polynucleotides

Abstract

The present invention relates to polynucleotides comprising a nucleotide sequence comprising an expression cassette and a transcription termination site, wherein the nucleotide sequence further comprises: (a) one or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette and / or downstream from the transcription termination site; (b) one or more miRNA target sequences having the opposite orientation as the expression cassette; and (c) one or more insulator elements.

Description

[0001]POLYNUCLEOTIDES FIELD OF THE INVENTION The present invention relates to genetic elements and polynucleotides for improving safety of gene therapy, gene editing and cancer immunotherapy. The present invention also relates to vectors, cells, and pharmaceutical compositions comprising the same. BACKGROUND TO THE INVENTION Lentiviral Vectors (LVs) with self-inactivating (SIN) Long Terminal Repeats (LTRs) are widely used in gene therapy applications to efficiently transduce and insert therapeutic transgenes into the genome of cells from patients affected by genetic diseases. LVs with SIN LTRs have been shown to pose a lower risk of insertional mutagenesis. However, there is evidence that SIN LVs may upregulate the expression of genes flanking the integration site, especially when carrying strong enhancer / promoter sequences in internal positions or induce aberrant splicing and / or premature termination of endogenous transcripts of the targeted gene and cause loss-of-function or gain-of-function mutations (see e.g. Cesana, D., et al., 2014. Molecular therapy, 22(4), pp.774-785). Indications of genotoxicity mediated by LV integrations have been reported in clinical trials, as dominant clones with SIN LV integrations targeting HMGA2 and other cancer-related genes were observed in the Hematopoietic Stem and Progenitor Cell (HSPC) gene therapy clinical trials for beta-thalassemia (Cavazzana-Calvo, M., et al.2010. Nature 467, 318-322), X-linked severe immunodeficiency (De Ravin, S.S., et al.2016. Sci Transl Med 8, 335ra357) and adrenoleukodystrophy (ALD) (Eichler, F., et al. 2017. N Engl J Med 377, 1630-1638), and in CAR-T cancer immunotherapy trials (Shah, N.N., et al. 2019. Blood Adv 3, 2317- 2322). SIN LV integrations may also have induced cancer in three patients in a clinical trial for ALD (Tucci, F., et al.2022. Nat Commun 13, 1315). Several modifications of the LV backbone have been proposed to increase safety. These include the recoding of vector splice sites and the polyadenylation site of the LTR to avoid vector-induced aberrant splicing and read-through transcription from the integrated provirus into neighbouring genes or the inclusion of chromatin insulators to block the enhancer- mediated activation of cancer genes. However, escape mechanisms of genotoxicity, such as inactivation of tumour suppressors, may be present even when insulator sequences are used (see e.g. Cesana, D., et al., 2014. Molecular therapy, 22(4), pp.774-785). Thus, there is a need for further polynucleotides and genetic elements for improving safety of gene therapy, gene editing and cancer immunotherapy. SUMMARY OF THE INVENTION The present inventors have developed polynucleotides and genetic elements which may improve the safety of gene therapy, gene editing and cancer immunotherapy, for example by reducing the genotoxicity of lentiviral vectors comprising the same. The present inventors have identified that the mechanisms underlying genotoxicity events can be roughly divided into three classes: (i) enhancer-mediated genotoxicity, where vector enhancer sequences activate genes surrounding the insertion sites; (ii) promoter-insertion and read-through events, driven or captured by the vector sequences leading to chimeric fusion transcripts with host sequences; and (iii) aberrant splicing promoting gene truncation upon vector insertion. The present inventors have developed novel strategies to overcome all these insertional mutagenesis mechanisms using microRNA (miRNA) target sequences and chromatin insulators. The present inventors have surprisingly shown that vectors harbouring novel combinations of miRNA target sequences and chromatin insulators are produced efficiently and have an improved safety profile. miRNA target sequences are DNA sequence complementary to the sequence of micro-RNA sequences triggering degradation of the transcript carrying such target sites. miRNA target sequences have been used to target transgene expression to specific target cells in gene therapy, thereby reducing immune response related to the transgene or toxic effects in off- target cells. For this purpose, miRNA target sequences are typically placed in the expression cassette, downstream from the open reading frame, upstream from the transcription termination site, and in the same orientation as the open reading frame (see e.g. Brown, B.D. and Naldini, L., 2009. Nature Reviews Genetics, 10(8), pp.578-585). On the other hand, in the context of vector safety, the present inventors have exploited miRNA target sequences to promote the degradation of chimeric and potentially oncogenic transcript generated by fusion of vectors and host genomic RNA sequences. For this purpose, miRNA target sequences are introduced in specific orientations and configurations, e.g. (a) one or more miRNA target sequences having the same orientation as the expression cassette upstream from the expression cassette and / or downstream from the transcription termination site; and / or (b) one or more miRNA target sequences having the opposite orientation as the expression cassette upstream from the expression cassette and / or downstream from the expression cassette. The present inventors have surprisingly shown that by leveraging both antisense and sense oriented miRNA target sequences in the vector it is possible to overcome sense and antisense aberrant transcription / read-through phenomena. Chromatin insulators are DNA sequences that allow gene expression regulation of the host genome by mediating chromatin loops that physically separate genomic regions in functional domains, where sequences within the same domain interact and regulate each other, while such crosstalk is blocked in regions from different chromatin domains. The present inventors have surprisingly shown that introducing chromatin insulators within the lentiviral backbone can reduce the interactions between the integrated vector enhancers and cellular promoters, thereby improving the safety of the vectors in vivo. In one aspect, the present invention provides a polynucleotide comprising a nucleotide sequence comprising an expression cassette, wherein the nucleotide sequence further comprises: (a) one or more miRNA target sequences having the same orientation as the expression cassette; (b) one or more miRNA target sequences having the opposite orientation as the expression cassette; and (c) one or more insulator elements. Any suitable expression cassette may be used in the present invention. The expression cassette may comprise or consist of a promoter, an open reading frame, and a 3’ regulatory region. The polynucleotide may further comprise a transcription termination site. The polynucleotide may comprise the one or more miRNA target sequences having the same orientation as the expression cassette in any suitable configuration. In preferred embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette and / or downstream from the transcription termination site. In some embodiments, the nucleotide sequence comprises two or more, three or more, or four or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette and / or downstream from the transcription termination site. In some embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette; and / or one or more miRNA target sequences having the same orientation as the expression cassette, downstream from the transcription termination site. In some embodiments, the nucleotide sequence comprises two or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette, optionally wherein the two or more miRNA target sequences are two or more copies of the same miRNA target sequence arranged in tandem; and / or two or more miRNA target sequences having the same orientation as the expression cassette, downstream from the transcription termination site, optionally wherein the two or more miRNA target sequences are two or more copies of the same miRNA target sequence arranged in tandem. In some embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette. In some embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the same orientation as the expression cassette, directly upstream from the expression cassette. In some embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the same orientation as the expression cassette 100 bp or less upstream from the expression cassette. In some embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the same orientation as the expression cassette, downstream from the transcription termination site. In some embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the same orientation as the expression cassette, directly downstream from the transcription termination site. In some embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the same orientation as the expression cassette 100 bp or less downstream from the transcription termination site. Any suitable miRNA target sequence having the same orientation as the expression cassette may be used in the present invention. In some embodiments, the one or more miRNA target sequences having the same orientation as the expression cassette are selected from a miR223 target sequence, a miR142 target sequence, a miR22 target sequence, a miR122 target sequence, a miR126 target sequence, a miR124 target sequence, a miR133a target sequence, a miR133b target sequence, a let7f target sequence, a miR7 target sequence, or a combination thereof. In some embodiments, the one or more miRNA target sequences having the same orientation as the expression cassette are miR142-3p target sequences. In some embodiments, the one or more miRNA target sequences having the same orientation as the expression cassette each comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 10. In some embodiments, the one or more miRNA target sequences having the same orientation as the expression cassette each comprise or consist of the nucleotide sequence of SEQ ID NO: 1. The polynucleotide may comprise the one or more miRNA target sequences having the opposite orientation as the expression cassette in any suitable configuration. In some embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the nucleotide sequence comprises two or more, three or more, or four or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette; and / or one or more miRNA target sequences having the opposite orientation as the expression cassette, downstream from the expression cassette. In some embodiments, the nucleotide sequence comprises two or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette, optionally wherein the two or more miRNA target sequences are two or more copies of the same miRNA target sequence arranged in tandem; and / or two or more miRNA target sequences having the opposite orientation as the expression cassette, downstream from the expression cassette, optionally wherein the two or more miRNA target sequences are two or more copies of the same miRNA target sequence arranged in tandem. In some embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette. In some embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the opposite orientation as the expression cassette, directly upstream from the expression cassette. In some embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the opposite orientation as the expression cassette 100 bp or less upstream from the expression cassette. In some embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the opposite orientation as the expression cassette, downstream from the expression cassette. In some embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the opposite orientation as the expression cassette, directly downstream from the expression cassette. In some embodiments, the nucleotide sequence comprises one or more miRNA target sequences having the opposite orientation as the expression cassette 100 bp or less downstream from the expression cassette. Any suitable miRNA target sequence having the opposite orientation as the expression cassette may be used in the present invention. In some embodiments, the one or more miRNA target sequences having the opposite orientation as the expression cassette are selected from a miR223 target sequence, a miR142 target sequence, a miR22 target sequence, a miR122 target sequence, a miR126 target sequence, a miR124 target sequence, a miR133a target sequence, a miR133b target sequence, a let7f target sequence, a miR7 target sequence, or a combination thereof. In some embodiments, wherein the one or more miRNA target sequences having the opposite orientation as the expression cassette are miR223-3p target sequences. In some embodiments, the one or more miRNA target sequences having the opposite orientation as the expression cassette each comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 to 20. In some embodiments, the one or more miRNA target sequences having the opposite orientation as the expression cassette each comprise or consist of the nucleotide sequence of SEQ ID NO: 12. The polynucleotide may comprise the one or more insulator elements in any suitable configuration. In some embodiments, the nucleotide sequence comprises one or more insulator elements upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the nucleotide sequence comprises two or more insulator elements upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the nucleotide sequence comprises a pair of insulator elements flanking the expression cassette. In some embodiments, the pair of insulator elements is in a divergent orientation. In some embodiments, the pair of insulator elements is in a convergent orientation. In some embodiments, the pair of insulator elements is in a parallel orientation. Any suitable insulator element may be used in the present invention. In some embodiments, the one or more insulator elements are CTCF-based insulator elements. In some embodiments, the one or more insulator elements each comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 23 to 28. In some embodiments, the one or more insulator elements each comprise or consist of a core insulator sequence. In some embodiments, the one or more insulator elements each comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29 to 36. In some embodiments, the one or more insulator elements each comprise or consist of a full length insulator sequence. In some embodiments, the one or more insulator elements each comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 37 to 90, 102 or 103, or a nucleotide sequence having at least 90% identity thereto. In some embodiments, the nucleotide sequence further comprises a 3’ LTR, optionally wherein the 3’ LTR is a self-inactivating (SIN) 3’ LTR. The 3’LTR may comprise one or more of the genetic elements in any suitable configurations. By cloning the genetic elements within the 3’LTR, after reverse transcription, these genetic elements may be copied in the same orientation within the 5’LTR. In some embodiments, the 3’ LTR comprises one or more miRNA target sequences having the same orientation as the expression cassette. In preferred embodiments, the 3’ LTR does not comprise a miRNA target sequence having the same orientation as the expression cassette. In some embodiments, the 3’ LTR comprises one or more miRNA target sequences having the opposite orientation as the expression cassette. In other embodiments, the 3’ LTR does not comprise a miRNA target sequence having the opposite orientation as the expression cassette. In some embodiments, the 3’ LTR comprises one or more insulator elements. In other embodiments, the 3’ LTR does not comprise an insulator element. In some embodiments, the 3’ LTR does not comprise (a) one or more miRNA target sequences having the same orientation as the expression cassette; and the 3’ LTR comprises (b) one or more miRNA target sequences having the opposite orientation as the expression cassette and / or (c) one or more insulator elements. In some embodiments, the 3’ LTR comprises (b) one or more miRNA target sequences having the opposite orientation as the expression cassette; and (c) one or more insulator elements. In some embodiments, the nucleotide sequence further comprises a 5’ LTR, optionally wherein the 5’ LTR is a self-inactivating (SIN) 5’ LTR. The 5’ LTR may comprise one or more of the genetic elements in any suitable configurations. In some embodiments, the 5’ LTR comprises one or more miRNA target sequences having the same orientation as the expression cassette. In other embodiments, the 5’ LTR does not comprise a miRNA target sequence having the same orientation as the expression cassette. In some embodiments, the 5’ LTR comprises one or more miRNA target sequences having the opposite orientation as the expression cassette. In other embodiments, the 5’ LTR does not comprise a miRNA target sequence having the opposite orientation as the expression cassette. In some embodiments, the 5’ LTR comprises one or more insulator elements. In other embodiments, the 5’ LTR does not comprise an insulator element. In some embodiments, the 5’ LTR does not comprise (a) one or more miRNA target sequences having the same orientation as the expression cassette; (b) one or more miRNA target sequences having the opposite orientation as the expression cassette; or (c) one or more insulator elements. In another aspect, the present invention provides a vector comprising the polynucleotide of the present invention. The vector may be a viral vector, a plasmid, or a transposon. In some embodiments, the vector is a viral vector selected from the group consisting of a lentiviral vector, a retroviral vector, and an adeno-associated viral (AAV) vector. In some embodiments, the vector is a retroviral vector. In preferred embodiments, the vector is a lentiviral vector. In another aspect, the present invention provides a viral particle comprising the polynucleotide of the present invention. The viral particle may be selected from the group consisting of a lentiviral particle, a retroviral particle, and an adeno-associated viral (AAV) particle. In some embodiments, the viral particle is a retroviral particle. In some embodiments, the viral particle is a lentiviral particle. In another aspect, the present invention provides a retroviral vector comprising from 5’ to 3’ direction: a 5’ LTR; one or more sense-oriented miRNA target sequences; a sense-oriented expression cassette; and a 3’ LTR. The retroviral vector may further comprise any other genetic elements for improving vector safety, such as one or more antisense-oriented miRNA target sequences and / or one or more insulator elements. In some embodiments, the retroviral vector further comprises one or more antisense-oriented miRNA target sequences. In some embodiments, the 3’ LTR comprises one or more antisense-oriented miRNA target sequences. In some embodiments, the retroviral vector further comprises one or more insulator elements. In some embodiments, the 3’ LTR comprises one or more insulator elements. In some embodiments, the 3’ LTR comprises one or more antisense-oriented miRNA target sequences and one or more insulator elements. In some embodiments, the retroviral vector comprises from 5’ to 3’ direction: a 5’ LTR; one or more sense-oriented miRNA target sequences; a sense-oriented expression cassette; and a 3’ LTR, wherein the 3’ LTR comprises from 5’ to 3’ direction: a first insulator element, one or more antisense-oriented miRNA target sequences, and a second insulator element, wherein the first insulator element and the second insulator element are in a divergent orientation. In some embodiments, the first insulator element is an A2 insulator element and the second insulator element is an E2 insulator element. In some embodiments, the first insulator element and the second insulator element each comprise or consist of a full length insulator sequence. In some embodiments, the first insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103, or a nucleotide sequence having at least 90% identity thereto, and / or the second insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 55, or a nucleotide sequence having at least 90% identity thereto. In some embodiments, the first insulator element and the second insulator element each consist of a core insulator sequence. In some embodiments, the first insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 34 and / or the second insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 32. In other embodiments, the retroviral vector comprises from 5’ to 3’ direction: a 5’ LTR; one or more sense-oriented miRNA target sequences; a sense-oriented expression cassette; a first insulator element; and a 3’ LTR, wherein the 3’ LTR comprises from 5’ to 3’ direction: one or more antisense-oriented miRNA target sequences, and a second insulator element, wherein the first insulator element and the second insulator element are in a divergent orientation. In some embodiments, the first insulator element is an A2 insulator element and the second insulator element is an E2 insulator element. In some embodiments, the first insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103, or a nucleotide sequence having at least 90% identity thereto, and / or the second insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 55, or a nucleotide sequence having at least 90% identity thereto. In other embodiments, the retroviral vector comprises from 5’ to 3’ direction: a 5’ LTR; one or more sense-oriented miRNA target sequences; a sense-oriented expression cassette; and a 3’ LTR, wherein the 3’ LTR comprises from 5’ to 3’ direction: one or more insulator elements and one or more antisense-oriented miRNA target sequences. In some embodiments, the one or more insulator elements consist of an A1 insulator element and an A2 insulator element in a parallel orientation, wherein the A1 insulator element and the A2 insulator element each comprise or consist of a full length insulator sequence. In some embodiments, the A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 64, or a nucleotide sequence having at least 90% identity thereto, and / or the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103, or a nucleotide sequence having at least 90% identity thereto. In other embodiments, the one or more insulator elements consist of an A1 insulator element and an A2 insulator element in a parallel orientation, wherein the A1 insulator element and the A2 insulator element each consist of a core insulator sequence. In some embodiments, the A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 33 and / or the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 34. In other embodiments, the one or more insulator elements consist of an A2 insulator element, wherein the A2 insulator element comprises or consists of a full length insulator sequence. In some embodiments, the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103, or a nucleotide sequence having at least 90% identity thereto. In other embodiments, the one or more insulator elements consist of an A2 insulator element, wherein the A2 insulator element consists of a core insulator sequence. In some embodiments, wherein the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 34. In other embodiments, the one or more insulator elements consist of an A1 insulator element, wherein the A1 insulator element consists of a core insulator sequence. In some embodiments, wherein the A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 33. In other embodiments, the one or more insulator elements consist of a B2 insulator element, wherein the B2 insulator element consists of a core insulator sequence. In some embodiments, wherein the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 35. In another aspect, the present invention provides a retroviral vector comprising from 5’ to 3’ direction: a 5’ LTR; an antisense-oriented expression cassette; and a 3’ LTR, wherein the retroviral vector further comprises: (a) one or more antisense-oriented miRNA target sequences; (b) one or more sense-oriented miRNA target sequences; and (c) one or more insulator elements. In preferred embodiments, the retroviral vector comprises one or more antisense-oriented miRNA target sequences upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the retroviral vector comprises one or more sense-oriented miRNA target sequences upstream from the expression cassette and / or downstream from the expression cassette; and / or one or more insulator elements, upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the 3’ LTR does not comprise one or more antisense-oriented miRNA target sequences. In some embodiments, the 3’ LTR does not comprise one or more sense- oriented miRNA target sequences. Any suitable sense-oriented or antisense-oriented miRNA target sequence may be used in the present invention. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of two or more miR142-3p target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of two or more miR223-3p target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 22. The retroviral vector of the present invention may be derived from any type of retroviral vector. In preferred embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the lentiviral vector is a self-inactivating lentiviral vector. In some embodiments, the 3’ LTR is a self-inactivating (SIN) 3’ LTR. In some embodiments, the lentiviral vector is an integration-proficient lentiviral vector (IPLV). The retroviral vector of the present invention may have improved safety (e.g. reduced toxicity). The retroviral vector may be substantially non-genotoxic. The one or more sense- oriented miRNA target sequences and the one or more antisense-oriented miRNA target sequences may avoid and / or reduce aberrant splicing and / or read-through events. The one or more insulator elements may avoid and / or reduce enhancer-mediated gene activation. In another aspect, the present invention provides a cell comprising the polynucleotide of the present invention, the vector of the present invention, the viral particle of the present invention, or the retroviral vector of the present invention. In some embodiments, the cell is a hematopoietic stem and / or progenitor cell (HSPC). In another aspect, the present invention provides a pharmaceutical composition comprising the polynucleotide of the present invention, the vector of the present invention, the viral particle of the present invention, the retroviral vector of the present invention, or the cell of the present invention, in combination with a pharmaceutically acceptable carrier, diluent or excipient. In another aspect, the present invention provides the polynucleotide of the present invention, the vector of the present invention, the viral particle of the present invention, the retroviral vector of the present invention, the cell of the present invention, or the pharmaceutical composition of the present invention, for use as a medicament. In another aspect, the present invention provides a method of reducing the genotoxicity of a polynucleotide or vector comprising a nucleotide sequence comprising an expression cassette and a transcription termination site, wherein the method comprises introducing into the nucleotide sequence: (a) one or more miRNA target sequences having the same orientation as the expression cassette; (b) one or more miRNA target sequences having the opposite orientation as the expression cassette; and (c) one or more insulator elements. In some embodiments, the one or more miRNA target sequences having the same orientation as the expression cassette are introduced upstream from the expression cassette and / or downstream from the transcription termination site. In another aspect, the present invention provides a method of reducing the genotoxicity of a retroviral vector comprising a sense-oriented expression cassette, wherein the method comprises introducing into the retroviral vector: (a) one or more sense-oriented miRNA target sequences upstream from the sense-oriented expression cassette. In some embodiments, the method further comprises introducing into the retroviral vector: (b) one or more antisense-oriented miRNA target sequences upstream from the sense- oriented expression cassette and / or downstream from the sense-oriented expression cassette; and / or (c) one or more insulator elements upstream from the sense-oriented expression cassette and / or downstream from the sense-oriented expression cassette. In another aspect, the present invention provides a method of reducing the genotoxicity of a retroviral vector comprising an antisense-oriented expression cassette, wherein the method comprises introducing into the retroviral vector: (a) one or more antisense-oriented miRNA target sequences upstream from the antisense-oriented expression cassette and / or downstream from the antisense-oriented expression cassette; (b) one or more sense- oriented miRNA target sequences upstream from the antisense-oriented expression cassette and / or downstream from the antisense-oriented expression cassette; and (c) one or more insulator elements upstream from the antisense-oriented expression cassette and / or downstream from the antisense-oriented expression cassette. In another aspect, the present invention provides an insulator element comprising or consisting of a sequence having at least 95% sequence identity to SEQ ID NO: 103, wherein the insulator element comprises the nucleotides G and C at positions corresponding to