCCCCAACACCTACATCTGCATGCAGCGGACCGTG miR-20a pri-miRNA (SEQ ID NO:212) guagcacuaaagugcuuauagugcagguaguguuuaguuaucuacugcauuaugagcacuuaaaguacugc miR-92a-1 pri-miRNA (SEQ ID NO:213) cuuucuacacagguugggaucgguugcaaugcuguguuucuguaugguauugcacuugucccggccuguugaguuug g pri-miR-20a-derived pri-amiRNA (containing a TCR α-specific sequence) (SEQ ID NO:214) guagcaccaugucuagcacaguuuuguuaguguuuaguuaucuacaaaacugugcuagacaagaguacugc pri-mir-92-1-derived pri-amiRNA (containing a TCR α-specific sequence) (SEQ ID NO:215) cuuucuacacuggauccuaaccguaauuuacuguguuucuguaugguaaauucggguaggauccaguugaguuugg pri-miR-20a-derived pri-amiRNA (containing a TCR β-specific sequence) (SEQ ID NO:216) guagcacuccuuucucuugaccauggguaguguuuaguuaucuaccauggucaagagaaagcaaguacugc pri-miR-92-1-derived pri-amiRNA (containing a TCR β-specific sequence) (SEQ ID NO:217) cuuucuacacguucuacgggcaucuucggagacuguguuucuguauggucuccgagagcccguagaacguugaguuug g pri-miR-20a-derived pri-amiRNA (containing a CIITA-specific sequence) (SEQ ID NO:218) guagcacuagaugaauauacuggagaguaguguuuaguuaucuaucuccaguauauucaucaaaguacugc pri-miR-19a-derived pri-amiRNA (containing a β2M-specific sequence) (SEQ ID NO:219) gcaguccucuguucacagcccaagaguuaaguacaagaagaauguaguacuuaacuaucuugggcugugga miR-30a mature miRNA (SEQ ID NO:220) uguaaacauccucgacuggaag miR-30b mature miRNA (SEQ ID NO:221) uguaaacauccuacacucagcu miR-30c-1 mature miRNA (SEQ ID NO:222) uguaaacauccuacacucucagc miR-30c-2 mature miRNA (SEQ ID NO:223) uguaaacauccuacacucucagc miR-30d mature miRNA (SEQ ID NO:224) uguaaacauccccgacuggaag miR-30e mature miRNA (SEQ ID NO:225) uguaaacauccuugacuggaag miR-30a pri-miRNA (SEQ ID NO:226) gcgacuguaaacauccucgacuggaagcugugaagccacagaugggcuuucagucggauguuugcagcugc miR-30b pri-miRNA (SEQ ID NO:227) accaaguuucaguucauguaaacauccuacacucagcuguaauacauggauuggcugggagguggauguuuacuuca gcugacuugga miR-30c-1 pri-miRNA (SEQ ID NO:228) accaugcuguaguguguguaaacauccuacacucucagcugugagcucaagguggcugggagaggguuguuuacuccu ucugccaugga miR-30c-2 pri-miRNA (SEQ ID NO:229) agauacuguaaacauccuacacucucagcuguggaaaguaagaaagcugggagaaggcuguuuacucuuucu miR-30d pri-miRNA (SEQ ID NO:230) guuguuguaaacauccccgacuggaagcuguaagacacagcuaagcuuucagucagauguuugcugcuac miR-30e pri-miRNA (SEQ ID NO:231) gggcagucuuugcuacuguaaacauccuugacuggaagcuguaagguguucagaggagcuuucagucggauguuuac agcggcaggcugcca miR-30a loop region (SEQ ID NO:232) cugugaagccacagauggg miR-30b loop region (SEQ ID NO:233) guaauacauggauugg miR-30c-1 loop region (SEQ ID NO:234) ugugagcucaaggugg miR30c-2 loop region (SEQ ID NO:235) uguggaaaguaagaaag miR30d loop region (SEQ ID NO:236) cuguaagacacagcuaag miR-30e loop region (SEQ ID NO:237) cuguaagguguucagaggag miR-30a-derived amiRNA (CD247-specific sequence 1) (SEQ ID NO:238) aacaucguacuccucucuucgu miR-30a-derived amiRNA (CD247-specific sequence 2) (SEQ ID NO:239) uucaucccaaucucacuguagg miR-30a-derived amiRNA (CD247-specific sequence 3) (SEQ ID NO:240) aacuucacucucaggaacaagg miR-30a-derived amiRNA (CD247-specific sequence 4) (SEQ ID NO:241) uccgccaucuuaucuuucugca miR-30a-derived amiRNA (CD247-specific sequence 5) (SEQ ID NO:242) uugagcucguuauagagcuggu miR-17-derived amiRNA (CD247-specific sequence) (SEQ ID NO:243) uucaucccaaucucacuguaggc miR-17-derived amiRNA (CD247-specific sequence (SEQ ID NO:244)