nucleotides 45 and 46, respectively, wherein the nucleotides are numbered with reference to SEQ ID NO: 103. In some embodiments, the insulator element comprises SEQ ID NO: 34. In some embodiments, the insulator element comprises or consists of SEQ ID NO: 103. In another aspect, the present invention provides an insulator element comprising or consisting of a sequence having at least 95% sequence identity to SEQ ID NO: 102, wherein the insulator element comprises the nucleotides G and C at positions corresponding to nucleotides 221 and 222, respectively, wherein the nucleotides are numbered with reference to SEQ ID NO: 102. In some embodiments, the insulator element comprises SEQ ID NO: 30. In some embodiments, the insulator element comprises or consists of SEQ ID NO: 102. In another aspect, the present invention provides a polynucleotide comprising a nucleotide sequence comprising the insulator element of the present invention. In some embodiments, the nucleotide sequence further comprises an expression cassette and the insulator element flanks the expression cassette. In another aspect, the present invention provides a vector comprising the polynucleotide. In another aspect, the present invention provides a cell comprising the polynucleotide or the vector. In another aspect, the present invention provides a self-inactivating (SIN) 3’ LTR comprising one or more antisense-oriented miRNA target sequences and one or more insulator elements. In another aspect, the present invention provides a self-inactivating (SIN) 3’ LTR comprising or consisting of a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 104 to 112. In some embodiments, the SIN 3’ LTR comprises an antisense-oriented miRNA target sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the SIN 3’ LTR comprises an insulator element comprising or consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29 to 36. In another aspect, the present invention provides a polynucleotide comprising a nucleotide sequence comprising the SIN 3’ LTR of the present invention. In some embodiments, the nucleotide sequence further comprises an expression cassette. In another aspect, the present invention provides a vector comprising the polynucleotide. In another aspect, the present invention provides a cell comprising the polynucleotide or the vector. DESCRIPTION OF DRAWINGS Figure 1 - Generation of insulator-harbouring SIN.LVs and in vitro characterization A) Schematic diagram of SIN.LV vectors comprising chromatin insulators: SIN, self- inactivating LTR; SD, splice donor; SA, splice acceptor; SFFV, SFFV enhancer / promoter; GFP, green fluorescent protein; PRE, posttranscriptional regulatory element; A2, A2 chromatin insulator; E2, E2 chromatin insulator. B) Experimental scheme to generate K562 transduced clones and molecular characterisation. C) Analysis of LV-mediated interactions with genomic loci ± 0.5Mb from centre. All LV sequences are oriented 5’ to 3’. Two independent clones per vector constructs are shown. D) LV4C derived interactions (arches) are show for SIN LV (left) and A2 insulated LV (right). E) CTCF-enrichment analysis at the LV / genome interaction site show an enrichment of genomic CTCF sites in convergent orientation with the A2 insulator contained in the LV LTR. F) Insulation boundary analysis for the different vector types is shown. X-axis indicates distance in Kb; Y-axis indicates the insulation score; a single line indicates the interaction skewing as readout of insulation; the line with confidence limits indicates the distribution at sites without insertion sites. Figure 2 - In vivo safety study of CI-harbouring SIN.LVs in tumour prone mice A) Experimental scheme of the in vivo genotoxicity assays with tumour prone Cdkn2a+ / -mice. B) Survival kinetics of the Cdkn2a+ / -mice treated with SIN.LV (SIN.SF); A2-insulated SIN.LV (A2.LV); E2-insulated SIN.LV (E2.LV) and untreated Mock-controls. C) Common Integration Sites (CIS) of the different treatment groups in Cdkn2a+ / -mice with a CIS power ≥4 are shown. Figure 3 - In vivo safety evaluation of SIN.LVs with reduced aberrant-splicing / read- through potential A) Schematic diagram of miRT-containing SIN.LVs, mirT.LV and mirT.LTR.LV: SIN, self- inactivating LTR; SD, splice donor; SA, splice acceptor; cPPT, central polypurine tract; pA, polyadenylation sequence; GFP, green fluorescent protein; PGK, PGK enhancer-promoter; PRE, posttranscriptional regulatory element; 142.3pT, miR142-3p target sequence; 223T, miR223 target sequence. B) Experimental scheme of the in vivo genotoxicity assays with tumour prone Cdkn2a- / -mice. C) Survival kinetics of the Cdkn2a- / -mice treated with SIN.PGK; mirT.LV; mirT.LTR.LV and untreated Mock-controls. D) Common Integration Sites (CIS) of the different treatment groups in Cdkn2a- / -mice. CIS genes with power ≥4 are shown. Figure 4 - Combination of safety-features to enhance vector safety A) Schematic representation of the chromatin loops and insulation regulation by the convergent (CONV) or divergent (DIV) insulator configurations. B) Schematic representation of the inclusion of different micro-RNA-target sites (miRT) in the vectors. Two tandem copies of each of the hematopoietic- or liver-specific miRTs are included in the LTRs. Arrows indicate sense and antisense orientation of the miRT comparted to vector backbone transcription. C) Schematic diagram of control and CI- and miRT-containing SIN.LVs, ET.LTR, SIN.ETas, Combo CONV, and Combo DIV: SIN, self-inactivating LTR; ET, ET promoter; SD, splice donor; SA, splice acceptor; GA, truncated gag sequence; RRE, Rev responsive element; WPRE, woodchuck posttranscriptional regulatory element; pA, polyadenylation sequence; GFP, green fluorescent protein; miR142T, miR142 target sequence; A2, A2 chromatin insulator; E2, E2 chromatin insulator. D) Experimental scheme of the in vivo assay with WT mice treated with cancer-promoting regimen to evaluate the safety of the different test vectors. E) Results from histopathology analysis in liver from mice treated with the different vectors. Bars indicate the event frequency calculated as total occurrences per number of mice composing the treatment group. Numbers on top of the bars indicate the absolute number of events. F) Integration sites analysis and abundance analysis from healthy liver tissues (first column) and tumours (following columns) of the vector-treated mice. Stacked bars indicate vector integration sites. IS are sorted from most abundant to least abundant integration. Gene list highlights the top abundant IS. Genes known to be genotoxic or that could potentially induce transformation are highlighted. Mouse ID and the total number of insertions is shown at the top of the plot. Figure 5 - Optimisation of vector designs comprising a combination of safety-features A) Schematic diagram of the COMBO-LV designs and control vector tested in the in vitro assay: SF, SF enhancer-promoter; GFP, green fluorescent protein; PRE, posttranscriptional regulatory element; CI, chromatin enhancer; mirT AS, antisense-oriented miRNA target sequence; mirT sense, sense-oriented miRNA target sequence. Control LV is a SIN.LV with SF enhancer-promoter, GFP and mutated PRE. All test vectors contain the novel positioning of hematopoietic-active miRTs (223 and 142-3p) in the LV backbone and a combination of insulator elements. The Div.Conv LV contains E2 and A2mut CI in divergent and convergent orientations in the LTRs; the Div.Conv.Core LV contains A2 and E2 core sequences in divergent and convergent orientation cloned in the LTR; the Split.Conv LV displays a single internal copy of A2mut and two copies of E2 in the two LTRs. A1.A2 insulator combination placed in parallel orientation in the LTR were tested in full length or core versions. Individual core insulators A1, A2 and B2 were also tested in combination with mirTs. B) Mean fluorescence intensity (MFI) measured at FACS in technical triplicates for each vector preparation for and for each construct. C) Non-concentrated vector titre assayed for all tested vectors and controls. D) Vector infectivity measured for all test vectors and controls. The “*” indicates a threshold for good-quality lab-grade vector production performances. Figure 6 - Example configurations of miRNA target sequences (mirT) in lentiviral vector (LV) proviruses A-C) For sense-oriented expression cassettes in LVs. Sense-oriented mirT (yellow in scheme): preferred embodiment upstream the expression cassette. Antisense-oriented mirT: no preferred embodiment, can be placed in the LTRs, upstream and / or downstream the expression cassette. D-G) For antisense-oriented expression cassettes in LVs. Sense- oriented mirT: no preferred embodiment, can be placed in the LTRs, upstream and / or downstream the expression cassette, in single or combination (convergent and divergent orientation). Antisense-oriented mirT: no preferred embodiment, can be placed in the LTRs, upstream and / or downstream the expression cassette, in single or combination (convergent and divergent orientation). Figure legend: E / P: enhancer / promoter; T: transgene; O: other (e.g. WPRE site and / or any other transcriptional and / or post-transcriptional regulatory element); mirT AS: antisense oriented micro-RNA target sites; mirT sense: sense oriented micro-RNA target sites. Figure 7 - Example configurations of insulator elements in lentiviral vector (LV) proviruses A-F) Chromatin insulators within the LTRs for sense- and antisense- oriented expression cassettes. Embodiment within the LTR in combination (preferred embodiment divergent or parallel orientation) or single elements both in sense or antisense orientations. G-H and J-K) Chromatin insulators within the backbone for sense- and antisense- oriented expression cassettes. Within the LV backbone preferred embodiment upstream and downstream the expression cassette, in divergent and / or convergent orientation. Embodiment in parallel orientation is also possible. I, L) Insulators within LTRs and backbone for sense- and antisense- oriented expression cassettes. Within the LTR or backbone as described above. Preferred embodiment divergent in LTR, sense and / or antisense in the backbone, both upstream and / or downstream the expression cassette is possible. Figure legend: E / P: enhancer / promoter; T: transgene; O: other (e.g. WPRE site, polyA site and / or any other transcriptional and / or post-transcriptional regulatory element); CI: Chromatin Insulator. DETAILED DESCRIPTION Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed. It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes", "containing", or "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or steps. The terms "comprising", "comprises" and "comprised of" also include the term "consisting of". Numeric ranges are inclusive of the numbers defining the range. As used herein the term “about” means approximately, in the region of, roughly, or around. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxy orientation, respectively. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. All publications mentioned in the specification are herein incorporated by reference. Polynucleotides In one aspect, the present invention provides a polynucleotide comprising an expression cassette, wherein the polynucleotide further comprises: (a) one or more miRNA target sequences having the same orientation as the expression cassette; (b) one or more miRNA target sequences having the opposite orientation as the expression cassette; and / or (c) one or more insulator elements. The polynucleotide of the present invention may be an isolated polynucleotide. The polynucleotide of the present invention may be double or single stranded. The polynucleotide of the invention may comprise or consist of DNA and / or RNA. In some embodiments, the polynucleotide is a DNA polynucleotide. In some embodiments, the polynucleotide is a RNA polynucleotide. The directionality of the polynucleotide may be used to define the orientation of the nucleotide sequences comprised therein. A DNA polynucleotide has one strand in the 5′ to 3′ direction with respect to the carbon atoms on the deoxyribose (referred to herein as the “sense” strand) and one complementary strand in the 3′ to 5′ direction (referred to herein as the “anti-sense strand). A RNA polynucleotide has one strand is in the 5′ to 3′ direction with respect to the carbon atoms on the ribose. Expression cassettes The polynucleotide of the present invention comprises an expression cassette. An “expression cassette” may refer to a nucleotide sequence consisting of a gene and one or more regulatory sequences controlling the expression of the gene. An expression cassette typically comprises a promoter or enhancer-promoter, an open reading frame, and a 3’ regulatory region. An expression cassette is transcribed from DNA from 5’ to 3’. The orientation of the expression cassette may be used to define the relative orientation of the other elements. In some embodiments, an expression cassette comprises or consists of from 5’ to 3’ direction: a promoter or enhancer-promoter, an open reading frame, and a 3’ regulatory region. An expression cassette may be sense-oriented or antisense-oriented. In the context of a DNA polynucleotide, a “sense-oriented” expression cassette is transcribed from the sense strand. In the context of a DNA polynucleotide, an “antisense-oriented” expression cassette is transcribed from the antisense strand. Promoters and enhancers A “promoter” may refer to a nucleotide sequence that leads to initiation of transcription of a gene. Promoters are located near the transcription start sites of genes, upstream from the gene. Any promoter may be used in the present invention, the selection of which may be readily made by the skilled person. In some embodiments, the promoter is a constitutive promoter. A “constitutive promoter” may refer to a promoter which is always active. Suitable constitutive promoters will be known to the skilled person. Example constitutive promoters include the CMV promoter, the EF1a promoter, the CAG promoter, the PGK promoter, the U6 promoter, the T7 promoter, the SV40 promoter, and the Sp6 promoter. In some embodiments, the promoter is an inducible promoter. An “inducible promoter” may refer to a promoter which is activated in response to specific stimuli (e.g. in response to chemicals, temperature, or light). Suitable inducible promoters will be known to the skilled person. Example inducible promoters include the TRE promoter, the lac promoter, the araBad promoter. In some embodiments, the promoter is a repressible promoter. A “repressible promoter” may refer to a promoter which is de-activated in response to specific stimuli. Suitable repressible promoters will be known to the skilled person. Example repressible promoters include the trp promoter. In some embodiments, the promoter is a ubiquitous promoter. As used herein, a “ubiquitous promoter” is a promoter which is active in a wide range of cells and tissues. Suitable ubiquitous promoters will be known to the skilled person and include, e.g. Phosphoglycerate kinase (PGK) promoter, Cytomegalovirus (CMV) promoter, EF1alpha promoter (short), EF1alpha promoter (long), Ubiquitin C (UbiC) promoter, Simian Virus 40 (SV40) enhancer / promoter, U6 promoter, H1 promoter, CAG promoter (CMV enhancer / chicken beta-actin promoter), or comprises a TRE (tetracyclin response element). In some embodiments, the promoter is a PGK promoter. In some embodiments, the promoter is a tissue-specific promoter. A “tissue-specific promoter” may refer to a promoter which preferentially facilitates expression of a gene in a specific cell and / or tissue. Suitable tissue-specific promoters will be known to the skilled person. In some embodiments, the promoter is a hematopoietic-specific promoter, erythroid- specific promoter, liver-specific promoter, or neuron-specific promoter. In some embodiments, the promoter is a hematopoietic-specific promoter. In some embodiments, the promoter comprises Universal chromatin opener element (UCOE) enhancer. In some embodiments, the promoter is a Gp91 phox promoter, Wiskott Aldrich- Syndrome (WAS) promoter, Murine-leukemia virus derived MND promoter, Spleen Focus Forming Virus (SFFV) enhancer / promoter, or Tyrosine protein kinase receptor (Tie2) promoter. In some embodiments, the promoter is a SFFV enhancer / promoter. In some embodiments, the promoter is an erythroid-specific promoter. In some embodiments, the promoter comprises ß globin LCR HS2 and HS3 or ß globin LCR HS2, HS3, and HS4 In some embodiments, the promoter is a liver-specific promoter. In some embodiments, the promoter is an Enhanced Transthyretin (ET) enhancer / promoter, Albumin promoter (Alb) promoter, or Apolipoprotein (Apo) promoter. In some embodiments, the promoter is an ET promoter. In some embodiments, the promoter is a neuron-specific promoter. In some embodiments, the promoter is a Nestin promoter or CaMKIIa promoter. In some embodiments, the promoter is a strong promoter. As used herein, a “strong promoter” may refer to a promoter which facilitates high expression of a protein-coding sequence. Genes regulated by strong promoters yield more mRNA and therefore more product protein than genes regulated by weak promoters. Example strong promoters include the spleen focus forming virus (SFFV) promoter. In some embodiments, the promoter is a moderate promoter. As used herein, a “moderate promoter” may refer to a promoter which facilitates moderate expression of a protein-coding sequence. Example moderate promoters include the PGK promoter. In some embodiments, the promoter is a viral promoter, e.g. a strong viral promoter. As used herein, a “viral promoter” may refer to a promoter derived from a viral genome. In some embodiments, the promoter is a cellular promoter. As used herein, a “cellular promoter” may refer to a promoter derived from a cell. In some embodiments, the promoter is a bacterial promoter. In some embodiments, the promoter is a eukaryotic promoter. In some embodiments, the promoter is a mammalian promoter. In some embodiments, the polynucleotide further comprises an enhancer. An “enhancer” may refer to a nucleotide sequence that can be bound by activators to enhance transcription. Enhancers are cis-acting and can be located up to 1 Mbp (1,000,000 bp) away from the promoter, upstream or downstream from the start site. Suitably, the enhancer is upstream from the promoter. Any suitable enhancer may be used, the selection of which may be readily made by the skilled person. In some embodiments, an expression cassette comprises an enhancer-promoter. In some embodiments, an expression cassette comprises or consists of from 5’ to 3’ direction: an enhancer-promoter, an open reading frame, and a 3’ regulatory region. In some embodiments, the enhancer-promoter is a strong enhancer-promoter. Example strong enhancer-promoters include the spleen focus forming virus (SFFV) enhancer- promoter. In some embodiments, the enhancer-promoter is a moderate enhancer-promoter. Example moderate enhancer-promoters include the PGK enhancer-promoter. In some embodiments, the enhancer-promoter is a viral enhancer-promoter, e.g. a strong viral enhancer-promoter. In some embodiments, the promoter is a cellular enhancer-promoter. In some embodiments, the promoter is a bacterial enhancer-promoter. In some embodiments, the promoter is a eukaryotic enhancer-promoter. In some embodiments, the promoter is a mammalian enhancer-promoter. 5’ regulatory region An expression cassette may further comprise a 5' regulatory region. In some embodiments, an expression cassette comprises or consists of from 5’ to 3’ direction: a promoter or enhancer-promoter, a 5' regulatory region, an open reading frame, and a 3’ regulatory region. A “5’ regulatory region” may refer to a nucleotide sequence that is downstream from the transcription start site and upstream from the start codon which controls expression of the open reading frame. The 5′ regulatory region is typically not translated, but forms a complex secondary structure in the mRNA to regulate translation. Any 5′ regulatory region may be used, the selection of which may be readily made by the skilled person. A prokaryotic 5′ regulatory region can contains a ribosome binding site (RBS), for example having the sequence AGGAGGT, which is usually 3–10 base pairs upstream from the initiation codon. A eukaryotic 5′ regulatory region usually contains a Kozak sequence, for example having the sequence ACCATGG, which contains the initiation codon. A eukaryotic 5′ regulatory region may also contain cis-acting regulatory elements. Open reading frame An “open reading frame” may refer to a nucleotide sequence between a start codon (e.g. ATG) and a stop codon (e.g. TAA, TAG, or TGA). An open reading frame typically encodes a polypeptide (i.e. a protein-coding sequence) or a functional RNA (i.e. a RNA-coding sequence). Any open reading frame may be used, the selection of which may be readily made by the skilled person. In some embodiments, the open reading frame is a protein-coding sequence. A “protein- coding sequence” or “transgene” may encode any polypeptide of interest. In some embodiments, the polypeptide is a therapeutic polypeptide. A protein-coding sequence may further encode a signal peptide and / or a protein tag. For example, a protein-coding sequence may encode from 5’ to 3’ direction: optionally a signal peptide; a polypeptide; and optionally a protein tag. Suitable signal peptides and protein tags will be known to the skilled person. For a protein-coding polynucleotide, numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. For example, Nucleotide substitutions may be made that do not affect the polypeptide sequence encoded by the protein coding sequence to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed. The protein-coding sequence may be codon-optimised. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available. Codon usage tables are known in the art for mammalian cells (e.g. humans), as well as for a variety of other organisms. In some embodiments, the open reading frame is a RNA-coding sequence. A “RNA-coding sequence” may encode any RNA of interest. For example, a miRNA, a siRNA, a piRNA, a snoRNA, a snRNA, an exRNA, a scaRNA, a transfer RNA (tRNA), a small hairpin RNA (shRNA), or a ribosomal RNA (rRNA). 3’ regulatory region A “3’ regulatory region” may refer to a nucleotide sequence that is downstream from the stop codon and upstream from the transcription termination which controls expression of the open reading frame. The 3′ regulatory region is typically not translated, but contains regulatory regions that post-transcriptionally influence gene expression. Any 3’ regulatory region may be used, the selection of which may be readily made by the skilled person. For example, a 3’ regulatory region may include one or more regulatory elements. For example, a 3’ regulatory region may include one or more post-transcriptional regulatory elements, an AU-rich element, one or more miRNA target sequences, and / or a polyadenylation signal. In some embodiments, a 3’ regulatory region comprises one or more post-transcriptional regulatory elements. A “post-transcriptional regulatory element” may control gene expression at the RNA level (i.e. post-transcription). Suitable post-transcriptional regulatory element will be known to the skilled person. Example post-transcriptional regulatory elements include a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE). In some embodiments, a 3’ regulatory region comprises a WPRE devoid of the X-protein ORF. In some embodiments, a 3’ regulatory region comprises an AU-rich element. An “AU-rich element” is typically from 50 to 150 bp in length and includes many copies of the sequence ATTTA. In some embodiments, a 3’ regulatory region comprises one or more miRNA target sequences. The one or more miRNA target sequences may be used to control expression of the open reading frame, for example to de-target the expression of the open reading frame from specific cells and / or tissues. In preferred embodiments, a 3’ regulatory region does not comprise one or more miRNA target sequences. In some embodiments, a 3’ regulatory region comprises a polyadenylation sequence. A polyadenylation sequence may regulate polyadenylation of the transcribed mRNA. A polyadenylation sequence typically comprises a polyadenylation signal, e.g. having the sequence AATAAA, a polyadenylation site and a GT-rich downstream element. Suitable polyadenylation sequences will be known to the skilled person. Example polyadenylation sequences include the early SV40 polyadenylation sequence, the human growth hormone (hGH) polyadenylation sequence, the bovine growth hormone (bGH) polyadenylation sequence, and the rbGlob polyadenylation sequence. In some embodiments, a 3’ regulatory region comprises or consists of from 5’ to 3’ direction: one or more post-transcriptional regulatory elements and a polyadenylation sequence. In some embodiments, a 3’ regulatory region does not comprise a polyadenylation sequence. In some embodiments, a 3’ regulatory region comprises or consists of one or more post-transcriptional regulatory elements. Transcription termination site The polynucleotide of the present invention may further comprise a transcription termination site. The transcription termination site may be downstream from the expression cassette. A “transcription termination site” or “transcription terminator” may refer to a nucleic acid sequence that marks the end of a gene or operon during transcription. A transcription termination site may mediate transcriptional termination by providing signals in the newly synthesized transcript RNA that trigger processes which release the transcript RNA from the transcriptional complex. For example, in eukaryotic transcription of mRNAs, terminator signals are recognised by protein factors that are associated with the RNA polymerase II and which trigger the termination process. These proteins factors then recruit other proteins to the site to cleave the transcript, freeing the mRNA from the transcription complex, and carrying out polyadenylation (see e.g. Proudfoot, N.J., 2016. Science, 352(6291), p.aad9926). The transcription termination site may be 10kb or less downstream from the expression cassette. In some embodiments, the transcription termination site is 5000 bp or less, 4000 bp or less, 3000 bp or less, 2500 bp or less, 2000 bp or less, 1500 bp or less, 1000 bp or less, 500 bp or less, 400 bp or less, 300 bp or less, 200 bp or less, 100 bp or less, 90 bp or less, 80 bp or less, 70 bp or less, 60 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, 10 bp, 5 bp or less, 4 bp or less, 3 bp or less, or 2 bp or less, downstream from the expression cassette. In some embodiments, the transcription termination site is immediately downstream from the expression cassette (i.e. there are no nucleotides between the expression cassette and the transcription termination site). In some embodiments, the polynucleotide of the present invention comprises from 5’ to 3’ direction: an expression cassette and a transcription termination site. The polynucleotide may comprise further elements between the expression cassette and the transcription termination site, such as one or more miRNA target sequences, one or more insulator elements, and / or one or more further regulatory elements. microRNA (miRNA) target sequences The polynucleotide of the present invention comprises one or more miRNA target sequences. A “miRNA target sequence” may refer to a nucleotide sequence to which a naturally- occurring miRNA binds (see e.g. Geisler, A. and Fechner, H., 2016. World journal of experimental medicine, 6(2), pp.37-54). MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression. miRNA target sequences may be provided using prediction tools and / or by experimental validation (see e.g. Riffo-Campos, Á.L., et al., 2016. International journal of molecular sciences, 17(12), p.1987; and Riolo, G., et al., 2020. Methods and protocols, 4(1), p.1). The miRBase database is a searchable database of published miRNA sequences and annotation (see Griffiths-Jones, S., et al., 2007. Nucleic acids research, 36(suppl_1), pp.D154-D158). Suitably, the miRNA target sequences are human miRNA target sequences. Example human miRNA target sequences include let-7a-1, let-7a-3, let-7a-2, let-7c, let-7f-2, let-7f-1, mir-143, let-7g, mir-21, let-7b, let-7e, mir-98, mir-26a-2, mir-145, mir-26a-1, mir-99a, mir- 26b, let-7i, mir-148a, mir-30a, mir-27b, mir-125b-1, mir-1-2, mir-103a-2, mir-103a-1, mir-107, mir-29a, mir-19b-2, mir-30d, mir-125b-2, mir-19b-1, mir-29c, mir-23b, mir-30e, mir-19a, mir- 16-1, mir-16-2, mir-199a-2, mir-23a, mir-100, mir-199a-1, mir-17, mir-199b, mir-191, mir- 92a-1, mir-27a, mir-29b-2, mir-29b-1, mir-106a, mir-10b, mir-20a, mir-1246, mir-92a-2, mir- 1-1, mir-205, mir-15b, mir-101-2, mir-101-1, mir-378a, mir-23c, mir-140, mir-320a, mir-320b- 2, mir-10a, mir-320b-1, mir-378i, mir-378c, mir-151a, mir-200c, mir-378d-2, mir-378f, mir- 320e, let-7d, mir-378g, mir-92b, mir-320d-1, mir-126, mir-221, mir-320c-2, mir-186, mir-15a, mir-125a, mir-378b, mir-451a, mir-200b, mir-22, mir-378h, mir-335, mir-24-2, mir-24-1, mir- 423, mir-424, mir-7-1, mir-142, mir-28, mir-181a-1, mir-181a-2, mir-320c-1, mir-222, mir-630, mir-375, mir-378d-1, mir-7-3, mir-7-2, mir-30c-2, mir-30c-1, mir-378e, mir-25, mir-18a, mir- 320d-2, mir-18b, mir-148b, mir-151b, mir-146a, mir-200a, mir-152, mir-30b, mir-146b, mir- 195, mir-93, mir-4429, mir-9985, mir-185, mir-106b, mir-130a, mir-99b, mir-141, mir-196b, mir-196a-1, mir-182, mir-374a, mir-31, mir-1234, mir-196a-2, mir-122, mir-218-1, mir-425, mir-374b, mir-218-2, mir-9-1, mir-9-2, mir-9-3, mir-223, mir-365b, mir-365a, mir-8485, mir- 4449, mir-340, mir-1260b, mir-1260a, mir-181d, mir-181b-2, mir-181b-1, mir-4264, mir-4443, mir-130b, mir-361, mir-4289, mir-7851, mir-1290, mir-888, mir-192, mir-574, mir-342, mir- 