auaaucugggcgucugcaggucu miR-17-derived amiRNA (CD247-specific sequence) (SEQ ID NO:245) aauuuacagcaggagugaagcca miR-20a-derived amiRNA (CD247-specific sequence 1) (SEQ ID NO:246) aacaucguacuccucucuucguc pri-miR-30a-derived pri-miRNA (containing CD247-specific sequence 1) (SEQ ID NO:247) gcgacaacaucguacuccucucuucgucugugaagccacagaugggacgaagagaaguacgauguugcugc pri-miR-30a-derived pri-miRNA (containing CD247-specific sequence 2) (SEQ ID NO:248) gcgacuucaucccaaucucacuguaggcugugaagccacagaugggccuacagugauugggaugaagcugc pri-miR-30a-derived pri-miRNA (containing CD247-specific sequence 3) (SEQ ID NO:249) gcgacaacuucacucucaggaacaaggcugugaagccacagaugggccuuguuccagagugaaguugcugc pri-miR-30a-derived pri-miRNA (containing CD247-specific sequence 4) (SEQ ID NO:250) gcgacuccgccaucuuaucuuucugcacugugaagccacagaugggugcagaaagaagauggcggagcugc pri-miR-30a-derived pri-miRNA (containing CD247-specific sequence 5) (SEQ ID NO:251) gcgacuugagcucguuauagagcuggucugugaagccacagaugggaccagcucuaacgagcucaagcugc pri-miR-30a-derived pri-miRNA (containing CD247-specific sequence 1) (SEQ ID NO:252) cuaaagaagguauauugcuguugacagugagcgacaacaucguacuccucucuucgucugugaagccacagaugggac gaagagaaguacgauguugcugccuacugccucggacuucaaggggcuacuuu pri-miR-30a-derived pri-miRNA (containing CD247-specific sequence 2) (SEQ ID NO:253) cuaaagaagguauauugcuguugacagugagcgacuucaucccaaucucacuguaggcugugaagccacagaugggcc uacagugauugggaugaagcugccuacugccucggacuucaaggggcuacuuu pri-miR-30a-derived pri-miRNA (containing CD247-specific sequence 3) (SEQ ID NO:254) cuaaagaagguauauugcuguugacagugagcgacaacuucacucucaggaacaaggcugugaagccacagaugggcc uuguuccagagugaaguugcugccuacugccucggacuucaaggggcuacuuu pri-miR-30a-derived pri-miRNA (containing CD247-specific sequence 4) (SEQ ID NO:255) cuaaagaagguauauugcuguugacagugagcgacuccgccaucuuaucuuucugcacugugaagccacagaugggug cagaaagaagauggcggagcugccuacugccucggacuucaaggggcuacuuu pri-miR-30a-derived pri-miRNA (containing CD247-specific sequence 5) (SEQ ID NO:256) cuaaagaagguauauugcuguugacagugagcgacuugagcucguuauagagcuggucugugaagccacagauggga ccagcucuaacgagcucaagcugccuacugccucggacuucaaggggcuacuuu pri-miR-17-derived pri-miRNA (containing CD247-specific sequence) (SEQ ID NO:257) gucagaauaauguaacaucguacuccucucuucgucugauaugugcaugacaagagugguguacgaugcccgcauuau ggugac pri-miR-17-derived pri-miRNA (containing CD247-specific sequence) (SEQ ID NO:258) gucagaauaauguuucaucccaaucucacuguaggcugauaugugcaugccacagucaguuugggaugcccgcauuau ggugac pri-miR-17-derived pri-miRNA (containing CD247-specific sequence) (SEQ ID NO:259) gucagaauaauguauaaucugggcgucugcaggucuugauaugugcauagacugcacacccccagauucccgcauuau ggugac pri-miR-17-derived pri-miRNA (containing CD247-specific sequence) (SEQ ID NO:260) gucagaauaauguaauuuacagcaggagugaagccaugauaugugcauugguucacaccagcuguaaacccgcauuau ggugac pri-miR-17-derived pri-miRNA (containing CD247-specific sequence) (SEQ ID NO:261) aucaccuuguaaaacugaagauugugaccagucagaauaauguaacaucguacuccucucuucgucugauaugugcau gacaagagugguguacgaugcccgcauuauggugacagcugccucgggaagccaaguugggcuuua pri-miR-17-derived pri-miRNA (containing CD247-specific sequence) (SEQ ID NO:262) aucaccuuguaaaacugaagauugugaccagucagaauaauguuucaucccaaucucacuguaggcugauaugugcau gccacagucaguuugggaugcccgcauuauggugacagcugccucgggaagccaaguugggcuuua pri-miR-17-derived pri-miRNA (containing CD247-specific sequence) (SEQ ID NO:263) aucaccuuguaaaacugaagauugugaccagucagaauaauguauaaucugggcgucugcaggucuugauaugugca uagacugcacacccccagauucccgcauuauggugacagcugccucgggaagccaaguugggcuuua pri-miR-17-derived pri-miRNA (containing CD247-specific sequence) (SEQ ID NO:264) aucaccuuguaaaacugaagauugugaccagucagaauaauguaauuuacagcaggagugaagccaugauaugugca uugguucacaccagcuguaaacccgcauuauggugacagcugccucgggaagccaaguugggcuuua pri-miR-20a-derived pri-miRNA (containing CD247-specific sequence) (SEQ ID NO:265) guagcacaacaucguacuccucucuucgucuguuuaguuaugacaagagaggaguacgaugaauguacugc pri-miR-20a-derived pri-miRNA (containing CD247-specific sequence) (SEQ ID NO:266) guagcacuucaucccaaucucacuguaggcuguuuaguuaugccacagugagauugggauguuaguacugc pri-miR-20a-derived pri-miRNA (containing CD247-specific sequence) (SEQ ID NO:267) guagcacaauuuacagcaggagugaagccauguuuaguuauugguucacuccugcuguaaaccuguacugc pri-miR-20a-derived pri-miRNA (containing CD247-specific sequence) (SEQ ID NO:268) cugccuggucuaucugaugugacagcuucuguagcacaacaucguacuccucucuucgucuguuuaguuaugacaaga gaggaguacgaugaauguacugcuagcuguagaacuccagcuucggccugucg pri-miR-20a-derived pri-miRNA (containing CD247-specific sequence) (SEQ ID NO:269) cugccuggucuaucugaugugacagcuucuguagcacuucaucccaaucucacuguaggcuguuuaguuaugccacag ugagauugggauguuaguacugcuagcuguagaacuccagcuucggccugucg pri-miR-20a-derived pri-miRNA (containing CD247-specific sequence) (SEQ ID NO:270) cugccuggucuaucugaugugacagcuucuguagcacaauuuacagcaggagugaagccauguuuaguuauugguuc acuccugcuguaaaccuguacugcuagcuguagaacuccagcuucggccugucg miR-17-92 TCRα specific pri-miRNA cluster (SEQ ID NO: 271) uaauguuggauuuagagucucucagguagugauaugugcaucuacugagugagucuaaaucguagcauuauggugac agcugccucgggaagccaaguugggcuuuaaagugcagggccugcugauguugagugcuuuuuguucaauaaugcug uuguugaaggcagugaaguagauuagcaucugcuuccacacuagcuuuaucuaagaaguuauguauucauccaauaa uucaagccaagcaaguauauagguguuuuaauaguuuuuguuugcaguccucuguuagguaaggauucauguguua caagaagaauguaguuacacaucagaauccuuaccugaugcuauuuccuucaaaugaaugauuuuuacuaauuuugu guacuuuuauugugucgauguagaaucugccuggucuaucugaugugacagcuucuguagcaccaugucuagcacag uuuuguuaguguuuaguuaucuacaaaacugugcuagacaagaguacugcuagcuguagaacuccagcuucggccug ucgcccaaucaaacuguccuguuacugaacacuguucuaugguuagcaacaaaugacuuugaccagcugugugauauu cugcugcaaagucagauuuguugcugacugugguagugaaaagucuguagaaaaguaagggaaacucaaaccccuuu cuacaacuggauccuaaccguaauuuacuguguuucuguaugguaaauucggguaggauccaguuugaguuuggu miR-17-92 β2M and CIITA-specific pri-miRNA cluster (SEQ ID NO:272) uaauguuacucucaccgaucacuucguagugauaugugcaucuagaagucaugggugagaggcagcauuauggugac agcugccucgggaagccaaguugggcuuuaaagugcagggccugcugauguugagugcuuuuuguucaaucuuugga guacgcuggauagugaaguagauuagcaucuaccagaguaugccauagaucuaagaaguuauguauucauccaauaa