363, mir-132, mir-150, mir-128-1, mir-128-2, mir-215, mir-193b, mir-4521, mir-486-1, mir- 486-2, mir-144, mir-3159, mir-135a-1, mir-7110, mir-514b, mir-20b, mir-133a-1, mir-133a-2, mir-193a, mir-203a, mir-660, mir-211, mir-183, mir-96, mir-8081, mir-214, mir-3192, mir- 4454, mir-12136, mir-135a-2, mir-484, mir-514a-2, mir-514a-3, mir-514a-1, mir-216a, and mir-9902-1. Suitably, the miRNA target sequences are -5p or -3p miRNA target sequences. miRNAs are usually transcribed from polymerase-II promoters, generating a so-called primary miRNA transcript (pri-miRNA). From the pri-miRNA, a stem loop of about 60 nucleotides in length, called miRNA precursor (pre-miRNA), is excised leaving a 5’ phosphate and a 2 bp long, 3’ overhang. The pre-miRNA is then actively transported from the nucleus to the cytoplasm. Dicer performs a double strand cut at the other end of the stem loop, generating a 19-24 bp duplex, which is composed of the mature miRNA and the opposite strand of the duplex, called miRNA*. One strand of the duplex is selectively loaded into the RNA-induced silencing complex (RISC), and accumulates as the mature microRNA. This strand is usually the one whose 5’ end is less tightly paired to its complement. However, there are some miRNAs that support accumulation of both duplex strands to similar extent. Once loaded into RISC, the guide strand of the mature microRNA interacts with mRNA target sequences. If the whole guide strand sequence is perfectly complementary to the mRNA target, the mRNA is endonucleolytically cleaved. If only the seed sequence (i.e. nucleotides 2-8 counted from the 5' end of the miRNA) is perfectly complementary to the target mRNA, RNAi may act through alternative mechanisms leading to translational repression. A miRNA target sequence may be fully or partially complementary to the corresponding miRNA. The term “fully complementary”, as used herein, may mean that the target sequence has a nucleic acid sequence which is 100% complementary to the sequence of the miRNA which recognises it. The term “partially complementary”, as used herein, may mean that the target sequence is only in part complementary to the sequence of the miRNA which recognises it, whereby the partially complementary sequence is still recognised by the miRNA. In other words, a partially complementary target sequence in the context of the present invention is effective in recognising the corresponding miRNA. Suitably, a partially complementary miRNA target sequence may be fully complementary to the miRNA seed sequence. A miRNA target sequence which suppresses transgene expression in specific cells and / or tissues will be known to the skilled person. Determining a miRNA with the desired expression profile may be achieved using techniques known to those skilled in the art. For example, a mammalian microRNA expression atlas is described in Landgraf, P., et al., 2007. Cell, 129(7), pp.1401-1414 and the distribution of miRNA expression across human tissues is described in Ludwig, N., et al., 2016. Nucleic acids research, 44(8), pp.3865-3877. Once a miRNA has been identified, the corresponding target sequence can readily be determined using, for example, a microRNA database, such as miRBase (see Griffiths-Jones, S., et al., 2007. Nucleic acids research, 36(suppl_1), pp.D154-D158). Examples of miRNAs specific to or enriched in cells and / or tissues are provided in the table below (see e.g. Dhungel, B., et al., 2018. Molecules, 23(7), p.1500; Geisler, A. and Fechner, H., 2016. World journal of experimental medicine, 6(2), pp.37-54; and Kelly, E.J. and Russell, S.J., 2009. Molecular Therapy, 17(3), pp.409-416). Cell / Tissue type miRNA Eye miR124, miR204, miR181 Heart miR1, miR206, miR126, miR134, miR133, miR208, miR302 miR338, miR219, miR124a, miR9, miR218, miR7, miR128, miR125, Brain / Nervous miR138, miR132, miR212, miR137, miR31, miR127, miR143, miR346, system miR708 Kidney miR10a, miR192, miR204, miR194, miR215, miR216 Liver miR22, miR122, miR192, miR92a, miR483 Lung miR126, mir200c, miR141 Hematopoietic and miR223, miR142, miR126, miR130a, miR302, miR292 pluripotent cells Pancreas miR216, miR217, mir375, mir200c Spleen miR142, miR150 Muscle miR133a, miR1, miR206, miR134, miR193a, miR128a Immune system miR150, miR181a, miR155, miR142 The polynucleotide may comprise more than one miRNA target sequence, which may or may not be different. The polynucleotide may, for example, comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 miRNA target sequences. The miRNA target sequences may be the same or different. The miRNA target sequences may be in any orientation relative to the expression cassette. A miRNA target sequence may be a sense-oriented miRNA target sequence or an antisense-oriented miRNA target sequence. A “sense-oriented” miRNA target sequence may refer to a nucleotide sequence to which a miRNA binds, e.g. a sense-oriented miRNA target sequence may be a reverse complement of a miRNA. When a sense strand comprising a sense-oriented miRNA sequence is transcribed, the resulting mRNA may be degraded. An “antisense-oriented” miRNA target sequence may refer to a nucleotide sequence to which a miRNA binds the reverse complement thereof, e.g. an antisense-oriented miRNA target sequence may be identical to a miRNA (except U may be replaced by T). When a sense strand comprising an antisense-oriented miRNA sequence is transcribed, the mRNA is not degraded, however, when an antisense strand comprising an antisense-oriented miRNA sequence is transcribed, the resulting mRNA may be degraded. In some embodiments, the polynucleotide of the present invention does not comprise an miRNA target sequence upstream of a splice donor site or downstream of a splice acceptor site. miRNA target sequences outside expression cassette The polynucleotide of the present invention may comprise one or more miRNA target sequences upstream from the expression cassette and / or downstream from the expression cassette (e.g. an expression cassette comprising a promoter, an open reading frame, and a 3’ regulatory region). miRNA target sequences upstream from the expression cassette and / or downstream from the expression cassette may improve safety of the polynucleotide by avoiding and / or reducing aberrant transcription, aberrant splicing, and / or read-through events. The miRNA target sequences may specifically degrade aberrant transcripts. The miRNA target sequences may be any distance upstream from the expression cassette and / or downstream from the expression cassette, for example 5000 bp or less, 4000 bp or less, 3000 bp or less, 2000 bp or less, 1000 bp or less, 500 bp or less, 400 bp or less, 300 bp or less, 200 bp or less, or 100 bp or less upstream from the expression cassette and / or downstream from the expression cassette. The miRNA target sequences may be directly upstream and / or directly downstream from the expression cassette. As used herein, “directly upstream” or “directly downstream” may mean that there are no further regulatory elements between the elements. In some embodiments, the miRNA target sequences are 100 bp or less, 90 bp or less, 80 bp or less, 70 bp or less, 60 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less upstream from the expression cassette and / or downstream from the expression cassette. The miRNA target sequence may be downstream from the transcription termination site. In some embodiments, the polynucleotide comprises one or more miRNA target sequences downstream from the transcription termination site. The miRNA target sequences may be any distance downstream from the transcription termination site, for example 5000 bp or less, 4000 bp or less, 3000 bp or less, 2000 bp or less, or 1000 bp or less, or 500 bp or less, 400 bp or less, 300 bp or less, 200 bp or less, or 100 bp or less downstream from the transcription termination site. In some embodiments, the polynucleotide comprises one or more miRNA target sequences directly downstream from the transcription termination site. In some embodiments, the miRNA target sequences are 100 bp or less, 90 bp or less, 80 bp or less, 70 bp or less, 60 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less downstream from the transcription termination site. Suitably, the miRNA target sequences are active in target cells allowing selective degradation, through the miRNA pathway, of aberrant transcripts. In some embodiments, the miRNA target sequences suppress transgene expression in hematopoietic lineages. Suitable miRNA target sequences for suppressing transgene expression in hematopoietic lineages include miR223 target sequences and mir142 target sequences. In some embodiments, the miRNA target sequences are mir223-3p target sequences. In some embodiments, the miRNA target sequences are miR142-3p target sequences. In some embodiments, the miRNA target sequences suppress transgene expression in liver. Suitable miRNA target sequences for suppressing transgene expression in liver include mir122 target sequences, miR22 target sequences and mir126 target sequences. In some embodiments, the miRNA target sequences are miR122-5p target sequences. In some embodiments, the miRNA target sequences are mir22-3p target sequences. In some embodiments, the miRNA target sequences are mir126-3p target sequences. In some embodiments, the miRNA target sequences suppress transgene expression in neurons. Suitable miRNA target sequences for suppressing transgene expression in neurons include miR124 target sequences. In some embodiments, the miRNA target sequences are miR124-3p target sequences. In some embodiments, the miRNA target sequences suppress transgene expression in muscle. Suitable miRNA target sequences for suppressing transgene expression in muscle include miR133a and miR133b target sequences. In some embodiments, the miRNA target sequences are miR133a-3p target sequences. In some embodiments, the miRNA target sequences are miR133b target sequences. In some embodiments, the miRNA target sequences suppress transgene expression in lung. Suitable miRNA target sequences for suppressing transgene expression in lung include let7f and miR7 target sequences. In some embodiments, the miRNA target sequences are let7f- 5p target sequences. In some embodiments, the miRNA target sequences are miR7-5p target sequences. In some embodiments, the miRNA target sequences suppress transgene expression in multiple tissues. Suitable miRNA target sequences for suppressing transgene expression in multiple tissues include miR22 target sequences and. In some embodiments, the miRNA target sequences are mir22-3p target sequences. Example sense-oriented miRNA target sequences are provided by SEQ ID NOs: 1 to 10. Example antisense-oriented miRNA target sequences are provided by SEQ ID NOs: 11 to 20. TCCATAAAGTAGGAAACACTACA mirT 142-3p sense-oriented (SEQ ID NO: 1) GGGGTATTTGACAAACTGACA mirT 223-3p sense-oriented (SEQ ID NO: 2) ACAAACACCATTGTCACACTCCA mirT 122-5p sense-oriented (SEQ ID NO: 3) ACAGTTCTTCAACTGGCAGCTT mirT 22-3p sense-oriented (SEQ ID NO: 4) CGCATTATTACTCACGGTACGA mirT 126-3p sense-oriented (SEQ ID NO: 5) GGCATTCACCGCGTGCCTTA mirT 124-3p sense-oriented (SEQ ID NO: 6) CAGCTGGTTGAAGGGGACCAAA mirT 133a-3p sense-oriented (SEQ ID NO: 7) TAGCTGGTTGAAGGGGACCAAA mirT 133b sense-oriented (SEQ ID NO: 8) AACTATACAATCTACTACCTCA let 7f-5p sense-oriented (SEQ ID NO: 9) ACAACAAAATCACTAGTCTTCCA mirT 7-5p sense-oriented (SEQ ID NO: 10) TGTAGTGTTTCCTACTTTATGGA mirT 142-3p antisense-oriented (SEQ ID NO: 11) TGTCAGTTTGTCAAATACCCC mirT 223-3p antisense-oriented (SEQ ID NO: 12) TGGAGTGTGACAATGGTGTTTGT mirT 122-5p antisense-oriented (SEQ ID NO: 13) AAGCTGCCAGTTGAAGAACTGT mirT 22-3p antisense-oriented (SEQ ID NO: 14) TCGTACCGTGAGTAATAATGCG mirT 126-3p antisense-oriented (SEQ ID NO: 15) TAAGGCACGCGGTGAATGCC mirT 124-3p antisense-oriented (SEQ ID NO: 16) TTTGGTCCCCTTCAACCAGCTG mirT 133a-3p antisense-oriented (SEQ ID NO: 17) TTTGGTCCCCTTCAACCAGCTA mirT 133b antisense-oriented (SEQ ID NO: 18) TGAGGTAGTAGATTGTATAGTT let 7f-5p antisense-oriented (SEQ ID NO: 19) TGGAAGACTAGTGATTTTGTTGT mirT 7-5p antisense-oriented (SEQ ID NO: 20) Variant miRNA target sequences may have one or two nucleotide variations (e.g. substitutions, insertions, additions or deletions) whilst retaining the ability to bind the corresponding miRNA. As described above, a partially complementary target sequence may be effective in recognising the corresponding miRNA. Suitably, a variant miRNA target sequence may be fully complementary to the miRNA seed sequence (i.e. nucleotides 2-8 counted from the 5' end of the miRNA). Including more than one copy of a miRNA target sequence may increase the effectiveness of the miRNA target sequence. Also, different miRNA target sequences can be combined. The polynucleotide may, for example, comprise 1, 2, 3, 4, 5, 6, 7, 8 or more copies of the same or different miRNA target sequences. In some embodiments, the polynucleotide comprises at least 2 copies of each miRNA target sequence. In some embodiments, the polynucleotide comprises from 2 to 4 copies of each miRNA target sequence. Copies of miRNA target sequences may be arranged in tandem, but other arrangements are envisaged. Copies of miRNA target sequences may be separated by a spacer sequence. A spacer sequence may comprise any suitable number of nucleotide bases, for example, at least one, at least two, at least three, at least four or at least five nucleotide bases. Suitably, the polynucleotide comprises at least 2 copies of each miRNA target sequence arranged in tandem. Suitably, the polynucleotide comprises from 2 to 4 copies of each miRNA target sequence arranged in tandem. Example tandem miRNA target sequences are provided by SEQ ID NOs: 21 and 22. TCCATAAAGTAGGAAACACTACACGATTCCATAAAGTAGGAAACACTACA 2x mirT 142-3p sense-oriented (SEQ ID NO: 21) TGTCAGTTTGTCAAATACCCCATCGTGTCAGTTTGTCAAATACCCC 2x mirT 223-3p antisense-oriented (SEQ ID NO: 22) The one or more miRNA target sequences may have any orientation relative to the expression cassette. The polynucleotide of the present invention may comprise: (a) one or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette and / or downstream from the transcription termination site; and / or (b) one or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the polynucleotide comprises: (a) one or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette and / or downstream from the transcription termination site; and (b) one or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the polynucleotide of the present invention does not comprise an miRNA target sequence having the same orientation as the expression cassette upstream from the expression cassette. In some embodiments, the polynucleotide of the present invention does not comprise an miRNA target sequence having the opposite orientation as the expression cassette upstream from the expression cassette. miRNA target sequences inside expression cassette The polynucleotide of the present invention may further comprise one or more miRNA target sequences within the expression cassette (e.g. an expression cassette comprising a promoter, an open reading frame, and a 3’ regulatory region). The one or more miRNA target sequences preferably have the same orientation as the expression cassette. For example, the 3’ regulatory region may comprise one or more miRNA target sequences having the same orientation as the expression cassette. Expression of the open reading frame may be regulated by one or more endogenous miRNAs using one or more corresponding miRNA target sequences within the expression cassette. Using this method, one or more miRNAs endogenously expressed in a cell may prevent or reduce transgene expression in that cell by interacting with the corresponding miRNA target sequence positioned within the expression cassette (see e.g. Brown, B.D. and Naldini, L., 2009. Nature Reviews Genetics, 10(8), pp.578-585). Suitable miRNA target sequences which suppress transgene expression in specific cells are known. The one or more miRNA target sequence may suppress transgene expression in hematopoietic-lineage and / or antigen-presenting cells. By preventing transgene expression in antigen-presenting cells, while permitting high levels of expression in other cells, miRNA regulation may enable strong and stable gene transfer in the absence of an immune response. Suitable miRNA target sequences for suppressing transgene expression in hematopoietic-lineage cells and / or antigen-presenting cells include miR223 target sequences and mir142 target sequences. Other miRNA target sequences which suppress transgene expression in hematopoietic-lineage cells and / or antigen-presenting cells are known in the art (see e.g. Ghafouri-Fard, S., et al., 2021. Non-coding RNA research, 6(1), pp.8-14). Further miRNA target sequences that suppress transgene expression in hematopoietic-lineage cells and / or antigen-presenting cells can be identified by any suitable method, for example miRNA expression analysis as described in Monticelli, S., et al., 2005. Genome biology, 6(8), pp.1-15. Including more than one copy of a miRNA target sequence may increase the effectiveness of the miRNA target sequence. Also, different miRNA target sequences can be combined. The polynucleotide may, for example, comprise 1, 2, 3, 4, 5, 6, 7, 8 or more copies of the same or different miRNA target sequences. In some embodiments, the polynucleotide comprises at least 2 copies of each miRNA target sequence. In some embodiments, the polynucleotide comprises from 2 to 4 copies of each miRNA target sequence. Copies of miRNA target sequences may be arranged in tandem, but other arrangements are envisaged. Copies of miRNA target sequences may be separated by a spacer sequence. A spacer sequence may comprise any suitable number of nucleotide bases, for example, at least one, at least two, at least three, at least four or at least five nucleotide bases. Suitably, the polynucleotide comprises at least 2 copies of each miRNA target sequence arranged in tandem. Suitably, the polynucleotide comprises from 2 to 4 copies of each miRNA target sequence arranged in tandem. In some embodiments, the polynucleotide of the present invention does not comprise one or more miRNA target sequences within the expression cassette. In some embodiments, the polynucleotide of the present invention does not comprise one or more miRNA target sequences having the same orientation as the expression cassette within the expression cassette. In some embodiments, the polynucleotide of the present invention does not comprise one or more miRNA target sequences within the 3’ regulatory region. In some embodiments, the polynucleotide of the present invention does not comprise one or more miRNA target sequences having the same orientation as the expression cassette within the 3’ regulatory region. Same orientation miRNA target sequences In some embodiments, the polynucleotide of the present invention comprises one or more miRNA target sequences having the same orientation as the expression cassette. The one or more miRNA target sequences having the same orientation as the expression cassette may be upstream from the expression cassette and / or downstream from the transcription termination site. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: one or more miRNA target sequences having the same orientation as the expression cassette; and the expression cassette. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: the expression cassette; and one or more miRNA target sequences having the same orientation as the expression cassette. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: one or more miRNA target sequences having the same orientation as the expression cassette; the expression cassette; and one or more miRNA target sequences having the same orientation as the expression cassette. The polynucleotide may comprise two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette and / or downstream from the transcription termination site. In some embodiments, the polynucleotide comprises two or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette; and / or two or more miRNA target sequences having the same orientation as the expression cassette, downstream from the transcription termination site. In some embodiments, the polynucleotide comprises two or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette. In some embodiments, the polynucleotide comprises two or more miRNA target sequences having the same orientation as the expression cassette, downstream from the transcription termination site. In some embodiments, the polynucleotide comprises two miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette; and / or two miRNA target sequences having the same orientation as the expression cassette, downstream from the transcription termination site. In some embodiments, the polynucleotide comprises two miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette. In some embodiments, the polynucleotide comprises two miRNA target sequences having the same orientation as the expression cassette, downstream from the transcription termination site. The miRNA target sequences having the same orientation as the expression cassette may be 5000 bp or less, 4000 bp or less, 3000 bp or less, 2000 bp or less, 1000 bp or less, 500 bp or less, 400 bp or less, 300 bp or less, 200 bp or less, or 100 bp or less upstream from the expression cassette and / or downstream from the transcription termination site. The miRNA target sequences having the same orientation as the expression cassette may be directly upstream from the expression cassette and / or directly downstream from the transcription termination site. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette and upstream from the expression cassette are 100 bp or less, 90 bp or less, 80 bp or less, 70 bp or less, 60 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less upstream from the expression cassette and / or the miRNA target sequences having the same orientation as the expression cassette and downstream from the transcription termination site are 100 bp or less, 90 bp or less, 80 bp or less, 70 bp or less, 60 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less downstream from the transcription termination site. The miRNA target sequences having the same orientation as the expression cassette may be 5000 bp or less, 4000 bp or less, 3000 bp or less, 2000 bp or less, 1000 bp or less, 500 bp or less, 400 bp or less, 300 bp or less, 200 bp or less, or 100 bp or less upstream from the expression cassette. In some embodiments, the polynucleotide comprises one or more miRNA target sequences having the same orientation as the expression cassette, directly upstream from the expression cassette. In some embodiments, the polynucleotide comprises one or more miRNA target sequences having the same orientation as the expression cassette 100 bp, 90 bp or less, 80 bp or less, 70 bp or less, 60 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less upstream from the expression cassette. The miRNA target sequences having the same orientation as the expression cassette may be 5000 bp or less, 4000 bp or less, 3000 bp or less, 2000 bp or less, 1000 bp or less, 500 bp or less, 400 bp or less, 300 bp or less, 200 bp or less, or 100 bp or less downstream from the transcription termination site. In some embodiments, the polynucleotide comprises one or more miRNA target sequences having the same orientation as the expression cassette, directly downstream from the transcription termination site. In some embodiments, the polynucleotide comprises one or more miRNA target sequences having the same orientation as the expression cassette 100 bp, 90 bp or less, 80 bp or less, 70 bp or less, 60 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less downstream from the transcription termination site. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette suppress transgene expression in hematopoietic lineages and / or liver. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette are selected from a miR223 target sequence, a miR142 target sequence, a miR22 target sequence, a miR122 target sequence, a miR126 target sequence, a miR124 target sequence, a miR133a target sequence, a miR133b target sequence, a let7f target sequence, a miR7 target sequence, and a combination thereof. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette are selected from miR223 target sequences, miR142 target sequences, miR22 target sequences, and miR122 target sequences. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 20, or a variant thereof having one or two nucleotide variations. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 20. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 10, or a variant thereof having one or two nucleotide variations. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 10. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette suppress transgene expression in hematopoietic lineages. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette are miR142 target sequences. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette are miR142-3p target sequences. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 or 11, or a variant thereof having one or two nucleotide variations. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 or 11. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette comprise or consist of the nucleotide sequence of SEQ ID NO: 1, or a variant thereof having one or two nucleotide variations. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette comprise or consist of the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette comprise or consist of the nucleotide sequence of SEQ ID NO: 21, or a variant thereof having one to four nucleotide variations. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette comprise or consist of the nucleotide sequence of SEQ ID NO: 21, or a variant thereof having one or two nucleotide variations. In some embodiments, the miRNA target sequences having the same orientation as the expression cassette comprise or consist of the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the polynucleotide does not comprise an miRNA target sequence having the same orientation as the expression cassette upstream of a splice donor site or downstream of a splice acceptor site. In some embodiments, the polynucleotide does not comprise an miRNA target sequence having the same orientation as the expression cassette upstream of the expression cassette. Opposite orientation miRNA target sequences In some embodiments, the polynucleotide of the present invention comprises one or more miRNA target sequences having the opposite orientation as the expression cassette. The one or more miRNA target sequences having the opposite orientation as the expression cassette may be upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: one or more miRNA target sequences having the opposite orientation as the expression cassette; and the expression cassette. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: the expression cassette; and one or more miRNA target sequences having the opposite orientation as the expression cassette. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: one or more miRNA target sequences having the opposite orientation as the expression cassette; the expression cassette; and one or more miRNA target sequences having the opposite orientation as the expression cassette. The polynucleotide may comprise two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the polynucleotide comprises two or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette; and / or two or more miRNA target sequences having the opposite orientation as the expression cassette, downstream from the expression cassette. In some embodiments, the polynucleotide comprises two or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette. In some embodiments, the polynucleotide comprises two or more miRNA target sequences having the opposite orientation as the expression cassette, downstream from the expression cassette. In some embodiments, the polynucleotide comprises two miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette; and / or two miRNA target sequences having the opposite orientation as the expression cassette, downstream from the expression cassette. In some embodiments, the polynucleotide comprises two miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette. In some embodiments, the polynucleotide comprises two miRNA target sequences having the opposite orientation as the expression cassette, downstream from the expression cassette. The miRNA target sequences having the opposite orientation as the expression cassette may be 5000 bp or less, 4000 bp or less, 3000 bp or less, 2000 bp or less, 1000 bp or less, 500 bp or less, 400 bp or less, 300 bp or less, 200 bp or less, or 100 bp or less upstream from the expression cassette and / or downstream from the expression cassette. The miRNA target sequences having the opposite orientation as the expression cassette may be directly upstream and / or directly downstream from the expression cassette. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette are 100 bp or less, 90 bp or less, 80 bp or less, 70 bp or less, 60 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less upstream from the expression cassette and / or downstream from the expression cassette. The miRNA target sequences having the opposite orientation as the expression cassette may be 5000 bp or less, 4000 bp or less, 3000 bp or less, 2000 bp or less, 1000 bp or less, 500 bp or less, 400 bp or less, 300 bp or less, 200 bp or less, or 100 bp or less upstream from the expression cassette. In some embodiments, the polynucleotide comprises one or more miRNA target sequences having the opposite orientation as the expression cassette, directly upstream from the expression cassette. In some embodiments, the polynucleotide comprises one or more miRNA target sequences having the opposite orientation as the expression cassette 100 bp, 90 bp or less, 80 bp or less, 70 bp or less, 60 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less upstream from the expression cassette. The miRNA target sequences having the opposite orientation as the expression cassette may be 5000 bp or less, 4000 bp or less, 3000 bp or less, 2000 bp or less, 1000 bp or less, 500 bp or less, 400 bp or less, 300 bp or less, 200 bp or less, or 100 bp or less downstream from the expression cassette. In some embodiments, the polynucleotide comprises one or more miRNA target sequences having the opposite orientation as the expression cassette, directly downstream from the expression cassette. In some embodiments, the polynucleotide comprises one or more miRNA target sequences having the opposite orientation as the expression cassette 100 bp, 90 bp or less, 80 bp or less, 70 bp or less, 60 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less downstream from the expression cassette. Alternatively, or additionally, the polynucleotide may comprise one or more miRNA target sequences having the opposite orientation as the expression cassette within the expression cassette (e.g. within the 3’ regulatory region). In some embodiments, the polynucleotide comprises one or more miRNA target sequences having the opposite orientation as the expression cassette within the expression cassette. In some embodiments, the one or more miRNA target sequences having the opposite orientation as the expression cassette are within the 3’ regulatory region. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette suppress transgene expression in hematopoietic lineages and / or liver. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette are selected from a miR223 target sequence, a miR142 target sequence, a miR22 target sequence, a miR122 target sequence, a miR126 target sequence, a miR124 target sequence, a miR133a target sequence, a miR133b target sequence, a let7f target sequence, a miR7 target sequence, and a combination thereof. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette are selected from miR223 target sequences, miR142 target sequences, miR22 target sequences, and miR122 target sequences. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 20, or a variant thereof having one or two nucleotide variations. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 20. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 to 20, or a variant thereof having one or two nucleotide variations. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 to 20. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette suppress transgene expression in hematopoietic lineages. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette are miR223 target sequences. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette are miR223-3p target sequences. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2 or 12, or a variant thereof having one or two nucleotide variations. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette comprise or consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2 or 12. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette comprise or consist of the nucleotide sequence of SEQ ID NO: 12, or a variant thereof having one or two nucleotide variations. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette comprise or consist of the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette comprise or consist of the nucleotide sequence of SEQ ID NO: 22, or a variant thereof having one to four nucleotide variations. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette comprise or consist of the nucleotide sequence of SEQ ID NO: 22, or a variant thereof having one or two nucleotide variations. In some embodiments, the miRNA target sequences having the opposite orientation as the expression cassette comprise or consist of the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the polynucleotide does not comprise an miRNA target sequence having the opposite orientation as the expression cassette upstream of a splice donor site or downstream of a splice acceptor site. In some embodiments, the polynucleotide of the present invention does not comprise an miRNA target sequence having the opposite orientation as the expression cassette upstream of the expression cassette. Insulator elements The polynucleotide of the present invention may comprise one or more insulator elements. The one or more insulator elements may be upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: an insulator element; and the expression cassette. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: the expression cassette; and an insulator element. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: an insulator element; the expression cassette; and an insulator element. An “insulator element”, “chromatin insulator”, or “chromatin insulator element” may refer to a genetic element that functions to prevent activation of a promoter by nearby heterologous enhancers and / or functions as a barrier against repressive effects of neighbouring heterochromatin (see e.g. Valenzuela, L. and Kamakaka, R.T., 2006. Annu. Rev. Genet., 40, pp.107-138). Insulator elements may, for example, be used in integrating vectors to reduce the interactions between the integrated vector enhancers and cellular promoters. In a genomic context, insulator elements allow gene expression regulation of the host genome by mediating chromatin loops that physically separate genomic regions in functional domains, where sequences within the same domain interact and regulate each other, while such crosstalk is blocked in regions from different chromatin domains. An insulator element may bind to an insulator-binding protein (IBP). Examples of known insulator-binding proteins include CTCF, Su(Hw), Ibf1, Ibf2, ZIPIC, Cp190, Mod(mdg4), and BEAF-32 (see e.g. Özdemir, I. and Gambetta, M.C., 2019. Genes, 10(10), p.767). Suitably, an insulator element binds to a human IBP, such as CTCF. The insulator elements may be in any orientation relative to the expression cassette. An insulator element may be a forward insulator element or a reverse insulator element. A “forward” insulator element may refer to a nucleotide sequence to which an insulator-binding protein binds. A “reverse” insulator element may refer to a nucleotide sequence to which an insulator-binding protein binds the reverse-complement thereof. Both forward and reverse insulator elements may function to prevent activation of a promoter by nearby heterologous enhancers and / or as a barrier against repressive effects of neighbouring heterochromatin. Insulator elements may improve safety of the polynucleotide by avoiding and / or reducing enhancer-mediated gene activation. CTCF-binding motifs In some embodiments, an insulator element binds CCCTC-binding factor (CTCF). The best characterized insulator protein described to date is the CCCTC-binding factor (CTCF), which is one of the master regulators of chromatin structure and which is involved in long-range interactions via chromatin loops formation. Such loops are formed by the action of Cohesin that binds to DNA and extrude chromatin through a loop until a pair of convergent CTCF sites is found. By virtue of this ability, CTCF-bound insulators can block interactions between regulatory elements like enhancers and promoters. Suitably, an insulator element comprises a CTCF-binding motif. Example CTCF-binding motifs are provided in SEQ ID NOs: 23 to 28. Suitably, an insulator element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 23 to 28, or a nucleotide sequence having one or two nucleotide variations. In some embodiments, an insulator element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 23 to 28, or a nucleotide sequence having one nucleotide variation. In some embodiments, an insulator element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 23 to 28. CACCAGGTGGCGCT Example CTCF-binding motif A (SEQ ID NO: 23) CCACCAGGGGGAGC Example CTCF-binding motif B (SEQ ID NO: 24) TCAGTAGAGGGCGC Example CTCF-binding motif C (SEQ ID NO: 25) CCACTAGGGGGCAG Example CTCF-binding motif D (SEQ ID NO: 26) CAGCAGAGGGCGCT Example CTCF-binding motif E (SEQ ID NO: 27) CAGTAGAGGGCGCT Example CTCF-binding motif F (SEQ ID NO: 28) In some embodiments, an insulator element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 23, 24, and 27, or a nucleotide sequence having one or two nucleotide variations. In some embodiments, an insulator element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 23, 24, and 27, or a nucleotide sequence having one nucleotide variation. In some embodiments, an insulator element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 23, 24, and 27. In some embodiments, an insulator element comprises the nucleotide sequence of SEQ ID NO: 23, or a nucleotide sequence having one or two nucleotide variations. In some embodiments, an insulator element comprises the nucleotide sequence of SEQ ID NO: 23, or a nucleotide sequence having one nucleotide variation. In some embodiments, an insulator element comprises the nucleotide sequence of SEQ ID NO: 23. In some embodiments, an insulator element comprises the nucleotide sequence of SEQ ID NO: 24, or a nucleotide sequence having one or two nucleotide variations. In some embodiments, an insulator element comprises the nucleotide sequence of SEQ ID NO: 24, or a nucleotide sequence having one nucleotide variation. In some embodiments, an insulator element comprises the nucleotide sequence of SEQ ID NO: 24. In some embodiments, an insulator element comprises the nucleotide sequence of SEQ ID NO: 27, or a nucleotide sequence having one or two nucleotide variations. In some embodiments, an insulator element comprises the nucleotide sequence of SEQ ID NO: 27, or a nucleotide sequence having one nucleotide variation. In some embodiments, an insulator element comprises the nucleotide sequence of SEQ ID NO: 27. Core insulator sequences Suitably, an insulator element comprises a core insulator sequence. As used herein, a “core insulator sequence” may refer to a nucleic acid sequence comprising a binding motif and minimal flanking sequences. The flanking sequences may each independently comprise from 5 to 20 nucleotides, from 10 to 20 nucleotides, or from 10 to 15 nucleotides. A core insulator sequence may have any suitable length, for example a total length of from 30 to 50 nucleotides, from 35 to 45 nucleotides, or from 38 to 42 nucleotides. In some embodiments, an insulator element consists of a core insulator sequence. Using insulator elements consisting of core insulator sequences may allow the polynucleotide length to be minimised. Example forward core insulator sequence are provided in SEQ ID NOs: 29 to 32. Example reverse core insulator sequence are provided in SEQ ID NOs: 33 to 36. TCTGCTTGATGTGGACACCAGGTGGCGCTGGACATCAGAT Example core A1 insulator sequence (forward) (SEQ ID NO: 29) GCACAGACAAATTACCACCAGGTGGCGCTCAGAGTCTGCG Example core A2 insulator sequence (forward) (SEQ ID NO: 30) AGCGTTTGCTTCTGGCCACCAGGGGGAGCTGCTGGGACGCAG Example core B2 insulator sequence (forward) (SEQ ID NO: 31) ATCAAACCAGGAAACAGCAGAGGGCGCTGGTGCTGTGCT Example core E2 insulator sequence (forward) (SEQ ID NO: 32) ATCTGATGTCCAGCGCCACCTGGTGTCCACATCAAGCAGA Example core A1 insulator sequence (reverse) (SEQ ID NO: 33) CGCAGACTCTGAGCGCCACCTGGTGGTAATTTGTCTGTGC Example core A2 insulator sequence (reverse) (SEQ ID NO: 34) CTGCGTCCCAGCAGCTCCCCCTGGTGGCCAGAAGCAAACGCT Example core B2 insulator sequence (reverse) (SEQ ID NO: 35) AGCACAGCACCAGCGCCCTCTGCTGTTTCCTGGTTTGAT Example core E2 insulator sequence (reverse) (SEQ ID NO: 36) In some embodiments, an insulator element comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29 to 36, or variants thereof having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise a CTCF-binding motif (e.g. any of SEQ ID NOs: 23 to 28) and one or more variation is made in the flanking sequences. In some embodiments, an insulator element comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29 to 36. Full-length insulator sequences In some embodiments, an insulator element comprises or consists of one or more full-length insulator sequences. As used herein, a “full-length insulator sequence” may refer to a nucleic acid sequence comprising a binding motif and flanking sequences. The flanking sequences may each independently comprise from 21 to 500 nucleotides, from 30 to 400 nucleotides, from 40 to 300 nucleotides, or from 50 to 250 nucleotides. A full-length insulator sequence may have any suitable length, for example a total length of from 100 to 1000 nucleotides, from 150 to 800 nucleotides, or from 200 to 600 nucleotides. In some embodiments, the full-length insulator sequence has a length of 400 nucleotides or more, 410 nucleotides or more, 420 nucleotides or more, 430 nucleotides or more, 440 nucleotides or more, 450 nucleotides or more, 460 nucleotides or more, 470 nucleotides or more, 480 nucleotides or more, 490 nucleotides or more, 500 nucleotides or more, 550 nucleotides or more, or 600 nucleotides or more. In some embodiments, an insulator element consists of one or more full-length insulator sequence. Example (forward) full-length insulator sequence are provided in SEQ ID NOs: 37 to 63 and 102. Example (reverse) full-length insulator sequence are provided in SEQ ID NOs: 64 to 90 and 103. CTGGTTCTACTCATTACATTCCAATCGTGGCATATCCTCTAAACTTTCTTTTCCCTTCATAA ATCCTCTTTCTTTTTTTTCCCCCTCACAGTTTTCCTGAACAGGTTGACTATTAATTGTGTCT GCTTGATGTGGACACCAGGTGGCGCTGGACATCAGATTTGGAGAGGCAGTTGTCTAGGGAAC CGGGCTCTGTGCCAGCGCAGGAGGCAGGCTGGCTCTCCTATTCCAGGGATGCTCATCCAGGA AGGAAAGGTTGCATGCTGGACACACTAACCTTGAAGAATTCTTCTGTCTCTCTCGTCATTTA GAAAGGAAGG Example A1 insulator sequence (forward) (SEQ ID NO: 37) ACAATGGCTGGCCCATAGTAAATGCCGTGTTAGTGTGTTAGTTGCTGTTCTTCCACGTCAGA AGAGGCACAGACAAATTACCACCAGGTGGCGCTCAGAGTCTGCGGAGGCATCACAACAGCCC TGAATTTGAATCCTGCTCTGCCACTGCCTAGTTGAGACCTTTTACTACCTGACTAGCTGAGA CATTTACGACATTTACTGGCTCTAGGACTCATTTTATTCATTTCATTACTTTTTTTTTCTTT GAGACGGAATCTCGCTCT Example A2 insulator sequence (forward) (SEQ ID NO: 38) AGGGGTTGGTCTCCCTATGTTCCCCAGGCTGGTCTCCAACCCCTGGGCTCAAGCAATGCTCC TGCCTCAACCACCCAAAGTGCTGAGATTACAGGTGTGAACCACTGCGCCCGGCTTCAGAGGA GTTTTGATGCACCAGGTGGCGCTGGTAATTTAAGGTTCTCTCGTGAAATTGGTCTTTTCACC TGGCCAGCATCTTATTCCTTCTTCTGGTAACAGCATCCCATTGTTCTTTGTTGAACCACCTC TCCC Example A3 insulator sequence (forward) (SEQ ID NO: 39) GAACTCCTGACCCCTCACAAAGTGCTGGGATTACAAGCGTGAGCCACCGCGCCCGGCCAGAA CTTTTGAAAGGAAATTTACAAAGGTAGTTATTGGATAAAAGCGTGATAGAGTAGTTAGCACC AGGTGGCGCTAAAGGACTGAAATTGGAAACGGCTACGGTATTTGGTCCTCTTACCTGAGGGC CGGGGAAGGGCGATGCTCCTGGTTTAATGGCTGTGTTACAGGAAGGAAGGACAAGCA Example A4 insulator sequence (forward) (SEQ ID NO: 40) TCCCACTCCATCACCTTCAAGAATGTTCAAAATCCAGAGATTCTAGGATTCTGTTTCCCAGG ACTTGGACGACTCCGTGTCTCCATAGCTCCACCAGGTGGCGCTGCCGGGCCTCGTGACCACT TGGAAAAAGCAGTGTCACCAGAACGCTGCGGAGACCTGCCAGCAGGGGTCACACTGAGGTTG CCTCGTGGTGTCCCTGCCTCTGCAGGTGACCTATGAAATTCCTGGCAGGCCCAGCACAGTGG CTCACGCCTGTAATCCCAGCACTTTGGGTGGCCGAGGCGGGTGGATCACCTGAGGTCAGGAG TTCGAGACCAGCCTGGCCAACATGGTGAAACCCCGTCTCTAC Example A5 insulator sequence (forward) (SEQ ID NO: 41) AGGCATGACTGGGAAGAACAAATCACATTTCTCTGTGTACTTAGGGGAGAAAGAATTAATTC AAATGCAGGGTCTGCCGCTTCAGGTCCATTTACGGAAGCCGCAATCCTGCACTATGACCACC AGGGGGAGCGCTAGGCCCATCCCTGCACAGGTAGCTATTTTTAGAGGTTGCTTCTAGCCTGG AGGAAGAAACGAAAGAAATAAAAGACTATGATACTTTTTTTTTTTAACCGGACCAACTTATA AATTATTTGTAAGTCCAATAAAAGGCAATTATCAACATTTCCTACTATTTCAGGAGTAATAT ATTCTATGTTAGAAAAAGATGCACTTTATTCCATGGCT Example B1 insulator sequence (forward) (SEQ ID NO: 42) AAGAGCCCCAAGAAGTGGATGCGACGTGTGTGCCCCTGGCCTGTCCCTCATCACTCATCTGA CACATCACTCATATTTGCGGAGGAGCCTTGGTCTGAAAACTATTCCGGAATGGGAATCCAGG GGTAGGAGATGCTTTGGCTCTAGATGCAAGACATGAAGCGTTTGCTTCTGGCCACCAGGGGG AGCTGCTGGGACGCAGACGGACTCCAAGGACTGGAGCCAGAAGCCTGGTGTTGCCATGAGGG GTCTGATCTGCACACCTGTCTCAGGACCTAAACGTCAGGCCAGGCCTCCAAGGGCCTATTTG CAGGAGAGCCCTG Example B2 insulator sequence (forward) (SEQ ID NO: 43) CCATCCACCTGCTCTTCATTCTCCTCCCCTGCTTTTCCCAGCTTCTGGTAACCCCATTCCGC CCTCTATTGGAGGGTTGTGTTTTGATTGGCTGCGAAATGCACAGTGTGGCTTCTCTTCTCCC AGGAGTGGGAGGGGCCACCTCTAGGGTCCACCAGGGGGAGCTGCCCTGCAGCCCTGGGCTCC CATCACTCTGGTCCATCCCCTCTCCTCTCCCTCCTGAGCAAATGTGCTAGACAAGACTGTTC CAGGTATGGGCCAGCCAGATGGCAGCATGGTGGTGAGGGTGCAGATCTGCTGGAAGCAAGAG AATGTGGACTTCATGGGTTGTGGAGG Example B3 insulator sequence (forward) (SEQ ID NO: 44) CTCCCGTGTGGTACCTGAGGCCGGCTCCTGTGGCTCTGAGGGGGTCTGCAGCACCCCCTTAC ATCTGTCCACAGAAGGGCTGGGGAGCAGCTTTCCTGTCCCTCCTGTGAGTGGCCACCAGGGG GAGCGTGGACACAGCTGCCCGTGCAGTGACCACCTGCCCCCCACTCCCGCTACTCCAGCAGC AGCGGCTCCAGCCCTGGACACCCTCCCTGCCCCCACCAGCCTGGTCCTGAGCCAGGTGACCT CCTCCAGCATCC Example B4 insulator sequence (forward) (SEQ ID NO: 45) GCGGAAACTGAACCAAAAGACTCTAAGAGAGGAAGGGATAGTGACTTTTAGGCTGCATGGCT GGGGACAGTGATGGTGGCGTCTTCCTCCTCCTTTGGTTTCTCCGCTGCTTCCTCCCACATCT TTGACACTGGACAGTCACGCTGAGCTCGTTTCTGGTCTTGCCACCAGGGGGAGCCAGAGCTC ATATTTCTGGCCCCACTGCAGGAAGCCAGACCTGCTGGGGCAAGCCAGGGGCTTCCCACCCG GTCACTGGCACAAATCCAGGACCCCAGTTCAGAGGCAGCAGAGTGGTACAAGCAGCCCTGT Example B5 insulator sequence (forward) (SEQ ID NO: 46) GTCTGAATGGTGGCCGTAGTTTGCAGAGCCCTGGTTTCTTCTTGCCTCTCAGCTTCCAACTT CCCCGTGAGTGCCTGCTCCTTGATGGACTGGACTCTAAGCCCTTCTTTGCAGCAAGCACGAT ATCAAGCTTTGTCAGTAGAGGGCGCCGGAGGGACACTGTGGAGGAAGGGGCCTTTTCATGGT CCACAGAGCTCTGTTGTGCAATTTCTTGTTCCTGTTGCATCTTCTCTTAGGGTATGAACGCG GGGGGACATCCTCTGGGGCTTTTCCTCAGCTGTGCACCCAGAATGCATGGTCCCTCGACCAC CTCATAGCCCATCCT Example C1 insulator sequence (forward) (SEQ ID NO: 47) GTTTCGCATCCACCTTTCATTGCTTGCTCTGTGATAATGGAGAGGGACCCTGTAAACGTGTC CGCCTTGCCACTTTGTGCAATATTTAGCTCCTTCAGTAGAGGGCGCTGCAGGAACATGTCAG GAGGGGGCCTTCTCTTCCGGATTCTGGATTCTGGTATACACTTGGAGCGTGGCATTTGGGGG ACAGCCACTGGTGCGCAGCCCCATCAAGTTTTGGTGGCATCCCTGTGGACCATATTCTATTA ATCTTCAGTGGTACCCCTGTGGCAGATCCCCAGTGATTCTTATGGGCACTCAGGGCTAAC Example C3 insulator sequence (forward) (SEQ ID NO: 48) AGAAGCACTGCCTGGTAGGATTTCGTAGGAGGAATTCAATCATCAAGTGGGATGTGGATTGG AAATAGGAAGACATTAGATTAGCGTTTCTTTCAACCCAGTAAGTCTGTGAGTGTCCACGCCA CTTATCAATGCATTCTCAGCTCCAAAGGCATCCTCCTTTAACTGCTCTGTGATAATGGAGAT GGGCTTTGTAAATATTTCTTACCAGCTGGCATAATGTAAATCCTTGTCAGTAGAGGGCGCTA GAGAGAGAGAGACCAGAGGAGAAAAAAGTTTTATCTTCCAGATTCTGGGGTGCCTCATCTTT TTGCCTCCTCCAGAGCACAGCTTGCTTTCTCTGTTGCTGGGCTCCTATGCACATGCTTTCTT TTGAGCTAGGCCCA Example C5 insulator sequence (forward) (SEQ ID NO: 49) TCTTTTGCAATGCTCTTTGGGAAATTATATTAGCCTAATTACTAATTTCCTGGCCCTCAAGG TGACTTGCTTGAACTTGCCACCTTCGCCACTAGGGGGCAGCATTGGTTTACACAGGGTGAAC AAACGTTCAATTCTAAACTAATATTCTTTGGTGGGAAAGTGTGTTCATTTTGGTTTGTTTTG AAGAAGTCACTTCATACCTTTGAACTGGGGTTAAGCTGGTTAACTCCCAAAATTCCACCAGT TCCAATATCCTATGGAAAAACCCCAAAACCACT Example D1 insulator sequence (forward) (SEQ ID NO: 50) GAGACCCTCCACCCCCTACCACAGGGAGGCCTCAGGGATGCGCCCTATGGCCAGAGTGAGGG GGAGAGGGCCCTTGAGGGCCACTTTCCACCTTTGGTGTCCTTGACTGGCATGTCCTGCCCTG TTCTCCCGTCGCCACTAGGGGGCAGCCACGCAGCAGGAATTTTACGCCAAGAACTCGCGCTG GACCGAAGGCCTCATCTCGGCCTCCAAGGCTGTGGGCTGGGGAGCCACACAGCTGGTGTAGG TTGCCCTGGGTGGGGGGGGGCAGGGGGCTGCTTCCTGCCAGTTGGAGCAGTTTGGGGTTCAA CAG Example D3 insulator sequence (forward) (SEQ ID NO: 51) GGCGTGTTTGATTTGCTTTTCTCTAAAGCACTGCTCTGTAAGTTCCTATGGGGGGGGCGGGC AGAGCTGCTAGAAATACAGAAGCATAAGAGAGTAAATAATGGTACCTTCATATGCAAATGTG CTTTTCAGATTGGATCTGTCTGTGCTTGTTTTGAGCTATACCCGGTAAGGCTCCCTCCAGAA ACAGAATTCTTGTTTAGCTCCTGGGAGTGTGCAGAATCCACAACAGCCACTAGGGGGCAGGA GGCATGTGTCATATATACCGGGCAGTGAGAAAGAGTTAATGCAATTAACTAAGGGGCACTAT TTTGTACCCTCAGGCTTGAGAGGCCGCCAAGCTACTCCAGCCTCATACAAGTGGGGAAAGAC TTTGACGGAGTTTAGGGTGGACTGGCAGGCTCTCAACCATAGCAGGAGCACTCAAATTTCTC AGCTTATGTGGCAGGCCTCATT Example D4 insulator sequence (forward) (SEQ ID NO: 52) GTTTTCCCCAGTCCTCTTCCCTTCTCCACTGGTTGGGTAGCTCCGTATCTTCCTCTTGCATG TCAGGGTGGCGGCCACTAGGGGGCAGCAGGCACCGTGGTAACGAATGGGCTGCAGCTTTGGC ACTTGCGCTCGGGGACTTGGCCTGGCTTGGAGGAGCTCTGCTCCATCCTTCAAGATGACTTA CGTTGGAGGATGGCGATTCGAGCACCTCTTCACCCCCTTTTTCTCCAGTTTAGGGAAAGGGG Example D5 insulator sequence (forward) (SEQ ID NO: 53) GCCACCTTTGGTCTTGACATTCACTAAGGAGTGGGCCTGGAATAAAACCAGAAATCCCCATT CTCAGTCTGCCCCAAGCCCTCCAATTCCGAGGGCGCTGTATGTATAAGCTCGGGCAGGCAAA AGTCGACTGTGAGAACACGCCAGCAGAGGGCGCTGTGGCCCCATCAGTCCCTGCCCACTGAA CTCTCCAGAAGGAAAAGCGGCAAGGATGCAAACAAGAAAATCAGACCAGACTGGGAGTCTAG ACCCTGCGTCCCACGCGGTCCCACCATGGCTTCCTCTTTGGAATTTTCAGGGGGACCCAGGA AGACTGACACCGCTGAT Example E1 insulator sequence (forward) (SEQ ID NO: 54) TTTGATTTCCTTCACTCTGGAATATACTACAGAATGTACTAATATAAATGTGCTTGCCTATG TCTAATCTAGAATATAATTATCCTGAACTCTACATGGTATTTTAGTCTAGAAAGTACTGTAG CTTCTCTGAGTTCTAAATCAAACCAGGAAACAGCAGAGGGCGCTGGTGCTGTGCTTTTGCAA GTGAGATGTGTGCTCATTTCAAGGGTCCACAAAATTTTCTGGCATGCTGTTTCCATGGAGAG GAAAGTAGGTGTTTTCTTTTTCTTTTTTTTTTTCAGTCAAGCCACCTGCAAACT Example E2 insulator sequence (forward) (SEQ ID NO: 55) CACCCCCTTACTCCACTCAACCCCTCATTCCTTTAGGCAAGGTATTAAGAGCCCTATGCAAA GGCAATCTTCAAGTATGCGTTGGCAGAAAAACAACCAGTACAAATTGAGGCATAATGTAAAA CCGTTAGGCTGCTTTTCACCCAGCAGAGGGCGCTAAACAGCTGTGCCCAAGCCTCTGATTCA ACAAAGCAAACGAGGGCTGGTGAAGCAAGGGAAAGTCAGTTCAGACGCAAAAGCAGCTCTAC AATTGTCTCCCTTAATCTCCTAGTCAGTTCAGAAAGGCAGAGATTTATTGTTTAGTTCCACA GAGAGAGACTGACCTGCAAATAATCCAAGTGAAGAATATTTAGGATCATTTCAGACCATTTA AGCCAGCC Example E3 insulator sequence (forward) (SEQ ID NO: 56) TCAAAAGAATGTGCCAATGCATATTTCCTAACCCACTGTTATTAATAAGTATTAACCAACAA CATTGCAACATATAGTTTTAGTGTGATTCCTTTCATTCAGTGTTTTTCAAGAGTTTTAAAAC AACACCAGCTTTCCAATTGTACTGAATTTGTTTTTTTCCCTTTCATCACCACAAAAATCCAG TTTTGCCCAGTATATGGGGAAACCATTCTTACTGAACCAGCAGAGGGCGCTGATGCACTAGT GATGTCCGATATGCCTAAGACTTAAAACATATAGAAACATATTTATGAGAATGGAGAAGAGT GAGAGAAGAAAACCTTCAGAATCCCTTTTTAAAGGGAATTGTTAAAAATTCTATAGAAAGGA CGTTCCCTAATATGACATTAAATGTGAGCAGGATTAAATAGGTACTATTCAAAGTAATATTT ACAGTAAGAAGTTTCAATGTCAACACAAATTCCA Example E4 insulator sequence (forward) (SEQ ID NO: 57) ACACTTGGGCTGAGAGTGGTGGAGTCAAGAGGTCGGGTTGGGAAGTGCTAAATTTAAAATGC CCATTATATATCGTCTAAGGGTCTGAATGCAGGCGCCCAGGAACTCAGGGGAGAGGGTGGGA TGGAAAGAAAAATCCTGGCAGTCAGTCAGTGAACACTGGTCCCCAGAGCCTGCCCGGATTTG AGTTACTGTCAGTATTTGGGTTCAAACCCTAAAAGCTGAGGAGCTGATCCTGACCTGGAGAT CCGTTTCAGCAGGGATGTGTCTGTCTCCGTGCTGACTCTTAGTGAGGTTTAGAATAGTGTGA CAAACTCAGAGTTACAGCAGAGGGCGCTGTCAATCTAAAGTCCTAAAGCGCAGGAAAGGTTT GTGATATGCTGGGTGGATTTCAGCTTAAAACATAGTAAAATAATTGAGAAAGGGTCTACACC ATATCTTGGCTTATAACCTGAAATAAAACAGAATCTGTAATATTTTACCTTGAAATATCACT GTGTCCTGAAATGCA Example E5 insulator sequence (forward) (SEQ ID NO: 58) GAACAGAAGTGTGGGGGATGGGGGAGCCAAGCCTCTGCTTTGGGGCCACTTGGATGGGACCT GTGGCCCCTAGCGCCTGAGCGCTCCGCCTGTGTGCATCCTGCCTGGCAGCAATCGGTGGTCA CCTCCGTTGTCCAGCAGCTTCCCGCGGTAGATCTTGTCCTCCACCACGTCGGTCCAGTAGAG GGCGCTCTGGCTGAGGTGGAAGTCCAGGGCGATGGTGTTGCGCAGGCCGGGCACCAGGACGC TGTAGTCTCCTTTGTGAAGATCGATGCGCCGGATTTCATGGCGGTTGGAGAAAATGATGAAC GGCTTGAAGG Example F1 insulator sequence (forward) (SEQ ID NO: 59) TGCCTTTCAGCTCCAAATCTACCCTTATATTACCTGTTCTGAAATAACAGACAAGACTCTTT GAAGTCTTTCCTCTGTAGTAAGCATGATGCTAAGTTTGTCAGTAGAGGGCGCTGGAGGGCCG TGGCAGGAGGAAGGAGTTTCCTTTCACGGTTCCAGTAAGTTGCAGGTTTTTTGCTTCTTGCT CTTTCTGCGTTGCTGCCAAACTGCACTGTCCCAGCCATGCACCCAGACTGTGCTGCCTGACT TCCTGCAAACTCAAGGCCCCATCTTAGCTCAGTAATCACCTCACTGTGGCCC Example F2 insulator sequence (forward) (SEQ ID NO: 60) CAATCTCACCCAACCCTTTCATTTTACAGGAGACTAAAGTCCCGAGGATAAGTTAAATGAGG CTAGTGGCAGAGGCAGAATGGTCGATTTCTTAACATCTCAGCTCCCCGGCTCTGCCATCCTG TGAGTTTCTCCTTCACAGGTCGATGTTAAGTTTTGCCAGTAGAGGGCGCTGGCGGGACACGG TGGGAGGCGGGAACTCCTCTTTCTGCCTCCAGGGTGCAGTGTGCTGCTGCTGCTGCGTGTTC CTTTGCTGCTGCTGCAGGGTCTGTTCAGGGATGCTCTGCCCCAGCGGTGCACCCAGAGTTCC TCAGTGGCTGACC Example F3 insulator sequence (forward) (SEQ ID NO: 61) TCGGACATTTCCCTGTCTCTTAAACCCAGTTTTGCCGCTTTGTCCTGTCTTGGATTCCGCAC GCTGCACAAAAAATGGCCAGTAGAGGGCGCTGCTGGCTTACTTTTTAGAAAACTGCTCACAG CCTGATTTCAGCACTTTCAAAAACATGGAAATTATCTTAATTCAGTCAAAGCAATCAGAACG AGAAAACTGTCTTAAATTCATCAAAGTTGTTTCTTTGTGTCTTTTTCTATTTCGTTTCCCCA AAGGCATCAATA Example F4 insulator sequence (forward) (SEQ ID NO: 62) TTTCCCTACTTCTCCCACCATTTTAAAAACAAGGAAACGAAGCCTAGATTTGCAAGAATAAA AGGTTTGGGTTTATTTACCTGTTGGACTCTATACCAGTTATTGATTTATTGCCTCTCACCTC TGAATTCACCTTATTTGACTGCTTTGCGAAAATAAATCTGAACCTTTTCAAGAATTTGCCGT TGGCACCGAAGTTTTGCCAGTAGAGGGCGCTCGAGAAACATTGCAGGAGAAAGAGTTTTGCC TCCGGCTCGGTGGAGTCTCAGCGGGGTTGGCAGGGGCGCGTGGTTTCGCTGGCCCCCAGCGC CTTTGGCTCACAGGTTCTCCAGGGCCCTCCTGCTGCCATACACAGCCAGCAACACTCAGTGG CCAGTGGCTTTCCCCTGCAACCTCCTAAGGTGGTTTGTAGCCAAGGCGCGGTAGTGAGACAT CTTCCCATAAACAGCTTTCCCCAGCACCCTACAGGACTGATTTCTAGCAAGTTTCACCATTA TGGCATCACAGCAACTTGTCCGCCGTTCAGTAGCCATGACTGAGCCA Example F5 insulator sequence (forward) (SEQ ID NO: 63) CCTTCCTTTCTAAATGACGAGAGAGACAGAAGAATTCTTCAAGGTTAGTGTGTCCAGCATGC AACCTTTCCTTCCTGGATGAGCATCCCTGGAATAGGAGAGCCAGCCTGCCTCCTGCGCTGGC ACAGAGCCCGGTTCCCTAGACAACTGCCTCTCCAAATCTGATGTCCAGCGCCACCTGGTGTC CACATCAAGCAGACACAATTAATAGTCAACCTGTTCAGGAAAACTGTGAGGGGGAAAAAAAA GAAAGAGGATTTATGAAGGGAAAAGAAAGTTTAGAGGATATGCCACGATTGGAATGTAATGA GTAGAACCAG Example A1 insulator sequence (reverse) (SEQ ID NO: 64) AGAGCGAGATTCCGTCTCAAAGAAAAAAAAAGTAATGAAATGAATAAAATGAGTCCTAGAGC CAGTAAATGTCGTAAATGTCTCAGCTAGTCAGGTAGTAAAAGGTCTCAACTAGGCAGTGGCA GAGCAGGATTCAAATTCAGGGCTGTTGTGATGCCTCCGCAGACTCTGAGCGCCACCTGGTGG TAATTTGTCTGTGCCTCTTCTGACGTGGAAGAACAGCAACTAACACACTAACACGGCATTTA CTATGGGCCAGCCATTGT Example A2 insulator sequence (reverse) (SEQ ID NO: 65) GGGAGAGGTGGTTCAACAAAGAACAATGGGATGCTGTTACCAGAAGAAGGAATAAGATGCTG GCCAGGTGAAAAGACCAATTTCACGAGAGAACCTTAAATTACCAGCGCCACCTGGTGCATCA AAACTCCTCTGAAGCCGGGCGCAGTGGTTCACACCTGTAATCTCAGCACTTTGGGTGGTTGA GGCAGGAGCATTGCTTGAGCCCAGGGGTTGGAGACCAGCCTGGGGAACATAGGGAGACCAAC CCCT Example A3 insulator sequence (reverse) (SEQ ID NO: 66) TGCTTGTCCTTCCTTCCTGTAACACAGCCATTAAACCAGGAGCATCGCCCTTCCCCGGCCCT CAGGTAAGAGGACCAAATACCGTAGCCGTTTCCAATTTCAGTCCTTTAGCGCCACCTGGTGC TAACTACTCTATCACGCTTTTATCCAATAACTACCTTTGTAAATTTCCTTTCAAAAGTTCTG GCCGGGCGCGGTGGCTCACGCTTGTAATCCCAGCACTTTGTGAGGGGTCAGGAGTTC Example A4 insulator sequence (reverse) (SEQ ID NO: 67) GTAGAGACGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGACCTCAGGTGATCC ACCCGCCTCGGCCACCCAAAGTGCTGGGATTACAGGCGTGAGCCACTGTGCTGGGCCTGCCA GGAATTTCATAGGTCACCTGCAGAGGCAGGGACACCACGAGGCAACCTCAGTGTGACCCCTG CTGGCAGGTCTCCGCAGCGTTCTGGTGACACTGCTTTTTCCAAGTGGTCACGAGGCCCGGCA GCGCCACCTGGTGGAGCTATGGAGACACGGAGTCGTCCAAGTCCTGGGAAACAGAATCCTAG AATCTCTGGATTTTGAACATTCTTGAAGGTGATGGAGTGGGA Example A5 insulator sequence (reverse) (SEQ ID NO: 68) AGCCATGGAATAAAGTGCATCTTTTTCTAACATAGAATATATTACTCCTGAAATAGTAGGAA ATGTTGATAATTGCCTTTTATTGGACTTACAAATAATTTATAAGTTGGTCCGGTTAAAAAAA AAAAGTATCATAGTCTTTTATTTCTTTCGTTTCTTCCTCCAGGCTAGAAGCAACCTCTAAAA ATAGCTACCTGTGCAGGGATGGGCCTAGCGCTCCCCCTGGTGGTCATAGTGCAGGATTGCGG CTTCCGTAAATGGACCTGAAGCGGCAGACCCTGCATTTGAATTAATTCTTTCTCCCCTAAGT ACACAGAGAAATGTGATTTGTTCTTCCCAGTCATGCCT Example B1 insulator sequence (reverse) (SEQ ID NO: 69) CAGGGCTCTCCTGCAAATAGGCCCTTGGAGGCCTGGCCTGACGTTTAGGTCCTGAGACAGGT GTGCAGATCAGACCCCTCATGGCAACACCAGGCTTCTGGCTCCAGTCCTTGGAGTCCGTCTG CGTCCCAGCAGCTCCCCCTGGTGGCCAGAAGCAAACGCTTCATGTCTTGCATCTAGAGCCAA AGCATCTCCTACCCCTGGATTCCCATTCCGGAATAGTTTTCAGACCAAGGCTCCTCCGCAAA TATGAGTGATGTGTCAGATGAGTGATGAGGGACAGGCCAGGGGCACACACGTCGCATCCACT TCTTGGGGCTCTT Example B2 insulator sequence (reverse) (SEQ ID NO: 70) CCTCCACAACCCATGAAGTCCACATTCTCTTGCTTCCAGCAGATCTGCACCCTCACCACCAT GCTGCCATCTGGCTGGCCCATACCTGGAACAGTCTTGTCTAGCACATTTGCTCAGGAGGGAG AGGAGAGGGGATGGACCAGAGTGATGGGAGCCCAGGGCTGCAGGGCAGCTCCCCCTGGTGGA CCCTAGAGGTGGCCCCTCCCACTCCTGGGAGAAGAGAAGCCACACTGTGCATTTCGCAGCCA ATCAAAACACAACCCTCCAATAGAGGGCGGAATGGGGTTACCAGAAGCTGGGAAAAGCAGGG GAGGAGAATGAAGAGCAGGTGGATGG Example B3 insulator sequence (reverse) (SEQ ID NO: 71) GGATGCTGGAGGAGGTCACCTGGCTCAGGACCAGGCTGGTGGGGGCAGGGAGGGTGTCCAGG GCTGGAGCCGCTGCTGCTGGAGTAGCGGGAGTGGGGGGCAGGTGGTCACTGCACGGGCAGCT GTGTCCACGCTCCCCCTGGTGGCCACTCACAGGAGGGACAGGAAAGCTGCTCCCCAGCCCTT CTGTGGACAGATGTAAGGGGGTGCTGCAGACCCCCTCAGAGCCACAGGAGCCGGCCTCAGGT ACCACACGGGAG Example B4 insulator sequence (reverse) (SEQ ID NO: 72) ACAGGGCTGCTTGTACCACTCTGCTGCCTCTGAACTGGGGTCCTGGATTTGTGCCAGTGACC GGGTGGGAAGCCCCTGGCTTGCCCCAGCAGGTCTGGCTTCCTGCAGTGGGGCCAGAAATATG AGCTCTGGCTCCCCCTGGTGGCAAGACCAGAAACGAGCTCAGCGTGACTGTCCAGTGTCAAA GATGTGGGAGGAAGCAGCGGAGAAACCAAAGGAGGAGGAAGACGCCACCATCACTGTCCCCA GCCATGCAGCCTAAAAGTCACTATCCCTTCCTCTCTTAGAGTCTTTTGGTTCAGTTTCCGC Example B5 insulator sequence (reverse) (SEQ ID NO: 73) AGGATGGGCTATGAGGTGGTCGAGGGACCATGCATTCTGGGTGCACAGCTGAGGAAAAGCCC CAGAGGATGTCCCCCCGCGTTCATACCCTAAGAGAAGATGCAACAGGAACAAGAAATTGCAC AACAGAGCTCTGTGGACCATGAAAAGGCCCCTTCCTCCACAGTGTCCCTCCGGCGCCCTCTA CTGACAAAGCTTGATATCGTGCTTGCTGCAAAGAAGGGCTTAGAGTCCAGTCCATCAAGGAG CAGGCACTCACGGGGAAGTTGGAAGCTGAGAGGCAAGAAGAAACCAGGGCTCTGCAAACTAC GGCCACCATTCAGAC Example C1 insulator sequence (reverse) (SEQ ID NO: 74) GTTAGCCCTGAGTGCCCATAAGAATCACTGGGGATCTGCCACAGGGGTACCACTGAAGATTA ATAGAATATGGTCCACAGGGATGCCACCAAAACTTGATGGGGCTGCGCACCAGTGGCTGTCC CCCAAATGCCACGCTCCAAGTGTATACCAGAATCCAGAATCCGGAAGAGAAGGCCCCCTCCT GACATGTTCCTGCAGCGCCCTCTACTGAAGGAGCTAAATATTGCACAAAGTGGCAAGGCGGA CACGTTTACAGGGTCCCTCTCCATTATCACAGAGCAAGCAATGAAAGGTGGATGCGAAAC Example C3 insulator sequence (reverse) (SEQ ID NO: 75) TGGGCCTAGCTCAAAAGAAAGCATGTGCATAGGAGCCCAGCAACAGAGAAAGCAAGCTGTGC TCTGGAGGAGGCAAAAAGATGAGGCACCCCAGAATCTGGAAGATAAAACTTTTTTCTCCTCT GGTCTCTCTCTCTCTAGCGCCCTCTACTGACAAGGATTTACATTATGCCAGCTGGTAAGAAA TATTTACAAAGCCCATCTCCATTATCACAGAGCAGTTAAAGGAGGATGCCTTTGGAGCTGAG AATGCATTGATAAGTGGCGTGGACACTCACAGACTTACTGGGTTGAAAGAAACGCTAATCTA ATGTCTTCCTATTTCCAATCCACATCCCACTTGATGATTGAATTCCTCCTACGAAATCCTAC CAGGCAGTGCTTCT Example C5 insulator sequence (reverse) (SEQ ID NO: 76) AGTGGTTTTGGGGTTTTTCCATAGGATATTGGAACTGGTGGAATTTTGGGAGTTAACCAGCT TAACCCCAGTTCAAAGGTATGAAGTGACTTCTTCAAAACAAACCAAAATGAACACACTTTCC CACCAAAGAATATTAGTTTAGAATTGAACGTTTGTTCACCCTGTGTAAACCAATGCTGCCCC CTAGTGGCGAAGGTGGCAAGTTCAAGCAAGTCACCTTGAGGGCCAGGAAATTAGTAATTAGG CTAATATAATTTCCCAAAGAGCATTGCAAAAGA Example D1 insulator sequence (reverse) (SEQ ID NO: 77) CTGTTGAACCCCAAACTGCTCCAACTGGCAGGAAGCAGCCCCCTGCCCCCCCCCACCCAGGG CAACCTACACCAGCTGTGTGGCTCCCCAGCCCACAGCCTTGGAGGCCGAGATGAGGCCTTCG GTCCAGCGCGAGTTCTTGGCGTAAAATTCCTGCTGCGTGGCTGCCCCCTAGTGGCGACGGGA GAACAGGGCAGGACATGCCAGTCAAGGACACCAAAGGTGGAAAGTGGCCCTCAAGGGCCCTC TCCCCCTCACTCTGGCCATAGGGCGCATCCCTGAGGCCTCCCTGTGGTAGGGGGTGGAGGGT CTC Example D3 insulator sequence (reverse) (SEQ ID NO: 78) AATGAGGCCTGCCACATAAGCTGAGAAATTTGAGTGCTCCTGCTATGGTTGAGAGCCTGCCA GTCCACCCTAAACTCCGTCAAAGTCTTTCCCCACTTGTATGAGGCTGGAGTAGCTTGGCGGC