uucaagccaagcaaguauauagguguuuuaauaguuuuuguuugcaguccucuguucacagcccaagaguuaaguac aagaagaauguaguacuuaacuaucuugggcuguggaugcuauuuccuucaaaugaaugauuuuuacuaauuuugu guacuuuuauugugucgauguagaaucugccuggucuaucugaugugacagcuucuguagcacuagaugaauauacu ggagaguaguguuuaguuaucuaucuccaguauauucaucaaaguacugcuagcuguagaacuccagcuucggccug ucgcccaaucaaacuguccuguuacugaacacuguucuaugguuaguugcuaugucugggucauugcugugugauau ucugcaaacccagacacauagcaacugacugugguagugaaaagucuguagaaaaguaagggaaacucaaaccccuuu cuacacaaggcuaagccuguuacaauacuguguuucuguaugguauuguacaagcuuagccuuguugaguuugg miR-106a-363 β2M and CIITA-specific pri-miRNA cluster (SEQ ID NO:273) ccuuggccaugugcugaacuggucgcaguugauagcuuuuugagaucuacaacuucgaccaguucacgcacauuacca uggugauuuagucaauggcuacugagaacuguaguuugugcauaauuaaguaguugaugcuuuugagcugcuucuu auaaugugucucuuguguaaucuuuggaguacgcugguuagugaagcagcuuagaaucuacugcccagaguaugcca uagaucacaggcugccuaauauacagcauuuuaaaaguaugccuugaguaguaauuugaauaggacacauuucagug guuuguuuuuugccuuuuuauuguuuguugggaacagaugguggggacugugcaguguacaguuguguacagagga uaagauuggguccuaguaguacacuuaacuaucuugggcugugaguuuuggcaugacucuacugcacagcccaagag uuaaguacucuuggauaacaaaucucuuguugauggagagaauauucaaagacauugcuacuuacaauugggaggcu uugcaauaucuucagcguauauauguauauguggcugauauuggcauaagccucccgauugugauaaugugugcuuc cuacgucugugugaacacaccuucaugcguaucuccagcacucaugcccauucaucccuuugcuaugucugggucauuu acuuguguucuauauaaaguauaaacccagacacauagcaaggaagaaaggaggauuuuuaucgucuucuuauuuua acuuuuaaaagccguaaguucugauauuuagucauuguaaaaugaucuguuuugcuguuguggagcuucugcacuag cgauguugaugaguaucauaggagaaacaucgcuguuaagaagcuccuaaacc Human CXCR2 polypeptide (SEQ ID NO:274) MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIYALVFLLSLLGNSLVML VILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCKVVSLLKEVNFYSGILLLACISVD RYLAIVHATRTLTQKRYLVKFICLSIWGLSLLLALPVLLFRRTVYSSNVSPACYEDMGNNTANWRMLLRIL PQSFGFIVPLLIMLFCYGFTLRTLFKAHMGQKHRAMRVIFAVVLIFLLCWLPYNLVLLADTLMRTQVIQETC ERRNHIDRALDATEILGILHSCLNPLIYAFIGQKFRHGLLKILAIHGLISKDSLPKDSRPSFVGSSSGHTST TL

Claims

CLAIMS A polynucleotide construct comprising: (a) an artificial microRNA (amiRNA) coding region comprising a polynucleotide encoding an amiRNA; and (b) a protein-coding region comprising a polynucleotide encoding a chimeric antigen receptor (CAR). The construct according to claim 1, wherein the amiRNA coding region comprises a plurality of polynucleotides encoding a plurality of amiRNAs. The construct according to claim 1 or claim 2, wherein the amiRNA coding region comprises at least six polynucleotides each encoding an amiRNA. The construct according to claim 2 or claim 3, wherein the plurality of amiRNAs is derived from a miRNA cluster. The construct according to claim 4, wherein the miRNA cluster is a miR-17-92 or miR- 106a-363 cluster. The construct according to any one of the preceding claims, wherein the amiRNA is derived from miR-155, miR-17, miR-18, miR-19a, miR-19b, miR-20a and/or miR-92a-1. The construct according to any one of claims 1 to 5, wherein the amiRNA is derived from miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2 and/or miR-363. The construct according to any one of the preceding claims, wherein the amiRNA coding region comprises a polynucleotide encoding a pri-amiRNA which comprises an amiRNA. The construct according to claim 8, wherein the pri-amiRNA forms a stem-loop structure comprising a 5’ stem strand, a loop region and a 3’ stem strand. The construct according to claim 9, wherein the 5’ stem strand or 3’ stem strand comprises the amiRNA. The construct according to claim 10, wherein the pri-amiRNA is derived from a naturally occurring pri-miRNA. The construct according to claim 11, wherein the pri-amiRNA comprises a substitution in the 5’ stem strand or a portion thereof or the 3’ stem strand or a portion thereof of a naturally occurring pri-miRNA with a sequence specific to a target, optionally wherein the naturally occurring pri-miRNA is selected from the group consisting of pri-miR-155, pri-miR- 17, pri-miR-18, pri-miR-19a, pri-miR-19b, pri-miR-20a and pri-miR-92a-1. 13. The construct according to claim 12, wherein the pri-amiRNA comprises (i) a substitution of a 5’ stem strand or a portion thereof of the naturally occurring pri-miRNA with a sequence specific to a target site, and (ii) a substitution of a 3’ stem strand or a portion thereof of the naturally occurring pri-miRNA with a sequence complementary to the sequence specific to the target site. The construct according to any one of claims 9 to 13, wherein the 5’ stem strand comprises a sequence mismatch with the 3’ stem strand. The construct according to any one of claims 9 to 14, wherein the pri-amiRNA comprises a pri-miR-155, pri-miR-17, pri-miR-18, pri-miR-19a, pri-miR-19b, pri-miR-20a or pri-miR- 92a-1 loop region. The construct according to any one of claims 9 to 15, wherein the pri-amiRNA comprises a pri-miR-155 loop region. The construct according to any one of claims 9 to 15, wherein the pri-amiRNA comprises a pri-miR-17, pri-miR-18, pri-miR-19a, pri-miR-19b, pri-miR-20a or pri-miR-92a-1 loop region. The construct according to claim 17, wherein the pri-amiRNA comprises a pri-miR-19b loop region. The construct according to any one of claims 9 to 14, wherein the pri-amiRNA comprises a pri-miR-106a, pri-miR-18b, pri-miR-20b, pri-miR-19b-2, pri-miR-92a-2 or pri-miR-363 loop region. The construct according to any one of claims 9 to 19, wherein the amiRNA coding region comprises a plurality of polynucleotides encoding a plurality of pri-amiRNAs. 21. The construct according to claim 20, wherein each of the plurality of pri-amiRNAs is derived from a naturally occurring pri-miRNA. 22. The construct according to claim 21, wherein each of the plurality of pri-amiRNAs comprises a substitution of a 5’ stem strand or a portion thereof or a 3’ stem strand or a portion thereof of a naturally occurring pri-miRNA with a sequence specific to a target site, optionally wherein the naturally occurring pri-miRNA is selected from the group consisting of pri-miR-155, pri-miR-17, pri-miR-18, pri-miR-19a, pri-miR-19b, pri-miR-20a and pri-miR- 92a-1.

23. The construct according to claim 22, wherein each of the plurality of pri-amiRNAs comprises (i) a substitution of a 5’ stem strand or a portion thereof of the naturally occurring pri-miRNA with a sequence specific to a target site, and (ii) a substitution of a 3’ stem strand or a portion thereof of the naturally occurring pri-miRNA with a sequence complementary to the sequence specific to the target site. The construct according to any one of claims 20 to 23, wherein the amiRNA of each of the plurality of pri-amiRNAs is specific for a different target mRNA or a different target site. The construct according to any one of claims 20 to 24, wherein each pri-amiRNA of the plurality of pri-amiRNAs comprises a pri-miR-17, pri-miR-18, pri-miR-19a, pri-miR-19b, pri- miR-20a or pri-miR-92a-1 loop region. The construct according to any one of claims 20 to 24, wherein each pri-amiRNA of the plurality of pri-amiRNAs comprises a pri-miR-106a, pri-miR-18b, pri-miR-20b, pri-miR-19b- 2, pri-miR-92a-2 or pri-miR-363 loop region. The construct according to any one of the preceding claims, wherein the amiRNA is specific for a target mRNA selected from a tumour microenvironment (TME) mRNA, an endogenous TCR mRNA, an endogenous HLA mRNA, a CD3 subunit mRNA, a pro-apoptotic mRNA or any combination thereof. The construct according to claim 27, wherein the endogenous TCR mRNA is a αβ TCR mRNA. The construct according to claim 27 or claim 28, wherein the TME mRNA comprises a PD-1 mRNA, TIM-3 mRNA and/or LAG-3 mRNA. The construct according to any one of claims 27 to 29, wherein the endogenous HLA mRNA comprises an HLA Class I mRNA, optionally a Beta-2 microglobulin (β2M) mRNA. The construct according to any one of claims 27 to 29, wherein the endogenous HLA mRNA comprises an HLA Class II mRNA, optionally a Class II transactivator (CIITA) mRNA. The construct according to any one of claims 27 to 31, wherein the pro-apoptotic mRNA comprises Fas mRNA or TNFr mRNA. The construct according to any one of the preceding claims, wherein the amiRNA coding region is downstream of an ori and upstream of the protein-coding region. The construct according to any one of the preceding claims, wherein the construct comprises a promoter nucleotide sequence downstream of an ori and upstream of the amiRNA coding region. The construct according to any one of the preceding claims, wherein the amiRNA coding region of (a) is a first amiRNA coding region comprising a polynucleotide encoding a first amiRNA, and the construct further comprises a second amiRNA coding region comprising at least one polynucleotide encoding a second amiRNA. The construct according to claim 35, wherein the construct comprises, from 5’ to 3’, the first amiRNA coding region, the protein-coding region and the second amiRNA coding region. The construct according to any one of the preceding claims, further comprising two LTRs flanking the amiRNA coding region(s) and the protein-coding region. The construct according to claim 37, comprising from 5’ to 3’, (i) a 5’ LTR, (ii) the amiRNA coding region, (iii) the protein-coding region, and (iv) a 3’ LTR. The construct according to claim 37 or claim 38, comprising from 5’ to 3’, (i) the 5’ LTR, (ii) a splice donor, (iii) the amiRNA coding region, (iv) a splice acceptor, (v) the protein- coding region, and (vi) the 3’ LTR. 40. The construct according to any one of the preceding claims, wherein the construct does not comprise more than one promoter nucleotide sequence. The construct according to any one of the preceding claims, wherein the CAR comprises a second-generation CAR. The construct according to any one of the preceding claims, wherein the protein coding region comprises a polynucleotide encoding a DAP10 and/or DAP12 polypeptide, or variants thereof. The construct according to any one of the preceding claims, wherein the CAR comprises an NKG2D protein or variant thereof. The construct according to any one of the preceding claims, wherein the protein coding region further comprises a polynucleotide encoding a chimeric costimulatory receptor (CCR). The construct according to any one of the preceding claims, wherein the protein coding region further comprises a polynucleotide encoding a chimeric cytokine receptor, optionally a 4αβ chimeric cytokine receptor. The construct according to any one of the preceding claims, wherein the construct is in a retroviral or lentiviral vector, optionally an SFG retroviral vector. The construct according to any one of the preceding claims, wherein the CAR is not specific for MAGE-A4. A vector comprising the construct according to any one of the preceding claims. The vector of claim 48, wherein the vector is a retroviral or lentiviral vector, optionally an SFG retroviral vector. A host cell comprising the construct according to any one of claims 1-47 or the vector according to claim 48 or claim 49. The host cell of claim 50, wherein the host cell is an immuno-responsive cell. The host cell of claim 51, wherein the immuno-responsive cell is selected from the group consisting of a Natural Killer (NK) cell, a T-cell, a B-cell, a Natural Killer T-(NKT) cell or any combination thereof. The host cell according to claim 51 or claim 52, wherein the immuno-responsive cell is a T-cell. The host cell according to any one of claims 51 to 53, wherein the immuno-responsive cell is a CD8⁺ T-cell. The host cell according to any one of claims 51 to 54, wherein the immuno-responsive cell is a primary cell, optionally a human primary cell. A pharmaceutical composition comprising the construct according to any one of claims 1-47, the vector according to claim 48 or claim 49 and/or the host cell according to any one of claims 50 to 55. The pharmaceutical composition of claim 56, wherein the pharmaceutical composition further comprises a pharmaceutically or physiologically acceptable diluent and/or carrier. A kit comprising the comprising the construct according to any one of claims 1-47, the vector according to claim 48 or claim 49, the host cell according to any one of claims 50 to 55 and/or the pharmaceutical composition of claim 56 or 57. A method of preparing a host cell according to any one of claims 50 to 55, comprising the steps of: (i) introducing the construct according to any one of claims 1-47 or the vector according to claim 48 or claim 49 into a host cell, and (ii) culturing the host cell such that the CAR is expressed. The method of claim 59, wherein step (i) comprises introducing one vector according to claim 48 or claim 49 into a host cell. The construct according to any one of claims 1-47, vector according to claim 48 or claim 49, host cell according to any one of claims 50 to 55 and/or pharmaceutical composition of claim 56 or 57 for use in the treatment or prevention of a disease, optionally wherein the disease is cancer. Use of the construct according to any one of claims 1-47, vector according to claim 48 or claim 49, host cell according to any one of claims 50 to 55 and/or pharmaceutical composition of claim 56 or 57 for (i) therapy or (ii) the treatment of cancer. A method for generating an immune response to a target cell in a subject in need thereof, wherein the method comprises administering to the subject the construct according to any one of claims 1-47, vector according to claim 48 or claim 49, host cell according to any one of claims 50 to 55 and/or pharmaceutical composition of claim 56 or 57. A method of treating or preventing cancer in a subject, wherein the method comprises administering to the subject the construct according to any one of claims 1-47, vector according to claim 48 or claim 49, host cell according to any one of claims 50 to 55 and/or pharmaceutical composition of claim 56 or 57. The construct, vector, host cell and/or pharmaceutical composition for use of claim 61 or the method of claim 64, wherein the cancer is selected from ovarian cancer, breast cancer, optionally triple-negative breast cancer, pancreatic cancer, malignant mesothelioma and combinations of said cancers.