CTCTCAAGCCTGAGGGTACAAAATAGTGCCCCTTAGTTAATTGCATTAACTCTTTCTCACTG CCCGGTATATATGACACATGCCTCCTGCCCCCTAGTGGCTGTTGTGGATTCTGCACACTCCC AGGAGCTAAACAAGAATTCTGTTTCTGGAGGGAGCCTTACCGGGTATAGCTCAAAACAAGCA CAGACAGATCCAATCTGAAAAGCACATTTGCATATGAAGGTACCATTATTTACTCTCTTATG CTTCTGTATTTCTAGCAGCTCTGCCCGCCCCCCCCATAGGAACTTACAGAGCAGTGCTTTAG AGAAAAGCAAATCAAACACGCC Example D4 insulator sequence (reverse) (SEQ ID NO: 79) CCCCTTTCCCTAAACTGGAGAAAAAGGGGGTGAAGAGGTGCTCGAATCGCCATCCTCCAACG TAAGTCATCTTGAAGGATGGAGCAGAGCTCCTCCAAGCCAGGCCAAGTCCCCGAGCGCAAGT GCCAAAGCTGCAGCCCATTCGTTACCACGGTGCCTGCTGCCCCCTAGTGGCCGCCACCCTGA CATGCAAGAGGAAGATACGGAGCTACCCAACCAGTGGAGAAGGGAAGAGGACTGGGGAAAAC Example D5 insulator sequence (reverse) (SEQ ID NO: 80) ATCAGCGGTGTCAGTCTTCCTGGGTCCCCCTGAAAATTCCAAAGAGGAAGCCATGGTGGGAC CGCGTGGGACGCAGGGTCTAGACTCCCAGTCTGGTCTGATTTTCTTGTTTGCATCCTTGCCG CTTTTCCTTCTGGAGAGTTCAGTGGGCAGGGACTGATGGGGCCACAGCGCCCTCTGCTGGCG TGTTCTCACAGTCGACTTTTGCCTGCCCGAGCTTATACATACAGCGCCCTCGGAATTGGAGG GCTTGGGGCAGACTGAGAATGGGGATTTCTGGTTTTATTCCAGGCCCACTCCTTAGTGAATG TCAAGACCAAAGGTGGC Example E1 insulator sequence (reverse) (SEQ ID NO: 81) AGTTTGCAGGTGGCTTGACTGAAAAAAAAAAAGAAAAAGAAAACACCTACTTTCCTCTCCAT GGAAACAGCATGCCAGAAAATTTTGTGGACCCTTGAAATGAGCACACATCTCACTTGCAAAA GCACAGCACCAGCGCCCTCTGCTGTTTCCTGGTTTGATTTAGAACTCAGAGAAGCTACAGTA CTTTCTAGACTAAAATACCATGTAGAGTTCAGGATAATTATATTCTAGATTAGACATAGGCA AGCACATTTATATTAGTACATTCTGTAGTATATTCCAGAGTGAAGGAAATCAAA Example E2 insulator sequence (reverse) (SEQ ID NO: 82) GGCTGGCTTAAATGGTCTGAAATGATCCTAAATATTCTTCACTTGGATTATTTGCAGGTCAG TCTCTCTCTGTGGAACTAAACAATAAATCTCTGCCTTTCTGAACTGACTAGGAGATTAAGGG AGACAATTGTAGAGCTGCTTTTGCGTCTGAACTGACTTTCCCTTGCTTCACCAGCCCTCGTT TGCTTTGTTGAATCAGAGGCTTGGGCACAGCTGTTTAGCGCCCTCTGCTGGGTGAAAAGCAG CCTAACGGTTTTACATTATGCCTCAATTTGTACTGGTTGTTTTTCTGCCAACGCATACTTGA AGATTGCCTTTGCATAGGGCTCTTAATACCTTGCCTAAAGGAATGAGGGGTTGAGTGGAGTA AGGGGGTG Example E3 insulator sequence (reverse) (SEQ ID NO: 83) TGGAATTTGTGTTGACATTGAAACTTCTTACTGTAAATATTACTTTGAATAGTACCTATTTA ATCCTGCTCACATTTAATGTCATATTAGGGAACGTCCTTTCTATAGAATTTTTAACAATTCC CTTTAAAAAGGGATTCTGAAGGTTTTCTTCTCTCACTCTTCTCCATTCTCATAAATATGTTT CTATATGTTTTAAGTCTTAGGCATATCGGACATCACTAGTGCATCAGCGCCCTCTGCTGGTT CAGTAAGAATGGTTTCCCCATATACTGGGCAAAACTGGATTTTTGTGGTGATGAAAGGGAAA AAAACAAATTCAGTACAATTGGAAAGCTGGTGTTGTTTTAAAACTCTTGAAAAACACTGAAT GAAAGGAATCACACTAAAACTATATGTTGCAATGTTGTTGGTTAATACTTATTAATAACAGT GGGTTAGGAAATATGCATTGGCACATTCTTTTGA Example E4 insulator sequence (reverse) (SEQ ID NO: 84) TGCATTTCAGGACACAGTGATATTTCAAGGTAAAATATTACAGATTCTGTTTTATTTCAGGT TATAAGCCAAGATATGGTGTAGACCCTTTCTCAATTATTTTACTATGTTTTAAGCTGAAATC CACCCAGCATATCACAAACCTTTCCTGCGCTTTAGGACTTTAGATTGACAGCGCCCTCTGCT GTAACTCTGAGTTTGTCACACTATTCTAAACCTCACTAAGAGTCAGCACGGAGACAGACACA TCCCTGCTGAAACGGATCTCCAGGTCAGGATCAGCTCCTCAGCTTTTAGGGTTTGAACCCAA ATACTGACAGTAACTCAAATCCGGGCAGGCTCTGGGGACCAGTGTTCACTGACTGACTGCCA GGATTTTTCTTTCCATCCCACCCTCTCCCCTGAGTTCCTGGGCGCCTGCATTCAGACCCTTA GACGATATATAATGGGCATTTTAAATTTAGCACTTCCCAACCCGACCTCTTGACTCCACCAC TCTCAGCCCAAGTGT Example E5 insulator sequence (reverse) (SEQ ID NO: 85) CCTTCAAGCCGTTCATCATTTTCTCCAACCGCCATGAAATCCGGCGCATCGATCTTCACAAA GGAGACTACAGCGTCCTGGTGCCCGGCCTGCGCAACACCATCGCCCTGGACTTCCACCTCAG CCAGAGCGCCCTCTACTGGACCGACGTGGTGGAGGACAAGATCTACCGCGGGAAGCTGCTGG ACAACGGAGGTGACCACCGATTGCTGCCAGGCAGGATGCACACAGGCGGAGCGCTCAGGCGC TAGGGGCCACAGGTCCCATCCAAGTGGCCCCAAAGCAGAGGCTTGGCTCCCCCATCCCCCAC ACTTCTGTTC Example F1 insulator sequence (reverse) (SEQ ID NO: 86) GGGCCACAGTGAGGTGATTACTGAGCTAAGATGGGGCCTTGAGTTTGCAGGAAGTCAGGCAG CACAGTCTGGGTGCATGGCTGGGACAGTGCAGTTTGGCAGCAACGCAGAAAGAGCAAGAAGC AAAAAACCTGCAACTTACTGGAACCGTGAAAGGAAACTCCTTCCTCCTGCCACGGCCCTCCA GCGCCCTCTACTGACAAACTTAGCATCATGCTTACTACAGAGGAAAGACTTCAAAGAGTCTT GTCTGTTATTTCAGAACAGGTAATATAAGGGTAGATTTGGAGCTGAAAGGCA Example F2 insulator sequence (reverse) (SEQ ID NO: 87) GGTCAGCCACTGAGGAACTCTGGGTGCACCGCTGGGGCAGAGCATCCCTGAACAGACCCTGC AGCAGCAGCAAAGGAACACGCAGCAGCAGCAGCACACTGCACCCTGGAGGCAGAAAGAGGAG TTCCCGCCTCCCACCGTGTCCCGCCAGCGCCCTCTACTGGCAAAACTTAACATCGACCTGTG AAGGAGAAACTCACAGGATGGCAGAGCCGGGGAGCTGAGATGTTAAGAAATCGACCATTCTG CCTCTGCCACTAGCCTCATTTAACTTATCCTCGGGACTTTAGTCTCCTGTAAAATGAAAGGG TTGGGTGAGATTG Example F3 insulator sequence (reverse) (SEQ ID NO: 88) TATTGATGCCTTTGGGGAAACGAAATAGAAAAAGACACAAAGAAACAACTTTGATGAATTTA AGACAGTTTTCTCGTTCTGATTGCTTTGACTGAATTAAGATAATTTCCATGTTTTTGAAAGT GCTGAAATCAGGCTGTGAGCAGTTTTCTAAAAAGTAAGCCAGCAGCGCCCTCTACTGGCCAT TTTTTGTGCAGCGTGCGGAATCCAAGACAGGACAAAGCGGCAAAACTGGGTTTAAGAGACAG GGAAATGTCCGA Example F4 insulator sequence (reverse) (SEQ ID NO: 89) TGGCTCAGTCATGGCTACTGAACGGCGGACAAGTTGCTGTGATGCCATAATGGTGAAACTTG CTAGAAATCAGTCCTGTAGGGTGCTGGGGAAAGCTGTTTATGGGAAGATGTCTCACTACCGC GCCTTGGCTACAAACCACCTTAGGAGGTTGCAGGGGAAAGCCACTGGCCACTGAGTGTTGCT GGCTGTGTATGGCAGCAGGAGGGCCCTGGAGAACCTGTGAGCCAAAGGCGCTGGGGGCCAGC GAAACCACGCGCCCCTGCCAACCCCGCTGAGACTCCACCGAGCCGGAGGCAAAACTCTTTCT CCTGCAATGTTTCTCGAGCGCCCTCTACTGGCAAAACTTCGGTGCCAACGGCAAATTCTTGA AAAGGTTCAGATTTATTTTCGCAAAGCAGTCAAATAAGGTGAATTCAGAGGTGAGAGGCAAT AAATCAATAACTGGTATAGAGTCCAACAGGTAAATAAACCCAAACCTTTTATTCTTGCAAAT CTAGGCTTCGTTTCCTTGTTTTTAAAATGGTGGGAGAAGTAGGGAAA Example F5 insulator sequence (reverse) (SEQ ID NO: 90) In some embodiments, an insulator element comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 37 to 90, 102 or 103, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. In some embodiments, an insulator element comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 37 to 90, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. Suitably, the variants comprise a CTCF-binding motif (e.g. any of SEQ ID NOs: 23 to 28) and one or more variation is made in the flanking sequences. In some embodiments, an insulator element comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 37 to 90, 102 or 103. In some embodiments, an insulator element comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 37, 38, 43, 55, 64, 65, 70, 82, 102 or 103 or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. In some embodiments, an insulator element comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 37, 38, 43, 55, 64, 65, 70, 82, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. Suitably, the variants comprise a CTCF- binding motif (e.g. any of SEQ ID NOs: 23 to 28) and one or more variation is made in the flanking sequences. In some embodiments, an insulator element comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 37, 38, 43, 55, 64, 65, 70, 82, 102 or 103. A1 insulator elements In some embodiments, the polynucleotide of the present invention comprises one or more A1 insulator element. An A1 insulator element may comprise or consist of a core A1 insulator sequence. In some embodiments, an A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 29 or 33, or variants thereof having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif A (e.g. SEQ ID NO: 23) and one or more variation is made in the flanking sequences. In some embodiments, an A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 29 or 33. In some embodiments, an A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 37 or 64, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif A (e.g. SEQ ID NO: 23) and one or more variation is made in the flanking sequences. In some embodiments, an A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 37 or 64. In some embodiments, the polynucleotide of the present invention comprises one or more core A1 insulator sequence. In some embodiments, a core A1 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 29 or 33, or variants thereof having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF- binding motif A (e.g. SEQ ID NO: 23) and one or more variation is made in the flanking sequences. In some embodiments, a core A1 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 29 or 33. In some embodiments, the polynucleotide of the present invention comprises one or more full-length A1 insulator sequence. In some embodiments, a full-length A1 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 37 or 64, or variants thereof having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif A (e.g. SEQ ID NO: 23) and one or more variation is made in the flanking sequences. In some embodiments, a full-length A1 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 37 or 64. A2 insulator elements In some embodiments, the polynucleotide of the present invention comprises one or more A2 insulator element. An A2 insulator element may comprise or consist of a core A2 insulator sequence. In some embodiments, an A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 30 or 34, or variants thereof having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif A (e.g. SEQ ID NO: 23) and one or more variation is made in the flanking sequences. In some embodiments, an A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 30 or 34. In some embodiments, an A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 38 or 65, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif A (e.g. SEQ ID NO: 23) and one or more variation is made in the flanking sequences. In some embodiments, an A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 38 or 65. In some embodiments, the polynucleotide of the present invention comprises one or more core A2 insulator sequence. In some embodiments, a core A2 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 30 or 34, or variants thereof having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF- binding motif A (e.g. SEQ ID NO: 23) and one or more variation is made in the flanking sequences. In some embodiments, a core A2 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 30 or 34. In some embodiments, the polynucleotide of the present invention comprises one or more full-length A2 insulator sequence. In some embodiments, a full-length A2 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 38 or 65, or variants thereof having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. In some embodiments, a full- length A2 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 38 or 65. In some embodiments, a full-length A2 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 102 or 103, or variants thereof having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif A (e.g. SEQ ID NO: 23) and one or more variation is made in the flanking sequences. In some embodiments, a full-length A2 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 102 or 103. In some embodiments, the polynucleotide of the present invention comprises one or more core A2 insulator sequence described below in the section entitled “Variant A2 insulator elements”. B2 insulator elements In some embodiments, the polynucleotide of the present invention comprises one or more B2 insulator element. A B2 insulator element may comprise or consist of a core B2 insulator sequence. In some embodiments, a B2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 31 or 35, or variants thereof having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif B (e.g. SEQ ID NO: 24) and one or more variation is made in the flanking sequences. In some embodiments, a B2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 31 or 35. In some embodiments, a B2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 43 or 70, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif B (e.g. SEQ ID NO: 24) and one or more variation is made in the flanking sequences. In some embodiments, a B2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 43 or 70. In some embodiments, the polynucleotide of the present invention comprises one or more core B2 insulator sequence. In some embodiments, a core B2 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 31 or 35, or variants thereof having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF- binding motif B (e.g. SEQ ID NO: 24) and one or more variation is made in the flanking sequences. In some embodiments, a core B2 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 31 or 35. In some embodiments, the polynucleotide of the present invention comprises one or more full-length B2 insulator sequence. In some embodiments, a full-length B2 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 43 or 70, or variants thereof having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif B (e.g. SEQ ID NO: 24) and one or more variation is made in the flanking sequences. In some embodiments, a full-length B2 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 43 or 70. E2 insulator elements In some embodiments, the polynucleotide of the present invention comprises one or more E2 insulator element. An E2 insulator element may comprise or consist of a core E2 insulator sequence. In some embodiments, an E2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 32 or 36, or variants thereof having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif E (e.g. SEQ ID NO: 27) and one or more variation is made in the flanking sequences. In some embodiments, an E2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 32 or 36. In some embodiments, an E2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 55 or 82, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif E (e.g. SEQ ID NO: 27) and one or more variation is made in the flanking sequences. In some embodiments, an E2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 55 or 82. In some embodiments, the polynucleotide of the present invention comprises one or more core E2 insulator sequence. In some embodiments, a core E2 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 32 or 36, or variants thereof having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF- binding motif E (e.g. SEQ ID NO: 27) and one or more variation is made in the flanking sequences. In some embodiments, a core E2 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 32 or 36. In some embodiments, the polynucleotide of the present invention comprises one or more full-length E2 insulator sequence. In some embodiments, a full-length E2 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 55 or 82, or variants thereof having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif E (e.g. SEQ ID NO: 27) and one or more variation is made in the flanking sequences. In some embodiments, a full-length E2 insulator sequence consists of the nucleotide sequence of SEQ ID NO: 55 or 82. Insulator element pairs In some embodiments, the polynucleotide comprises a pair of insulator elements flanking the expression cassette. Suitably, the polynucleotide comprises from 5’ to 3’ direction: a first insulator element, an expression cassette, and a second insulator element. A pair of insulator elements may be used to flank the expression cassette, thereby generating a functionally independent domain, protected from position effects. Regulatory interactions can occur with the domain but the insulator elements may block external regulatory signals. The pair of insulator elements may have the same or different orientations. A pair of insulator elements may be arranged in different orientations, such as a convergent orientation, a parallel orientation or a divergent orientation (see e.g. Ghirlando, R. and Felsenfeld, G., 2016. Genes & development, 30(8), pp.881-891). In some embodiments, the pair of insulator elements is in a convergent orientation. As used herein, a “convergent orientation” may refer to a forward-reverse pair of insulator elements, e.g. in which the first insulator element is in a forward orientation and the second insulator element is in a reverse orientation. In some embodiments, the pair of insulator elements is in a parallel orientation. As used herein, a “parallel orientation” or “tandem orientation” may refer to a forward-forward pair of insulator elements, e.g. in which the first insulator element is in a forward orientation and the second insulator element is in a forward orientation; or a reverse-reverse pair of insulator elements, e.g. in which the first insulator element is in a reverse orientation and the second insulator element is in a reverse orientation. In some embodiments, the pair of insulator elements is in a divergent orientation. As used herein, a “divergent orientation” may refer to a reverse-forward pair of insulator elements, e.g. in which the first insulator element is in a reverse orientation and the second insulator element is in a forward orientation. The pair of insulator elements may both have the same or different sequences. Suitably, both of the insulator elements binds CTCF. Suitably, both of the insulator elements comprises a CTCF-binding motif. A1-A1 insulator pairs In some embodiments, the polynucleotide comprises an A1-A1 insulator element pair flanking the expression cassette. In some embodiments, the A1 insulator elements are in a parallel orientation. In some embodiments, the A1 insulator elements each comprise or consist of a core insulator sequence. In some embodiments, the A1 insulator elements each consist of a core insulator sequence. In some embodiments, the A1 insulator elements each comprise or consists of the nucleotide sequence of SEQ ID NO: 33, or a nucleotide sequence having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif A (e.g. SEQ ID NO: 23) and one or more variation is made in the flanking sequences. In some embodiments, the A1 insulator elements each comprise or consist of the nucleotide sequence of SEQ ID NO: 33. A2-A2 insulator pairs In some embodiments, the polynucleotide comprises an A2-A2 insulator element pair flanking the expression cassette. In some embodiments, the A2 insulator elements are in a parallel orientation. In some embodiments, the A2 insulator elements each comprise or consist of a core insulator sequence. In some embodiments, the A2 insulator elements each consist of a core insulator sequence. In some embodiments, the A2 insulator elements each comprise or consists of the nucleotide sequence of SEQ ID NO: 34, or a nucleotide sequence having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif A (e.g. SEQ ID NO: 23) and one or more variation is made in the flanking sequences. In some embodiments, the A2 insulator elements each comprise or consist of the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the A2 insulator elements each comprise or consist of a full length insulator sequence. In some embodiments, the A2 insulator elements each comprise or consist of the nucleotide sequence of SEQ ID NO: 103, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif A (e.g. SEQ ID NO: 23) and one or more variation is made in the flanking sequences. In some embodiments, the A2 insulator elements each comprise or consist of the nucleotide sequence of SEQ ID NO: 103. B2-B2 insulator pairs In some embodiments, the polynucleotide comprises an B2-B2 insulator element pair flanking the expression cassette. In some embodiments, the B2 insulator elements are in a parallel orientation. In some embodiments, the B2 insulator elements each comprise or consist of a core insulator sequence. In some embodiments, the B2 insulator elements each consist of a core insulator sequence. In some embodiments, the B2 insulator elements each comprise or consists of the nucleotide sequence of SEQ ID NO: 35, or a nucleotide sequence having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif B (e.g. SEQ ID NO: 24) and one or more variation is made in the flanking sequences. In some embodiments, the B2 insulator elements each comprise or consist of the nucleotide sequence of SEQ ID NO: 35. A1-A2 insulator pairs In some embodiments, the polynucleotide comprises an A1-A2 insulator element pair flanking the expression cassette. In some embodiments, the first insulator element is an A1 insulator element and the second insulator element is an A2 insulator element. In some embodiments, the A1 insulator element and the A2 insulator element are in a parallel orientation. In some embodiments, the A1 insulator element and the A2 insulator element each comprise or consist of a core insulator sequence. In some embodiments, the A1 insulator element and the A2 insulator element each consist of a core insulator sequence. In some embodiments, the A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 33, or a nucleotide sequence having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto; and / or the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 34, or a nucleotide sequence having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif A (e.g. SEQ ID NO: 23) and one or more variation is made in the flanking sequences. In some embodiments, the A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 33; and / or the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the A1 insulator element and the A2 insulator element each comprise or consist of a full length insulator sequence. In some embodiments, the A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 64, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto; and / or the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. Suitably, the variants comprise CTCF-binding motif A (e.g. SEQ ID NO: 23) and one or more variation is made in the flanking sequences. In some embodiments, the A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 64; and / or the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103. A2-E2 insulator pairs In some embodiments, the polynucleotide comprises an A2-E2 insulator element pair flanking the expression cassette. In some embodiments, the first insulator element is an A2 insulator element and the second insulator element is an E2 insulator element. In some embodiments, the A2 insulator element and the E2 insulator element are in a divergent orientation. In some embodiments, the A2 insulator element and the E2 insulator element each comprise or consist of a core insulator sequence. In some embodiments, the A2 insulator element and the E2 insulator element each consist of a core insulator sequence. In some embodiments, the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 34, or a nucleotide sequence having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto; and / or the E2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 32, or a nucleotide sequence having at least 80.0%, at least 82.5%, at least 85.0%, at least 87.5%, at least 90.0%, at least 92.5%, at least 95.0%, or at least 97.5% sequence identity thereto. Suitably, the A2 insulator element variants comprise CTCF- binding motif A (e.g. SEQ ID NO: 23) and one or more variation is made in the flanking sequences. Suitably, the E2 insulator element variants comprise CTCF-binding motif E (e.g. SEQ ID NO: 27) and one or more variation is made in the flanking sequences. In some embodiments, the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 34; and / or the E2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the A2 insulator element and the E2 insulator element each comprise or consist of a full length insulator sequence. In some embodiments, the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto; and / or the E2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 55, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. Suitably, the A2 insulator element variants comprise CTCF-binding motif A (e.g. SEQ ID NO: 23) and one or more variation is made in the flanking sequences. Suitably, the E2 insulator element variants comprise CTCF-binding motif E (e.g. SEQ ID NO: 27) and one or more variation is made in the flanking sequences. In some embodiments, the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103; and / or the E2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 55. Multiple insulator elements The polynucleotide may comprise any number of insulator elements in any arrangement provided that at least one of the insulator elements functions to prevent activation of a promoter by nearby heterologous enhancers and / or functions as a barrier against repressive effects of neighbouring heterochromatin. In some embodiments, the polynucleotide comprises one or more insulator elements. In some embodiments, the polynucleotide comprises one insulator element. A single insulator element may form a pair of insulator elements with an element from another polynucleotide, for example following integration of the polynucleotide in a genomic context. For example, the insulator elements may be upstream from the expression cassette or downstream from the expression cassette. Alternatively, if the polypeptide is a retrovirus genome and the insulator element is in the 3’ LTR of the retrovirus genome, it may be copied to the 5’ LTR during reverse transcription, thereby providing a pair of insulator elements. For example, the insulator element may be in a 3’ LTR. In some embodiments, the polynucleotide comprises two or more insulator elements. In some embodiments, the polynucleotide comprises two insulator elements. The two or more insulator elements may form a pair of insulator elements flanking the expression cassette. For example, one of the insulator elements may be upstream from the expression cassette and one of the insulator elements may be downstream from the expression cassette. Alternatively, each of the insulator elements may form a pair of insulator elements with an insulator element from another polynucleotide. For example, both of the insulator elements may be upstream from the expression cassette or both of the insulator elements may be downstream from the expression cassette. Alternatively, if the polynucleotide is a retrovirus genome and at least one of the insulator elements is in the 3’ LTR of the retrovirus genome, at least one of the insulator elements may be copied to the 5’ LTR during reverse transcription, thereby providing a pair of insulator elements. For example, one of the insulator elements may be downstream of the expression cassette and upstream of a 3’ LTR and one of the insulator elements may be in a 3’ LTR. For example, both of the insulator elements may be in a 3’ LTR (e.g. in parallel or divergent orientation). In some embodiments, the polynucleotide comprises three or more insulator elements. In some embodiments, the polynucleotide comprises three insulator elements. At least two of the insulator elements may form a pair of insulator elements flanking the expression cassette. For example, at least one of the insulator elements may be upstream from the expression cassette and at least one of the insulator elements may be downstream from the expression cassette. Alternatively, each of the insulator elements may form a pair of insulator elements with an insulator element from another polynucleotide. For example, all of the insulator elements may be upstream from the expression cassette or all of the insulator elements may be downstream from the expression cassette. Alternatively, if the polynucleotide is a retrovirus genome and at least one of the insulator elements is in the 3’ LTR of the retrovirus genome, at least one of the insulator elements may be copied to the 5’ LTR during reverse transcription, thereby providing a pair of insulator elements. For example, at least one of the insulator elements may be present in downstream of the expression cassette and upstream of a 3’ LTR and at least one of the insulator elements may present in a 3’ LTR. For example, one of the insulator elements may be present downstream of the expression cassette and upstream of a 3’ LTR and two of the insulator elements may present in a 3’ LTR (e.g. in parallel or divergent orientation). For example, one of the insulator elements may present in a 5’ LTR, one of the insulator elements may be present downstream of the expression cassette and upstream of a 3’ LTR, and one of the insulator elements may present in a 3’ LTR. In some embodiments, the polynucleotide comprises four or more insulator elements. In some embodiments, the polynucleotide comprises four insulator elements. For example, two of the insulator elements may present in a 5’ LTR and two of the insulator elements may present in a 3’ LTR. The insulator elements may each have the same or different sequences. Suitably, each of the insulator elements binds CTCF. Suitably, each of the insulator elements comprises a CTCF-binding motif. Example polynucleotides In some embodiments, the polynucleotide comprises: (a) one or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette and / or downstream from the transcription termination site; and (b) one or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the polynucleotide comprises: (a) one or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette and / or downstream from the transcription termination site; and (c) one or more insulator elements, upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the polynucleotide comprises: (b) one or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette and / or downstream from the expression cassette; and (c) one or more insulator elements, upstream from the expression cassette and / or downstream from the expression cassette. In preferred embodiments, the polynucleotide comprises: (a) one or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette and / or downstream from the transcription termination site; (b) one or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette and / or downstream from the expression cassette; and (c) one or more insulator elements, upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the polynucleotide comprises: (a) (i) one or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette; and (ii) optionally, one or more miRNA target sequences having the same orientation as the expression cassette, downstream from the transcription termination site; and (b) (i) one or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette; and (ii) one or more miRNA target sequences having the opposite orientation as the expression cassette, downstream from the expression cassette. In some embodiments, the polynucleotide comprises: (a) (i) one or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette; and (ii) optionally, one or more miRNA target sequences having the same orientation as the expression cassette, downstream from the transcription termination site; and (c) (i) one or more insulator elements upstream from the expression cassette; and (ii) one or more insulator elements downstream from the expression cassette. In some embodiments, the polynucleotide comprises: (b) (i) one or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette; and (ii) one or more miRNA target sequences having the opposite orientation as the expression cassette, downstream from the expression cassette; and (c) (i) one or more insulator elements upstream from the expression cassette; and (ii) one or more insulator elements downstream from the expression cassette. In some embodiments, the polynucleotide comprises: (a) (i) one or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette; and (ii) optionally, one or more miRNA target sequences having the same orientation as the expression cassette, downstream from the transcription termination site; (b) (i) one or more miRNA target sequences having the opposite orientation as the expression cassette, upstream from the expression cassette; and (ii) one or more miRNA target sequences having the opposite orientation as the expression cassette, downstream from the expression cassette; and (c) (i) one or more insulator elements upstream from the expression cassette; and (ii) one or more insulator elements downstream from the expression cassette. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: one or more miRNA target sequences having the same orientation as the expression cassette and / or one or more miRNA target sequences having the opposite orientation as the expression cassette; one or more insulator elements; an expression cassette; one or more insulator elements; and one or more miRNA target sequences having the opposite orientation as the expression cassette. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: one or more insulator elements; one or more miRNA target sequences having the same orientation as the expression cassette and / or one or more miRNA target sequences having the opposite orientation as the expression cassette; an expression cassette; one or more miRNA target sequences having the same orientation as the expression cassette; and one or more insulator elements. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: one or more insulator elements; one or more miRNA target sequences having the same orientation as the expression cassette and / or one or more miRNA target sequences having the opposite orientation as the expression cassette; an expression cassette; one or more insulator elements; and one or more miRNA target sequences having the opposite orientation as the expression cassette. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: one or more miRNA target sequences having the same orientation as the expression cassette and / or one or more miRNA target sequences having the opposite orientation as the expression cassette; one or more insulator elements; an expression cassette; one or more miRNA target sequences having the opposite orientation as the expression cassette; and one or more insulator elements. The one or more insulator elements and one or more miRNA target sequences having the opposite orientation as the expression cassette that are downstream from the expression cassette may be upstream and / or downstream from the transcription termination site. For example, the polynucleotide may comprise: (i) one or more insulator elements and one or more miRNA target sequences having the opposite orientation as the expression cassette downstream from the expression cassette and upstream from the transcription termination site; and / or (ii) one or more insulator elements and one or more miRNA target sequences having the opposite orientation as the expression cassette downstream from the transcription termination site. In some embodiments, the polynucleotide further comprises one or more miRNA target sequences having the same orientation as the expression cassette downstream from the transcription termination site. LTRs In some embodiments, the polynucleotide comprises one or more long terminal repeat (LTR). In some embodiments, the polynucleotide comprises a 5’ LTR and a 3’ LTR. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: a 5’ LTR, an expression cassette, and a 3’ LTR. A “long terminal repeat” may refer to a nucleotide sequence that forms a retrotransposon, an endogenous retrovirus, or a retroviral provirus. All retroviral genomes are flanked by LTRs, while there are some retrotransposons without LTRs. A lentivirus genome is typically flanked at both ends by LTRs. The LTRs are responsible for integration and transcription. LTRs may also serve as enhancer-promoter sequences and may contain signals for transcription termination and polyadenylation. The LTRs themselves are identical or near-identical sequences that can typically be divided into three regions: U3, R and U5. LTRs may be naturally-occurring or may be modified. For example, U3 and U5 modifications are described in Iwakuma, T., et al., 1999. Virology, 261(1), pp.120-132. In some embodiments, the 3’ LTR and the 5’ LTR are identical. Any LTR sequences may be used, the selection of which may be readily made by the skilled person. Suitable LTR sequences will be well known to those of skill in the art (see e.g. Frech, K., et al., 1996. Virology, 224(1), pp.256-267). An example LTR sequence is provided as SEQ ID NO: 91. In some embodiments, a LTR comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 91 or a fragment thereof. Suitably, a LTR comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 91 or a fragment thereof. In some embodiments, a LTR comprises or consists of the nucleotide sequence SEQ ID NO: 91 or a fragment thereof. TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTC TGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCC TCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTA ACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG Example LTR (SEQ ID NO: 91) In some embodiments, the polynucleotide comprises one or more self-inactivating long terminal repeat (SIN-LTR). A “SIN-LTR” may comprise a deletion that abolishes transcription of the full-length virus after it has incorporated into a host cell. For example, a 3’ SIN-LTR may comprise a deletion in the U3 region removing the promoter / enhancer elements (see e.g. Zufferey, R., et al., 1998. Journal of virology, 72(12), pp.9873-9880). This deletion is copied into the 5’ LTR after reverse transcription, thereby making the gene expression in target cells dependent on an internal promoter of choice. Any SIN-LTR sequences may be used, the selection of which may be readily made by the skilled person. Suitable SIN-LTR sequences will be well known to those of skill in the art (see e.g. Zufferey, R., et al., 1998. Journal of virology, 72(12), pp.9873-9880 and Miyoshi, H., et al., 1998. Journal of virology, 72(10), pp.8150-8157). 3’ LTR In some embodiments, the polynucleotide comprises a 3’ LTR. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: an expression cassette, and a 3’ LTR. The expression cassette and the 3’ LTR may overlap (see e.g. Guntaka, R.V., 1993. Microbiological reviews, 57(3), pp.511-521). The 3’ LTR may comprise part of the 3’ regulatory region, e.g. comprising regulatory sequences, such as a polyadenylations sequence. The 3’ LTR may comprise a transcription termination site. In some embodiments, it is preferred for the one or more miRNA target sequences and / or the one or more insulator elements to be present in the 3’ LTR, because the 3’ LTR may be copied to the 5’ LTR during reverse transcription, e.g. thereby providing multiple copies of the one or more miRNA target sequences and / or a pair of insulator element flanking the expression cassette. In some embodiments, the 3’ LTR is a SIN-LTR. In some embodiments, the 3’ LTR is any described in the section below entitled “SIN 3’ LTRs”. In some embodiments, the 3’ LTR comprises one or more miRNA target sequences having the same orientation as the expression cassette. In some embodiments, the 3’ LTR does not comprise a miRNA target sequence having the same orientation as the expression cassette. This is preferred when the expression cassette is sense-oriented. In some embodiments, the 3’ LTR comprises one or more miRNA target sequences having the opposite orientation as the expression cassette. In some embodiments, the 3’ LTR does not comprise a miRNA target sequence having the opposite orientation as the expression cassette. In some embodiments, the 3’ LTR comprises one or more miRNA target sequences having the same orientation as the expression cassette and one or more miRNA target sequences having the opposite orientation as the expression cassette. In some embodiments, the 3’ LTR comprises one or more insulator elements. In some embodiments, the 3’ LTR does not comprise an insulator element. In some embodiments, the 3’ LTR comprises one or more miRNA target sequences and one or more insulator elements. In some embodiments, the 3’ LTR comprises one or more miRNA target sequences having the same orientation as the expression cassette and one or more insulator elements. In some embodiments, the 3’ LTR does not comprise one or more miRNA target sequences having the opposite orientation as the expression cassette. In some embodiments, the 3’ LTR comprises one or more miRNA target sequences having the opposite orientation as the expression cassette and one or more insulator elements. In some embodiments, the 3’ LTR does not comprise one or more miRNA target sequences having the same orientation as the expression cassette. In some embodiments, the 3’ LTR comprises one or more miRNA target sequences having the same orientation as the expression cassette, one or more miRNA target sequences having the opposite orientation as the expression cassette, and one or more insulator elements. 5’ LTR In some embodiments, the polynucleotide comprises a 5’ LTR. In some embodiments, the polynucleotide comprises from 5’ to 3’ direction: a 5’ LTR, and an expression cassette. In some embodiments, the one or more miRNA target sequences and / or the one or more insulator elements are not present in the 5’ LTR, since a 3’ LTR comprising said sequences may be copied to the 5’ LTR during reverse transcription. In some embodiments, the 5’ LTR is a SIN-LTR. An example 5’LTR sequence is provided as SEQ ID NO: 92. In some embodiments, a 5’LTR comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 92 or a fragment thereof. Suitably, a 5’LTR comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 92 or a fragment thereof. In some embodiments, a 5’LTR comprises or consists of the nucleotide sequence SEQ ID NO: 92 or a fragment thereof. GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT GCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG Example 5’LTR (SEQ ID NO: 92) In some embodiments, the 5 LTR comprises one or more miRNA target sequences having the same orientation as the expression cassette. In some embodiments, the 5’ LTR does not comprise a miRNA target sequence having the same orientation as the expression cassette. In some embodiments, the 5’ LTR comprises one or more miRNA target sequences having the opposite orientation as the expression cassette. In some embodiments, the 5’ LTR does not comprise a miRNA target sequence having the opposite orientation as the expression cassette. In some embodiments, the 5’ LTR comprises one or more miRNA target sequences having the same orientation as the expression cassette and one or more miRNA target sequences having the opposite orientation as the expression cassette. In some embodiments, the 5’ LTR comprises one or more insulator elements. In some embodiments, the 5’ LTR does not comprise an insulator element. In some embodiments, the 5’ LTR comprises one or more miRNA target sequences and one or more insulator elements. In some embodiments, the 5’ LTR comprises one or more miRNA target sequences having the same orientation as the expression cassette and one or more insulator elements. In some embodiments, the 5’ LTR does not comprise a miRNA target sequence having the opposite orientation as the expression cassette. In some embodiments, the 5’ LTR comprises one or more miRNA target sequences having the opposite orientation as the expression cassette and one or more insulator elements. In some embodiments, the 5’ LTR does not comprise a miRNA target sequence having the same orientation as the expression cassette. In some embodiments, the 5’ LTR comprises one or more miRNA target sequences having the same orientation as the expression cassette, one or more miRNA target sequences having the opposite orientation as the expression cassette, and one or more insulator elements. Vectors In one aspect, the present invention provides a vector comprising the polynucleotide of the invention. A “vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the invention, and by way of example, some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g. a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. The vector may serve the purpose of maintaining the heterologous nucleic acid (DNA or RNA) within the cell, facilitating the replication of the vector comprising a segment of nucleic acid, or facilitating the expression of the protein encoded by a segment of nucleic acid. Examples of vectors used in recombinant nucleic acid techniques include, but are not limited to, plasmids, chromosomes, artificial chromosomes, transposons and viruses. The vector may be single stranded or double stranded. The vector may comprise or consist of DNA and / or RNA. In some embodiments, the vector is a DNA vector. In some embodiments, the vector is a RNA vector. The vector may be linear or circular. The vector may also be, for example, a naked nucleic acid (e.g. DNA). In its simplest form, the vector may itself be a nucleotide of interest. The term “vector" includes an expression vector i.e. a construct capable of in vivo, in vitro, or ex vivo expression. Expression may be controlled by a vector sequence, or, for example in the case of insertion at a target site, expression may be controlled by a target sequence. A vector may be integrated or tethered to the cell’s DNA. In some embodiments, the vector is a plasmid, a transposon or a viral vector. In some embodiments, the vector is an integrating vector. Integrating vectors, such as lentivirus and gammaretrovirus, fuse a fragment of their genetic material into the host cell genome. In some embodiments, the vector is an integrating viral vector. Viral vectors In some embodiments, the vector is a viral vector. The vector of the present invention may be in the form of a viral vector particle. Viral vectors include but are not limited to a lentiviral vector, a retroviral vector, an adenovirus vector, an adeno-associated viral (AAV) vector, a herpes simplex viral vector, a baculoviral vector, an alphaviral vector, a flaviviral vector, a rhabdoviral vector, a measles viral vector, a Newcastle disease viral vector, a poxviral vector, and a picornaviral vector (see e.g. Lundstrom, K., 2018. Diseases, 6(2), p.42). Methods of preparing and modifying viral vectors and viral vector particles, such as those derived from AAV, are well known in the art. Suitable methods are described in Ayuso, E., et al., 2010. Current gene therapy, 10(6), pp.423-436, Merten, O.W., et al., 2016. Molecular Therapy-Methods & Clinical Development, 3, p.16017; and Nadeau, I. and Kamen, A., 2003. Biotechnology advances, 20(7-8), pp.475-489. In some embodiments, the viral vector has a minimal viral genome. By “minimal viral genome” it is to be understood that the viral vector has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell. The viral vector genome may have a defined orientation. For example, a retroviral genome may be oriented from the 5’ LTR to the 3’ LTR. In the context of a viral vector, a “sense- oriented” expression cassette may have the same orientation as the viral vector genome. For example, following conversion of a lentiviral vector into double-stranded DNA, the expression cassette may be transcribed in the direction of from the 5’ LTR to the 3’ LTR. In the context of a viral vector, an “antisense-oriented” expression cassette may have the opposite orientation to the viral vector genome. For example, following conversion of a lentiviral vector into double-stranded DNA, the expression cassette may be transcribed in the direction of from the 3’ LTR to the 5’ LTR. Retroviral vectors In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is a retroviral vector particle. Retroviruses are RNA viruses with a life cycle different to that of lytic viruses. In this regard, a retrovirus is an infectious entity that replicates through a DNA intermediate. When a retrovirus infects a cell, its genome is converted to a DNA form by a reverse transcriptase enzyme. The DNA copy serves as a template for the production of new RNA genomes and virally encoded proteins necessary for the assembly of infectious viral particles. All retroviruses are characterized by the ability to synthesize, starting from the genomic RNA template, a double-stranded proviral DNA which stably integrates into the host genome. The genomic RNA molecules display at both ends two identical sequences called Repeats, ordinarily known as R regions, while unique U5 and U3 sequences are present specifically at the 5’ and 3’ end, correspondingly. During reverse-transcription of the RNA genome in double-stranded proviral DNA, the U5 and U3 sequences are duplicated at both ends of the vector sequence generating the Long Terminal Repeats (LTRs). A retroviral vector may be derived from or may be derivable from any suitable retrovirus. A large number of different retroviruses have been identified. Examples include human immunodeficiency virus (HIV), murine leukaemia virus (MLV), human T-cell leukaemia virus (HTLV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukaemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29) and avian erythroblastosis virus (AEV). In some embodiments, the vector is a gammaretrovirus. In some embodiments, the vector is a gammaretrovirus vector particle. Gammaretrovirus is a genus in the Retroviridae family. Example species are Moloney murine leukemia virus (Mo-MLV), xenotropic MuLB-related virus, feline leukemia virus (FLV), and feline sarcoma virus (FSV). The double stranded DNA is highly stable and easily integrated into a host genome (see e.g. Maetzig, T., et al., 2011. Viruses, 3(6), pp.677-713). In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is a lentiviral vector particle. Lentiviruses belong to the retrovirus family, but they can infect both dividing and non-dividing cells. The lentiviral vector may be any described below in the section entitled “Retroviral Vectors”. In some embodiments, the vector is an alpharetroviral vector. The most widely used alpharetroviral vector system is the RCAS (replication-competent avian leukosis virus LTR with a splice acceptor) system, which is based on Rous sarcoma virus (RSV) (see e.g. Suerth, J.D., et al., A., 2014. Viruses, 6(12), pp.4811-4838). The basic structure of retrovirus genomes share many common features such as a 5’ LTR and a 3’ LTR. Between or within these are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome, and gag, pol and env genes encoding the packaging components. In a defective retroviral vector genome gag, pol and env may be absent or not functional. In a typical retroviral vector, at least part of one or more protein coding regions essential for replication may be removed from the virus. This makes the viral vector replication-defective. Portions of the viral genome may also be replaced by a library encoding candidate modulating moieties operably linked to a regulatory control region and a reporter moiety in the vector genome in order to generate a vector comprising candidate modulating moieties which is capable of transducing a target host cell and / or integrating its genome into a host genome. Adeno-associated viral (AAV) vectors In some embodiments, the vector is an AAV vector. In some embodiments, the vector is an AAV vector particle. AAVs occurring in nature may be classified according to various biological systems. The AAV may any naturally derived serotype, isolate or clade of AAV. AAV may be referred to in terms of their serotype. A serotype corresponds to a variant subspecies of AAV which, owing to its profile of expression of capsid surface antigens, has a distinctive reactivity which can be used to distinguish it from other variant subspecies. Typically, an AAV vector particle having a particular AAV serotype does not efficiently cross- react with neutralising antibodies specific for any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11, and derivatives thereof. Clades or clones refers to the phylogenetic relationship of naturally derived AAVs, and typically to a phylogenetic group of AAVs which can be traced back to a common ancestor, and includes all descendants thereof. Additionally, AAVs may be referred to in terms of a specific isolate, i.e. a genetic isolate of a specific AAV found in nature. The term genetic isolate describes a population of AAVs which has undergone limited genetic mixing with other naturally occurring AAVs, thereby defining a recognisably distinct population at a genetic level. Typically, the AAV genome of a naturally derived serotype, isolate or clade of AAV comprises at least one inverted terminal repeat sequence (ITR). ITRs may be the only sequences required in cis next to the therapeutic gene. The AAV genome may also comprise packaging genes, such as rep and / or cap genes which encode packaging functions for an AAV particle. A promoter may be operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19 and p40 promoters. For example, the p5 and p19 promoters are generally used to express the rep gene, while the p40 promoter is generally used to express the cap gene. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof. The cap gene encodes one or more capsid proteins such as VP1, VP2 and VP3 or variants thereof. These proteins make up the capsid of an AAV particle, which determines the AAV serotype. VP1, VP2, and VP3 may be produced by alternate mRNA splicing (Trempe, J.P. and Carter, B.J., 1988. Journal of virology, 62(9), pp.3356-3363). Thus, VP1, VP2 and VP3 may have identical sequences, but wherein VP2 is truncated at the N-terminus relative to VP1, and VP3 is truncated at the N-terminus relative to VP2. Preferably, the AAV genome is derivatised for the purpose of administration to patients. Such derivatisation is standard in the art and the invention encompasses the use of any known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art. The AAV genome may be a derivative of any naturally occurring AAV. Suitably, the AAV genome is a derivative of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of a transgene from an AAV vector of the invention in vivo. Typically, it is possible to truncate the AAV genome significantly to include minimal viral sequence yet retain the above function. This is preferred for safety reasons to reduce the risk of recombination of the vector with wild-type virus, and also to avoid triggering a cellular immune response by the presence of viral gene proteins in the target cell. Typically, a derivative will include at least one inverted terminal repeat sequence (ITR), preferably more than one ITR, such as two ITRs or more. One or more of the ITRs may be derived from AAV genomes having different serotypes, or may be a chimeric or mutant ITR. A preferred mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows for continued replication of the genome to generate a single-stranded genome which contains both coding and complementary sequences, i.e. a self- complementary AAV (scAAV) genome. This allows for bypass of DNA replication in the target cell, and so enables accelerated transgene expression. The AAV genome may comprise one or more ITR sequences from any naturally derived serotype, isolate or clade of AAV or a variant thereof. The AAV genome may comprise at least one, such as two, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 ITRs, or variants thereof. The inclusion of one or more ITRs is preferred to aid concatamer formation of the AAV vector in the nucleus of a host cell, for example following the conversion of single-stranded vector DNA into double-stranded DNA by the action of host cell DNA polymerases. The formation of such episomal concatamers protects the AAV vector during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo. Suitably, ITR elements will be the only sequences retained from the native AAV genome in the derivative. A derivative will preferably not include the rep and / or cap genes of the native genome and any other sequences of the native genome. This is preferred for the reasons described above, and also to reduce the possibility of integration of the vector into the host cell genome. Additionally, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) within the vector in addition to the protein-coding sequence. The AAV vector particle may be encapsidated by capsid proteins. Suitably, the AAV vector particles may be transcapsidated forms wherein an AAV genome or derivative having an ITR of one serotype is packaged in the capsid of a different serotype. The AAV vector particle also includes mosaic forms wherein a mixture of unmodified capsid proteins from two or more different serotypes makes up the viral capsid. The AAV vector particle also includes chemically modified forms bearing ligands adsorbed to the capsid surface. For example, such ligands may include antibodies for targeting a particular cell surface receptor. Where a derivative comprises capsid proteins i.e. VP1, VP2 and / or VP3, the derivative may be a chimeric, shuffled or capsid-modified derivative of one or more naturally occurring AAVs. In particular, capsid protein sequences from different serotypes, clades, clones, or isolates of AAV may be used within the same vector (i.e. a pseudotyped vector). The AAV vector may be in the form of a pseudotyped AAV vector particle. Adenoviral vectors In some embodiments, the vector is an adenoviral vector. In some embodiments, the vector is an adenoviral vector particle. The adenovirus is a double-stranded, linear DNA virus that does not go through an RNA intermediate. There are over 50 different human serotypes of adenovirus divided into 6 subgroups based on the genetic sequence homology. The natural targets of adenovirus are the respiratory and gastrointestinal epithelia, generally giving rise to only mild symptoms. Serotypes 2 and 5 (with 95% sequence homology) are most commonly used in adenoviral vector systems and are normally associated with upper respiratory tract infections in the young. Adenoviruses have been used as vectors for gene therapy and for expression of heterologous genes. The large (36 kb) genome can accommodate up to 8 kb of foreign insert DNA and is able to replicate efficiently in complementing cell lines to produce very high titres of up to 1012. Adenovirus is thus one of the best systems to study the expression of genes in primary non-replicative cells. The expression of viral or foreign genes from the adenovirus genome does not require a replicating cell. Adenoviral vectors enter cells by receptor mediated endocytosis. Once inside the cell, adenovirus vectors rarely integrate into the host chromosome. Instead, they function episomally (independently from the host genome) as a linear genome in the host nucleus. Hence, the use of recombinant adenovirus alleviates the problems associated with random integration into the host genome. Other viral vectors Other viral vectors include those described in Lundstrom, K., 2018. Diseases, 6(2), p.42. In some embodiments, the vector is a herpes simplex vector. In some embodiments, the vector is a herpes simplex vector particle. Herpes simplex virus (HSV) is a neurotropic DNA virus with favorable properties as a gene delivery vector. HSV is highly infectious, so HSV vectors are efficient vehicles for the delivery of exogenous genetic material to cells. Viral replication is readily disrupted by null mutations in immediate early genes that in vitro can be complemented in trans enabling straightforward production of high-titre pure preparations of non-pathogenic vector. The genome is large (152 Kb) and many of the viral genes are dispensable for replication in vitro, allowing their replacement with large or multiple transgenes. Latent infection with wild-type virus results in episomal viral persistence in sensory neuronal nuclei for the duration of the host lifetime. The vectors are non-pathogenic, unable to reactivate and persist long-term. The latency active promoter complex can be exploited in vector design to achieve long-term stable transgene expression in the nervous system. HSV vectors transduce a broad range of tissues because of the wide expression pattern of the cellular receptors recognized by the virus. Increasing understanding of the processes involved in cellular entry has allowed targeting the tropism of HSV vectors. In some embodiments, the vector is baculoviral vector. In some embodiments, the vector is a baculoviral vector particle. Baculoviruses have been extensively studied as potential vectors for both in vitro and in vivo gene therapy. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is one of the most well-studied baculoviruses. Given the highly efficient gene delivery into many cell types, baculoviruses have captured increasing interest as vectors for in vivo gene delivery. In comparison with other viral vectors, baculoviruses possess a number of advantages, including a large cloning capacity (see e.g. Hu, Y.C., 2006. Advances in virus research, 68, pp.287-320). In some embodiments, the vector is an alphaviral vector. In some embodiments, the vector is an alphaviral vector particle. In some embodiments, the vector is a flaviviral vector. In some embodiments, the vector is a flaviviral vector particle. Self-amplifying ssRNA viruses comprise of alphaviruses (e.g. Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis virus, and M1) and flaviviruses (e.g. Kunjin virus, West Nile virus, and Dengue virus) possessing a genome of positive polarity. Alphaviruses have been mainly applied in preclinical gene therapy studies for cancer treatment. Alphavirus vectors can be delivered in the form of naked RNA, layered plasmid DNA vectors and recombinant replication-deficient or -proficient particles. In some embodiments, the vector is a rhabdoviral vector. In some embodiments, the vector is a rhabdoviral vector particle. In some embodiments, the vector is a measles viral vector. In some embodiments, the vector is a measles viral vector particle. Rhabdoviruses (e.g. rabies and vesicular stomatitis virus) and measles viruses carry negative strand genomes. Among rhabdoviruses, recombinant vesicular stomatitis virus (VSV) has been applied for preclinical gene therapy studies. Measles viruses (e.g. MV-Edm) have found a number of gene therapy applications. In some embodiments, the vector is a Newcastle disease viral vector. In some embodiments, the vector is a Newcastle disease viral vector particle. The ssRNA paramyxovirus Newcastle disease virus (NDV) replicates specifically in tumour cells and has therefore been frequently applied for cancer gene therapy. In some embodiments, the vector is a poxviral vector. In some embodiments, the vector is a poxviral vector particle. The characteristic feature of poxviruses is their dsDNA genome, which can generously accommodate more than 30 kb of foreign DNA. Poxviruses have found several applications as gene therapy vectors. For instance, vaccinia virus vectors have demonstrated potential for treatment of cancer. Vaccinia virus is large enveloped poxvirus that has an approximately 190 kb linear, double-stranded DNA genome. Vaccinia virus can accommodate up to approximately 25 kb of foreign DNA, which also makes it useful for the delivery of large genes. A number of attenuated vaccinia virus strains are known in the art that are suitable for gene therapy applications, for example the MVA and NYVAC strains. In some embodiments, the vector is a picornaviral vector. In some embodiments, the vector is a picornaviral vector particle. Picornaviruses are non-enveloped ssRNA viruses. Coxsackieviruses belonging to Picornaviridae, have been applied as oncolytic vectors. Transposons In some embodiments, the vector is a transposon vector. In some embodiments, a transposon system is provided comprising a transposon vector and a vector encoding a transposase. Transposon vectors are integrating non-viral vectors based on mobile DNA elements that can stably integrate a therapeutic gene in the target cell genome (see e.g. Tipanee, J., et al., 2017. Human gene therapy, 28(11), pp.1087-1104). A vector comprising transposable elements and a vector comprising the corresponding transposase may be provided separately to provide a transposition system. DNA transposons translocate via a “non-replicative mechanism,” where two Terminal Inverted Repeats (TIRs) are recognized and cleaved by a transposase enzyme, releasing the cognate DNA transposons with free DNA ends. The excised DNA transposons can then integrate into a new genomic region where target sites are recognized and cut by the same transposase. This cut-and-paste mechanism usually duplicates DNA target sites upon insertion, leaving target site duplications (TSDs) (see e.g. Tipanee, J., et al., 2017. Human gene therapy, 28(11), pp.1087-1104). DNA transposons can be classified into diverse families, which differ in terms of target site recognition, TSDs, TIRs, and transposon DNA sequences. Some of these DNA transposon subgroups are employed for human gene therapy to achieve sustained gene expression of the therapeutic transgene (see e.g. Di Matteo, M., et al., 2012. Expert opinion on biological therapy, 12(7), pp.841-858). The most commonly used transposon systems in gene therapy applications are the Sleeping Beauty (SB) and PiggyBac transposon systems. In some embodiments, the vector is a Sleeping Beauty transposon vector. Sleeping Beauty (SB) is a member of the Tc1 / mariner superfamily of transposable elements. A SB transposon vector typically comprises two inverted repeat / direct repeats (IR / DRs) flanking an expression cassette (see e.g. Izsvák, Z., et al., 2000. Journal of molecular biology, 302(1), pp.93-102). A SB transposon system may further comprise a vector encoding a SB transposase, for example a SB3, SB10, SB11, or SB100X transposase. In some embodiments, the vector is a PiggyBac (PB) transposon vector. A PB transposon vector typically comprises an expression cassette flanked by a 311 bp 5′ end and 235 bp 3′ end, each containing TIR sequences. A PB transposon system may further comprise vector encoding PB transposase, for example a mPB or hyPB transposase. The PB transposase recognizes the two TIRs of the PB transposon and excises the DNA, leaving the free 3′-OH group, which forms the hairpin structure with 5′-TTAA overhangs of the transposon. The hairpin transposome complex is then released from the genome and binds to the site of integration in which the TTAA consensus sequence is present. PB elements have been used to transform the germline of more than a dozen species (see e.g. Ding, S., et al., 2005. Cell, 122(3), pp.473-483). In some embodiments, the vector is a Tol2 transposon vector. The Tol2 transposable element belongs to the hAT (hobo / Ac / Tam3) superfamily and is composed of a gene encoding the transposase flanked by 12 bp TIRs (see e.g. Kwan, K.M., et al., 2007. Developmental dynamics, 236(11), pp.3088-3099). A Tol2 transposon vector typically comprises two TIRs flanking an expression cassette. A Tol2 transposon system may further comprise a vector encoding a Tol2 transposase. Retroviral vectors In one aspect, the present invention provides a retroviral vector comprising an expression cassette and a transcription termination site, wherein the polynucleotide further comprises: (a) one or more miRNA target sequences having the same orientation as the expression cassette, optionally upstream from the expression cassette and / or downstream from the transcription termination site, preferably upstream from the expression cassette; (b) one or more miRNA target sequences having the opposite orientation as the expression cassette, optionally upstream from the expression cassette and / or downstream from the expression cassette; and / or (c) one or more insulator elements, optionally upstream from the expression cassette and / or downstream from the expression cassette. A “retroviral vector” may comprise a retroviral genome, optionally wherein the retroviral genome is enveloped. As used herein, a “retroviral genome” may refer to a genome that comprises at least one element derived or derivable from a retrovirus genome. Suitably, a retroviral genome comprises at least one element that is involved in the mechanisms by which a retrovirus infects cells, expresses genes, and / or is replicated. The retroviral vector may be a retroviral particle. A “retroviral particle” may refer to an enveloped retroviral genome. Retroviral particles may be generated by co-transfection of a plasmid containing a retroviral genome (e.g. a “transfer vector”) with helper plasmids (e.g. “packaging vectors” and “envelope vectors”) into host cells and harvesting of the retrovirus - containing supernatant afterwards. In preferred embodiments, the retroviral vector is a lentiviral vector. A “lentiviral vector” may comprise a lentiviral genome, optionally wherein the lentiviral genome is enveloped. As used herein, a “lentiviral genome” may refer to a genome that comprises at least one element derived or derivable from a lentivirus genome. Suitably, a lentiviral genome comprises at least one element that is involved in the mechanisms by which a lentivirus infects cells, expresses genes, and / or is replicated. Lentivirus is a genus of retroviruses, which contain an RNA genome that is converted to DNA in the transduced cell by a virally encoded reverse transcriptase. Lentiviral vectors can transduce a wide range of cell types and integrate into the host genome in both dividing and post-mitotic cells, resulting in long-term expression of the protein-coding sequence both in vitro and in vivo (see e.g. Tiscornia, G., et al., 2006. Nature protocols, 1(1), pp.241-245). The basic genes required for lentivirus survival and function are the gag, pol, and env genes: gag encodes structural proteins; pol encodes enzymes required for reverse transcription and integration into the host cell genome; and env encodes the viral envelope glycoprotein (see e.g. Milone, M.C. and O’Doherty, U., 2018. Leukemia, 32(7), pp.1529-1541). Lentiviruses may also have additional cis-acting elements, such as a rev response element (RRE), which enables the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell; a retroviral psi packaging element, which is involved in regulating the essential process of packaging the retroviral RNA genome into the viral capsid during replication; a primer binding site (PBS), where reverse transcription is initiated; the TAT activation region (TAR); splice donor and acceptor sites; and central and terminal polypurine tracts, which allow initiation of plus-strand synthesis. In a lentivirus genome, these elements are typically flanked at both ends by long terminal repeats (LTRs). The LTRs are responsible for integration and transcription. A lentiviral vector may comprise a minimal lentiviral genome. As used herein, a “minimal lentiviral genome” may mean that the lentiviral genome has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell (see e.g. Kim, V.N., et al., 1998. Journal of virology, 72(1), pp.811-816 and Sertkaya, H., et al., 2021. Scientific reports, 11(1), pp.1-15). A lentiviral genome may comprise from 5’ to 3’ direction: a 5’ LTR, one or more lentiviral- derived cis-acting elements, and a 3’ LTR. Suitably, a lentiviral genome may comprise from 5’ to 3’ direction: a 5’ LTR, a RRE, and a 3’ LTR. Suitably, a lentiviral genome may comprise from 5’ to 3’ direction: a 5’ LTR, a retroviral psi packaging element, a RRE, and a 3’ LTR. Suitably, a lentiviral genome may comprise from 5’ to 3’ direction: a 5’ LTR, a retroviral psi packaging element, a RRE, a cPPT, and a 3’ LTR. Suitably, a lentiviral genome may comprise from 5’ to 3’ direction: a 5’ LTR, a PBS, a retroviral psi packaging element, a RRE, a cPPT, and a 3’ LTR. Suitably, a lentiviral genome may comprise from 5’ to 3’ direction: a 5’ LTR, a RRE, an expression cassette, and a 3’ LTR. Suitably, a lentiviral genome may comprise from 5’ to 3’ direction: a 5’ LTR, a retroviral psi packaging element, a RRE, an expression cassette, and a 3’ LTR. Suitably, a lentiviral genome may comprise from 5’ to 3’ direction: a 5’ LTR, a retroviral psi packaging element, a RRE, a cPPT, an expression cassette, and a 3’ LTR. Suitably, lentiviral genome may comprise from 5’ to 3’ direction: a 5’ LTR, a PBS, a retroviral psi packaging element, a RRE, a cPPT, an expression cassette, and a 3’ LTR. In some embodiments, the lentiviral vector is replication-defective. Typically, at least part of one or more protein coding regions essential for replication may be removed from the lentiviral genome. This makes the lentiviral vector “replication-defective” or “replication- incompetent”. Suitably, one or more of gag, pol, rev, and env genes are deleted (at least partially) in a replication-defective lentiviral vector. Suitably, each of the gag, pol, rev, and env genes are deleted (at least partially) in a replication-defective lentiviral vector. Optionally, the lentiviral vector lacks a functional gag-pol and / or env gene and / or other genes essential for replication. A lentiviral vector may be derived from any lentivirus. As used herein “lentivirus-derived” or “lentivirus-based” may mean that the lentiviral genome comprises one or more elements from said lentivirus. For example, the coding regions of viral proteins may be deleted, but one or more cis-acting element may be retained from said lentivirus. A lentiviral vector may be derived from a primate lentivirus. Examples of “primate” lentiviruses include, but are not limited to, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). A lentiviral vector may be derived from a non-primate lentivirus (i.e. derived from a lentivirus which does not primarily infect primates, especially humans). Examples of “non-primate” lentiviruses include, but are not limited to, the prototype “slow virus” visna / maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV). Suitably, the lentiviral vector is a HIV-derived lentiviral vector. As used herein “HIV-derived” or “HIV-based” may mean that the lentiviral genome comprises one or more element from HIV. For example, the coding regions of HIV viral proteins may be deleted, and one or more HIV cis-acting element may retained in the lentiviral genome (see e.g. Johnson, N.M., et al., 2021. Molecular Therapy-Methods & Clinical Development, 21, pp.451-465). A HIV-derived lentiviral genome may comprise from 5’ to 3’ direction: a 5’ LTR, one or more HIV-derived cis-acting elements (e.g. RRE and / or cPPT), and a 3’ LTR. Suitably, the lentiviral vector is a HIV-1-derived lentiviral vector. The prototype lentiviral vector system is based on HIV-1 (see e.g. Merten, O.W., et al., 2016. Molecular Therapy- Methods & Clinical Development, 3, p.16017). It has been shown that sequences that extend into the gag open reading frame may be important for packaging of HIV-1. Therefore, HIV-1 vectors often contain the relevant portion of gag in which the translational initiation codon has been mutated. In addition, HIV-1 vectors often also contain a portion of the env gene that includes the RRE. Rev binds to RRE, which permits the transport of full-length or singly spliced mRNAs from the nucleus to the cytoplasm. In the absence of rev and / or a RRE, full- length HIV-1 RNAs may accumulate in the nucleus. Alternatively, a constitutive transport element from certain simple retroviruses such as Mason-Pfizer monkey virus can be used to relieve the requirement for rev and a RRE. A HIV-1-derived lentiviral genome may comprise from 5’ to 3’ direction: a 5’ LTR, one or more HIV-1-derived cis-acting elements (e.g. a PBS, a retroviral psi packaging element, a RRE and / or a cPPT), and a 3’ LTR. The lentiviral vector may be a self-inactivating lentiviral vector. As used herein, “self- inactivating” or “SIN” lentiviral vectors may comprise lentiviral genomes in which the lentiviral enhancer and promoter sequences have been deleted (see e.g. Zufferey, R., et al., 1998. Journal of virology, 72(12), pp.9873-9880 and Miyoshi, H., et al., 1998. Journal of virology, 72(10), pp.8150-8157). SIN lentiviral vectors can be generated and transduce non-dividing cells in vivo with an efficacy similar to that of wild-type vectors. The transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus can prevent mobilisation by replication-competent virus. This can also enable the regulated expression of genes from internal promoters by eliminating any cis-acting effects of the LTR. In preferred embodiments, the 3’ LTR is a self-inactivating (SIN) 3’ LTR. In preferred embodiments, the lentiviral vector is integration proficient. As used herein, an “integration proficient” lentiviral vector is capable of integrating into the genome of a host cell. In some embodiments, the lentiviral vector is an integrase-proficient lentiviral vector. The one or more miRNA target sequences and / or the one or more insulator elements may reduce the genotoxicity of an integration proficient lentiviral vector. In other embodiments, the lentiviral vector is integration defective. Integration defective lentiviral vectors can be produced, for example, either by packaging the lentiviral vector with catalytically inactive integrase (such as an HIV integrase bearing the D64V mutation in the catalytic site) or by modifying or deleting essential att sequences from the lentiviral genome LTR, or by a combination of the above (see e.g. Wanisch, K. and Yáñez-Muñoz, R.J., 2009. Molecular Therapy, 17(8), pp.1316-1332). In some embodiments, the lentiviral vector is integrase-defective (i.e. integrase-deficient). The lentiviral vector may be a lentiviral particle. A “lentiviral particle” may refer to an enveloped lentiviral genome. Lentiviral particles may be generated by co-transfection of a plasmid containing a lentiviral genome (e.g. a “transfer vector”) with helper plasmids (e.g. “packaging vectors” encoding gag-pol and / or rev and “envelope vectors” encoding env) into host cells and harvesting of the lentivirus-containing supernatant afterwards. The lentiviral vector of may be pseudotyped. Pseudotyping lentiviral vectors with naturally occurring or engineered lentiviral envelopes can allow targeted transduction of specific cell types (see e.g. Joglekar, A.V. and Sandoval, S., 2017. Human Gene Therapy Methods, 28(6), pp.291-301). For example, the lentiviral vector may be VSV-G pseudotyped. VSV-G is a trimeric protein that binds phosphatidylserine and low-density lipoprotein receptors on a cell surface to endocytose into the cell. In some embodiments, the retroviral vector of the present invention does not comprise an miRNA target sequence upstream of a splice donor site or downstream of a splice acceptor site. Retroviral vector (sense-oriented expression cassette) In one aspect, the present invention provides a retroviral vector comprising from 5’ to 3’ direction: a 5’ LTR; one or more sense-oriented miRNA target sequences; a sense-oriented expression cassette; and a 3’ LTR. In preferred embodiments, the retroviral vector is a lentiviral vector. It is preferred that the sense-oriented miRNA target sequence is upstream from a sense- oriented expression cassette, to prevent degradation of mRNA transcribed from the expression cassette. As described above, in the context of a retroviral vector, a “sense- oriented” expression cassette may have the same orientation as the retroviral viral vector genome. For example, following conversion of the retroviral vector into double-stranded DNA, the expression cassette may be transcribed in the direction of from the 5’ LTR to the 3’ LTR. In some embodiments, the retroviral vector further comprises one or more antisense- oriented miRNA target sequences. In some embodiments, the retroviral vector further comprises one or more antisense-oriented miRNA target sequences upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the retroviral vector comprises two or more antisense-oriented miRNA target sequences upstream from the expression cassette and / or downstream from the expression cassette. The antisense-oriented miRNA target sequences may be in any position relative to the expression cassette, since it will not cause degradation of mRNA transcribed from the expression cassette. In some embodiments, the retroviral vector further comprises one or more insulator elements upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, it is preferred for the one or more antisense-oriented miRNA target sequences and / or the one or more insulator elements to be comprised in the 3’ LTR, because the 3’ LTR may be copied to the 5’ LTR during reverse transcription. In some embodiments, the 3’ LTR comprises one or more antisense-oriented miRNA target sequences. In some embodiments, the 3’ LTR comprises one or more insulator elements. In some embodiments, the 3’ LTR comprises one or more antisense-oriented miRNA target sequences and one or more insulator elements. The miRNA target sequences and the one or more insulator elements may be in any suitable configuration and may be any type described above in the section entitled “Polynucleotides”. Further, example configurations are shown in Figures 6A-C, Figures 7A-C, and Figures 7G-I. Figures 6 and 7 are schematics of the provirus (e.g. after the 3’ LTR has been copied to the 5’ LTR during reverse transcription). The 5’LTR of the retroviral vector may comprise none of the genetic elements present in the provirus (e.g. the 5’LTR of the retroviral vector may comprise only R and U5 regions). In some embodiments, the retroviral vector does not comprise a sense-oriented miRNA target sequence upstream of a splice donor site or downstream of a splice acceptor site. In some embodiments, the retroviral vector does not comprise a sense-oriented miRNA target sequence or an antisense-oriented miRNA target sequence upstream of a splice donor site or downstream of a splice acceptor site. Example convergent-divergent retroviral vectors In one aspect, the present invention provides a retroviral vector comprising from 5’ to 3’ direction: a 5’ LTR; one or more sense-oriented miRNA target sequences; a sense-oriented expression cassette; and a 3’ LTR, wherein the 3’ LTR comprises from 5’ to 3’ direction: a first insulator element, one or more antisense-oriented miRNA target sequences, and a second insulator element. In some embodiments, the first insulator element and the second insulator element are in a divergent orientation. In preferred embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the one or more sense-oriented miRNA target sequences are sense- oriented miR-142 target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences are sense-oriented miR-142-3p target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 1, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the one or more sense-oriented miRNA target sequences comprise two or more sense-oriented miRNA target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences comprises two sense-oriented miRNA target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences comprise two or more sense-oriented miR-142 target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences two or more sense- oriented miR-142-3p target sequences. In some embodiments, the one or more sense- oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 21, or a variant thereof having one to four nucleotide variations. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 21, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the one or more antisense-oriented miRNA target sequences are sense-oriented miR-223 target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences are sense-oriented miR-223-3p target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 12, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise two or more antisense-oriented miRNA target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences comprises two antisense- oriented miRNA target sequences. In some embodiments, the one or more antisense- oriented miRNA target sequences comprise two or more antisense-oriented miR-223 target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences two or more antisense-oriented miR-223-3p target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 22, or a variant thereof having one to four nucleotide variations. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 22, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the first insulator element is an A2 insulator element and / or the second insulator element is an E2 insulator element. In some embodiments, the first insulator element is an A2 insulator element and the second insulator element is an E2 insulator element. In some embodiments, the first insulator element comprises CTCF-binding motif A (e.g. SEQ ID NO: 23) and the second insulator element comprises CTCF-binding motif E (e.g. SEQ ID NO: 27). In some embodiments, the first insulator element and the second insulator element each comprise or consist of a full length insulator sequence. In some embodiments, the first insulator element is a full-length A2 insulator sequence. In some embodiments, the first insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103, or a nucleotide sequence having at least 70% identity thereto. In some embodiments, the first insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103. In some embodiments, the second insulator element is a full-length E2 insulator sequence. In some embodiments, the second insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 55, or a nucleotide sequence having at least 70% identity thereto. In some embodiments, the second insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 55. An example retroviral vector sequence is provided as SEQ ID NO: 93, wherein (X)n encodes an expression cassette. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 93. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 93. In some embodiments, the retroviral vector comprises or consists of the nucleotide sequence SEQ ID NO: 93. In some embodiments, n is from 1000 to 5000, from 1000 to 4000 or from 1000 to 3000. GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT GCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGC GCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCTCTCGACGCAGGACTCGGCTT GCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGAC TAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTA GATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACAT ATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATC AGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAAC TTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAA GACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACA GCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAA TTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAG AAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGG GAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTA TTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCT GTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGAT ACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACT GCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGAC CTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAG AATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGT TTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGT AGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGC AGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCC GAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGG ATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGG AAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACA AAAATTCAAAATTTTATCGATCACGAGACTAGCCTCGAGAAGCTTGTCCATAAAGTAGGAAA CACTACACGATTCCATAAAGTAGGAAACACTACA(X)TGGAAGGGCTAATTCACTCCCAACG AAGACAAAGAGCGAGATTCCGTCTCAAAGAAAAAAAAAGTAATGAAATGAAGCAAATGAGTC CTAGAGCCAGTAAATGTCGTAAATGTCTCAGCTAGTCAGGTAGTAAAAGGTCTCAACTAGGC AGTGGCAGAGCAGGATTCAAATTCAGGGCTGTTGTGATGCCTCCGCAGACTCTGAGCGCCAC CTGGTGGTAATTTGTCTGTGCCTCTTCTGACGTGGAAGAACAGCAACTAACACACTAACACG GCATTTACTATGGGCCAGCCATTGTATGCATTGTCAGTTTGTCAAATACCCCATCGTGTCAG TTTGTCAAATACCCCACCGCGGTTTGATTTCCTTCACTCTGGAATATACTACAGAATGTACT AATATAAATGTGCTTGCCTATGTCTAATCTAGAATATAATTATCCTGAACTCTACATGGTAT TTTAGTCTAGAAAGTACTGTAGCTTCTCTGAGTTCTAAATCAAACCAGGAAACAGCAGAGGG CGCTGGTGCTGTGCTTTTGCAAGTGAGATGTGTGCTCATTTCAAGGGTCCACAAAATTTTCT GGCATGCTGTTTCCATGGAGAGGAAAGTAGGTGTTTTCTTTTTCTTTTTTTTTTTCAGTCAA GCCACCTGCAAACTGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGA GCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTG AGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGAC CCTTTTAGTCAGTGTGGAAAATCTCTAGCAG wherein n is from 1000 to 6000 Example Div-Conv LV (full CI) (SEQ ID NO: 93) In some embodiments, the first insulator element and the second insulator element each consist of a core insulator sequence. In some embodiments, the first insulator element is a core A2 insulator sequence. In some embodiments, the first insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 34, or a nucleotide sequence having at least 80.0% identity thereto. In some embodiments, the first insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the second insulator element is a core E2 insulator sequence. In some embodiments, the second insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 32, or a nucleotide sequence having at least 80.0% identity thereto. In some embodiments, the second insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 42. An example retroviral vector sequence is provided as SEQ ID NO: 94, wherein (X)n encodes an expression cassette. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 94. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 94. In some embodiments, the retroviral vector comprises or consists of the nucleotide sequence SEQ ID NO: 94. In some embodiments, n is from 1000 to 5000, from 1000 to 4000 or from 1000 to 3000. GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT GCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGC GCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCTCTCGACGCAGGACTCGGCTT GCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGAC TAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTA GATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACAT ATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATC AGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAAC TTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAA GACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACA GCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAA TTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAG AAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGG GAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTA TTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCT GTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGAT ACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACT GCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGAC CTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAG AATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGT TTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGT AGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGC AGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCC GAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGG ATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGG AAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACA AAAATTCAAAATTTTATCGATCACGAGACTAGCCTCGAGAAGCTTGTCCATAAAGTAGGAAA CACTACACGATTCCATAAAGTAGGAAACACTACA(X)TGGAAGGGCTAATTCACTCCCAACG AAGACAACGCAGACTCTGAGCGCCACCTGGTGGTAATTTGTCTGTGCATGCATTGTCAGTTT GTCAAATACCCCATCGTGTCAGTTTGTCAAATACCCCACCGCGGATCAAACCAGGAAACAGC AGAGGGCGCTGGTGCTGTGCTGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCA GATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCT TGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCC CTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG wherein n is from 1000 to 6000 Example Div-Conv LV (core CI) (SEQ ID NO: 94) Example convergent retroviral vectors In one aspect, the present invention provides a retroviral vector comprising from 5’ to 3’ direction: a 5’ LTR; one or more sense-oriented miRNA target sequences; a sense-oriented expression cassette; a first insulator element; and a 3’ LTR, wherein the 3’ LTR comprises from 5’ to 3’ direction: one or more antisense-oriented miRNA target sequences, and a second insulator element. In some embodiments, the first insulator element and the second insulator element are in a divergent orientation. In preferred embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the one or more sense-oriented miRNA target sequences are sense- oriented miR-142 target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences are sense-oriented miR-142-3p target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 1, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the one or more sense-oriented miRNA target sequences comprise two or more sense-oriented miRNA target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences comprises two sense-oriented miRNA target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences comprise two or more sense-oriented miR-142 target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences two or more sense- oriented miR-142-3p target sequences. In some embodiments, the one or more sense- oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 21, or a variant thereof having one to four nucleotide variations. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 21, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the one or more antisense-oriented miRNA target sequences are sense-oriented miR-223 target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences are sense-oriented miR-223-3p target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 12, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise two or more antisense-oriented miRNA target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences comprises two antisense- oriented miRNA target sequences. In some embodiments, the one or more antisense- oriented miRNA target sequences comprise two or more antisense-oriented miR-223 target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences two or more antisense-oriented miR-223-3p target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 22, or a variant thereof having one to four nucleotide variations. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 22, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the first insulator element is an A2 insulator element and / or the second insulator element is an E2 insulator element. In some embodiments, the first insulator element is an A2 insulator element and the second insulator element is an E2 insulator element. In some embodiments, the first insulator element comprises CTCF-binding motif A (e.g. SEQ ID NO: 23) and the second insulator element comprises CTCF-binding motif E (e.g. SEQ ID NO: 27). In some embodiments, the first insulator element and the second insulator element each comprise or consist of a full length insulator sequence. In some embodiments, the first insulator element is a full-length A2 insulator sequence. In some embodiments, the first insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103, or a nucleotide sequence having at least 70% identity thereto. In some embodiments, the first insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103. In some embodiments, the second insulator element is a full-length E2 insulator sequence. In some embodiments, the second insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 55, or a nucleotide sequence having at least 70% identity thereto. In some embodiments, the second insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 55. An example retroviral vector sequence is provided as SEQ ID NO: 95, wherein (X)n encodes an expression cassette. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 95. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 95. In some embodiments, the retroviral vector comprises or consists of the nucleotide sequence SEQ ID NO: 95. In some embodiments, n is from 1000 to 5000, from 1000 to 4000 or from 1000 to 3000. GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT GCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGC GCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCTCTCGACGCAGGACTCGGCTT GCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGAC TAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTA GATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACAT ATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATC AGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAAC TTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAA GACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACA GCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAA TTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAG AAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGG GAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTA TTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCT GTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGAT ACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACT GCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGAC CTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAG AATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGT TTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGT AGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGC AGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCC GAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGG ATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGG AAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACA AAAATTCAAAATTTTATCGATCACGAGACTAGCCTCGAGTCCATAAAGTAGGAAACACTACA CGATTCCATAAAGTAGGAAACACTACA(X)AGAGCGAGATTCCGTCTCAAAGAAAAAAAAAG TAATGAAATGAAGCAAATGAGTCCTAGAGCCAGTAAATGTCGTAAATGTCTCAGCTAGTCAG GTAGTAAAAGGTCTCAACTAGGCAGTGGCAGAGCAGGATTCAAATTCAGGGCTGTTGTGATG CCTCCGCAGACTCTGAGCGCCACCTGGTGGTAATTTGTCTGTGCCTCTTCTGACGTGGAAGA ACAGCAACTAACACACTAACACGGCATTTACTATGGGCCAGCCATTGTGAATTCGAGCTCGG TACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAG GGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTATGCATTGTCA GTTTGTCAAATACCCCATCGTGTCAGTTTGTCAAATACCCCACCGCGGTTTGATTTCCTTCA CTCTGGAATATACTACAGAATGTACTAATATAAATGTGCTTGCCTATGTCTAATCTAGAATA TAATTATCCTGAACTCTACATGGTATTTTAGTCTAGAAAGTACTGTAGCTTCTCTGAGTTCT AAATCAAACCAGGAAACAGCAGAGGGCGCTGGTGCTGTGCTTTTGCAAGTGAGATGTGTGCT CATTTCAAGGGTCCACAAAATTTTCTGGCATGCTGTTTCCATGGAGAGGAAAGTAGGTGTTT TCTTTTTCTTTTTTTTTTTCAGTCAAGCCACCTGCAAACTGAAGACAAGATCTGCTTTTTGC TTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGA ACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTG TTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAG CAG wherein n is from 1000 to 6000 Example Split-Conv LV (full CI) (SEQ ID NO: 95) Example parallel retroviral vectors In one aspect, the present invention provides a retroviral vector comprising from 5’ to 3’ direction: a 5’ LTR; one or more sense-oriented miRNA target sequences; a sense-oriented expression cassette; and a 3’ LTR, wherein the 3’ LTR comprises from 5’ to 3’ direction: one or more insulator elements and one or more antisense-oriented miRNA target sequences. In preferred embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the one or more sense-oriented miRNA target sequences are sense- oriented miR-142 target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences are sense-oriented miR-142-3p target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 1, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the one or more sense-oriented miRNA target sequences comprise two or more sense-oriented miRNA target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences comprises two sense-oriented miRNA target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences comprise two or more sense-oriented miR-142 target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences two or more sense- oriented miR-142-3p target sequences. In some embodiments, the one or more sense- oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 21, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the one or more antisense-oriented miRNA target sequences are sense-oriented miR-223 target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences are sense-oriented miR-223-3p target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 12, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise two or more antisense-oriented miRNA target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences comprises two antisense- oriented miRNA target sequences. In some embodiments, the one or more antisense- oriented miRNA target sequences comprise two or more antisense-oriented miR-223 target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences two or more antisense-oriented miR-223-3p target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 22, or a variant thereof having one to four nucleotide variations. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 22, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the one or more insulator elements consist of an A1 insulator element and an A2 insulator element in a parallel orientation. In some embodiments, the first insulator element comprises CTCF-binding motif A (e.g. SEQ ID NO: 23) and the second insulator element comprises CTCF-binding motif A (e.g. SEQ ID NO: 23). In some embodiments, the A1 insulator element and the A2 insulator element each comprise or consist of a full length insulator sequence. In some embodiments, the A1 insulator element is a full-length A1 insulator sequence. In some embodiments, the A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 64, or a nucleotide sequence having at least 70% identity thereto. In some embodiments, the A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 64. In some embodiments, the A2 insulator element is a full-length A2 insulator sequence. In some embodiments, the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103, or a nucleotide sequence having at least 70% identity thereto. In some embodiments, the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103. An example retroviral vector sequence is provided as SEQ ID NO: 96, wherein (X)nencodes an expression cassette. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 96. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 96. In some embodiments, the retroviral vector comprises or consists of the nucleotide sequence SEQ ID NO: 96. In some embodiments, n is from 1000 to 5000, from 1000 to 4000 or from 1000 to 3000. GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT GCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGC GCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCTCTCGACGCAGGACTCGGCTT GCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGAC TAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTA GATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACAT ATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATC AGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAAC TTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAA GACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACA GCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAA TTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAG AAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGG GAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTA TTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCT GTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGAT ACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACT GCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGAC CTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAG AATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGT TTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGT AGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGC AGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCC GAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGG ATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGG AAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACA AAAATTCAAAATTTTATCGATCACGAGACTAGCCTCGAGAAGCTTGTCCATAAAGTAGGAAA CACTACACGATTCCATAAAGTAGGAAACACTACA(X)TGGAAGGGCTAATTCACTCCCAACG AAGACAAATGACGAGAGAGACAGAAGAATTCTTCAAGGTTAGTGTGTCCAGCATGCAACCTT TCCTTCCTGGATGAGCATCCCTGGAATAGGAGAGCCAGCCTGCCTCCTGCGCTGGCACAGAG CCCGGTTCCCTAGACAACTGCCTCTCCAAATCTGATGTCCAGCGCCACCTGGTGTCCACATC AAGCAGACACAATTAATAGTCAACCTGTTCAGGAAAACTGTGAGGGGGAAAAAAAAGAAAGA GGATTTATGAAGGGAAAAGAAAGTTTAGAGGATATGCCACGATAGAGCGAGATTCCGTCTCA AAGAAAAAAAAAGTAATGAAATGAAGCAAATGAGTCCTAGAGCCAGTAAATGTCGTAAATGT CTCAGCTAGTCAGGTAGTAAAAGGTCTCAACTAGGCAGTGGCAGAGCAGGATTCAAATTCAG GGCTGTTGTGATGCCTCCGCAGACTCTGAGCGCCACCTGGTGGTAATTTGTCTGTGCCTCTT CTGACGTGGAAGAACAGCAACTAACACACTAACACGGCATTTACTATGGGCCAGCCATTGTA TGCATTGTCAGTTTGTCAAATACCCCATCGTGTCAGTTTGTCAAATACCCCAGATCTGCTTT TTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTA GGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCG TCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCT CTAGCAG wherein n is from 1000 to 6000 Example CI-COMBO (A1 and A2 full CI) (SEQ ID NO: 96) In some embodiments, the A1 insulator element and the A2 insulator element each comprise or consist of a core insulator sequence. In some embodiments, the A1 insulator element is a core A1 insulator sequence. In some embodiments, the A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 33, or a nucleotide sequence having at least 80.0% identity thereto. In some embodiments, the A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the A2 insulator element is a core A2 insulator sequence. In some embodiments, the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 34, or a nucleotide sequence having at least 80.0% identity thereto. In some embodiments, the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 34. An example retroviral vector sequence is provided as SEQ ID NO: 97, wherein (X)nencodes an expression cassette. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 97. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 97. In some embodiments, the retroviral vector comprises or consists of the nucleotide sequence SEQ ID NO: 97. In some embodiments, n is from 1000 to 5000, from 1000 to 4000 or from 1000 to 3000. GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT GCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGC GCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCTCTCGACGCAGGACTCGGCTT GCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGAC TAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTA GATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACAT ATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATC AGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAAC TTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAA GACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACA GCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAA TTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAG AAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGG GAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTA TTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCT GTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGAT ACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACT GCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGAC CTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAG AATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGT TTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGT AGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGC AGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCC GAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGG ATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGG AAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACA AAAATTCAAAATTTTATCGATCACGAGACTAGCCTCGAGAAGCTTGTCCATAAAGTAGGAAA CACTACACGATTCCATAAAGTAGGAAACACTACA(X)TGGAAGGGCTAATTCACTCCCAACG AAGACAAATCTGATGTCCAGCGCCACCTGGTGTCCACATCAAGCAGACGCAGACTCTGAGCG CCACCTGGTGGTAATTTGTCTGTGCTTAATTAAATGCATTGTCAGTTTGTCAAATACCCCAT CGTGTCAGTTTGTCAAATACCCCAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGA CCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAA GCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGA TCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG wherein n is from 1000 to 6000 Example CI-COMBO (A1 and A2 core CI) (SEQ ID NO: 97) In some embodiments, the one or more insulator elements consist of an A2 insulator element. In some embodiments, the A2 insulator element comprises CTCF-binding motif A (e.g. SEQ ID NO: 23). In some embodiments, the A2 insulator element is a full-length A2 insulator sequence. In some embodiments, the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103, or a nucleotide sequence having at least 70% identity thereto. In some embodiments, the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 103. An example retroviral vector sequence is provided as SEQ ID NO: 98, wherein (X)nencodes an expression cassette. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 98. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 98. In some embodiments, the retroviral vector comprises or consists of the nucleotide sequence SEQ ID NO: 98. In some embodiments, n is from 1000 to 5000, from 1000 to 4000 or from 1000 to 3000. GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT GCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGC GCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCTCTCGACGCAGGACTCGGCTT GCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGAC TAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTA GATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACAT ATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATC AGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAAC TTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAA GACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACA GCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAA TTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAG AAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGG GAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTA TTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCT GTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGAT ACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACT GCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGAC CTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAG AATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGT TTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGT AGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGC AGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCC GAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGG ATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGG AAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACA AAAATTCAAAATTTTATCGATCACGAGACTAGCCTCGAGAAGCTTGTCCATAAAGTAGGAAA CACTACACGATTCCATAAAGTAGGAAACACTACA(X)TGGAAGGGCTAATTCACTCCCAACG AAGACAAAGAGCGAGATTCCGTCTCAAAGAAAAAAAAAGTAATGAAATGAAGCAAATGAGTC CTAGAGCCAGTAAATGTCGTAAATGTCTCAGCTAGTCAGGTAGTAAAAGGTCTCAACTAGGC AGTGGCAGAGCAGGATTCAAATTCAGGGCTGTTGTGATGCCTCCGCAGACTCTGAGCGCCAC CTGGTGGTAATTTGTCTGTGCCTCTTCTGACGTGGAAGAACAGCAACTAACACACTAACACG GCATTTACTATGGGCCAGCCATTGTATGCATTGTCAGTTTGTCAAATACCCCATCGTGTCAG TTTGTCAAATACCCCAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCT GAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCT TGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAG ACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG wherein n is from 1000 to 6000 Example CI-COMBO (A2 full CI) (SEQ ID NO: 98) In some embodiments, the A2 insulator element is a core A2 insulator sequence. In some embodiments, the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 34, or a nucleotide sequence having at least 80.0% identity thereto. In some embodiments, the A2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 34. An example retroviral vector sequence is provided as SEQ ID NO: 99, wherein (X)nencodes an expression cassette. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 99. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 99. In some embodiments, the retroviral vector comprises or consists of the nucleotide sequence SEQ ID NO: 99. In some embodiments, n is from 1000 to 5000, from 1000 to 4000 or from 1000 to 3000. GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT GCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGC GCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCTCTCGACGCAGGACTCGGCTT GCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGAC TAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTA GATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACAT ATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATC AGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAAC TTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAA GACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACA GCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAA TTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAG AAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGG GAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTA TTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCT GTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGAT ACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACT GCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGAC CTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAG AATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGT TTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGT AGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGC AGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCC GAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGG ATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGG AAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACA AAAATTCAAAATTTTATCGATCACGAGACTAGCCTCGAGAAGCTTGTCCATAAAGTAGGAAA CACTACACGATTCCATAAAGTAGGAAACACTACA(X)TGGAAGGGCTAATTCACTCCCAACG AAGACAACGCAGACTCTGAGCGCCACCTGGTGGTAATTTGTCTGTGCATGCATTGTCAGTTT GTCAAATACCCCATCGTGTCAGTTTGTCAAATACCCCAGATCTGCTTTTTGCTTGTACTGGG TCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACT CTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG wherein n is from 1000 to 6000 Example CI-COMBO (A2 core CI) (SEQ ID NO: 99) In some embodiments, the one or more insulator elements consist of an A1 insulator element. In some embodiments, the A1 insulator element comprises CTCF-binding motif A (e.g. SEQ ID NO: 23). In some embodiments, the A1 insulator element is a core A1 insulator sequence. In some embodiments, the A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 33, or a nucleotide sequence having at least 80.0% identity thereto. In some embodiments, the A1 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 33. An example retroviral vector sequence is provided as SEQ ID NO: 100, wherein (C)nencodes an expression cassette. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 100. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 100. In some embodiments, the retroviral vector comprises or consists of the nucleotide sequence SEQ ID NO: 100. In some embodiments, n is from 1000 to 5000, from 1000 to 4000 or from 1000 to 3000. GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT GCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGC GCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCTCTCGACGCAGGACTCGGCTT GCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGAC TAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTA GATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACAT ATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATC AGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAAC TTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAA GACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACA GCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAA TTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAG AAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGG GAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTA TTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCT GTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGAT ACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACT GCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGAC CTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAG AATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGT TTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGT AGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGC AGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCC GAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGG ATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGG AAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACA AAAATTCAAAATTTTATCGATCACGAGACTAGCCTCGAGAAGCTTGTCCATAAAGTAGGAAA CACTACACGATTCCATAAAGTAGGAAACACTACA(X)TGGAAGGGCTAATTCACTCCCAACG AAGACAAATCTGATGTCCAGCGCCACCTGGTGTCCACATCAAGCAGAATGCATTGTCAGTTT GTCAAATACCCCATCGTGTCAGTTTGTCAAATACCCCAGATCTGCTTTTTGCTTGTACTGGG TCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACT CTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG wherein n is from 1000 to 6000 Example CI-COMBO (A1 core CI) (SEQ ID NO: 100) In some embodiments, the one or more insulator elements consist of a B2 insulator element. In some embodiments, the B2 insulator element comprises CTCF-binding motif B (e.g. SEQ ID NO: 24). In some embodiments, the B2 insulator element is a core B2 insulator sequence. In some embodiments, B2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 35, or a nucleotide sequence having at least 80.0% identity thereto. In some embodiments, the B2 insulator element comprises or consists of the nucleotide sequence of SEQ ID NO: 35. An example retroviral vector sequence is provided as SEQ ID NO: 101, wherein (X)n encodes an expression cassette. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 101. In some embodiments, the retroviral vector comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 101. In some embodiments, the retroviral vector comprises or consists of the nucleotide sequence SEQ ID NO: 101. In some embodiments, n is from 1000 to 5000, from 1000 to 4000 or from 1000 to 3000. GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT GCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGC GCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCTCTCGACGCAGGACTCGGCTT GCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGAC TAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTA GATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACAT ATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATC AGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAAC TTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAA GACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACA GCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAA TTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAG AAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGG GAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTA TTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCT GTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGAT ACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACT GCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGAC CTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAG AATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGT TTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGT AGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGC AGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCC GAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGG ATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGG AAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACA AAAATTCAAAATTTTATCGATCACGAGACTAGCCTCGAGAAGCTTGTCCATAAAGTAGGAAA CACTACACGATTCCATAAAGTAGGAAACACTACA(X)TGGAAGGGCTAATTCACTCCCAACG AAGACAACTGCGTCCCAGCAGCTCCCCCTGGTGGCCAGAAGCAAACGCTATGCATTGTCAGT TTGTCAAATACCCCATCGTGTCAGTTTGTCAAATACCCCAGATCTGCTTTTTGCTTGTACTG GGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTG CTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGA CTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG wherein n is from 1000 to 6000 Example CI-COMBO (B2 core CI) (SEQ ID NO: 101) Retroviral vector (antisense-oriented expression cassette) In one aspect, the present invention provides a retroviral vector comprising from 5’ to 3’ direction: a 5’ LTR; an antisense-oriented expression cassette; and a 3’ LTR, wherein the retroviral vector further comprises: (a) one or more antisense-oriented miRNA target sequences; (b) one or more sense-oriented miRNA target sequences; and (c) one or more insulator elements. In preferred embodiments, the retroviral vector is a lentiviral vector. As described above, in the context of a retroviral vector, an “antisense-oriented” expression cassette may have the opposite orientation to the retroviral vector genome. For example, following conversion of a retroviral vector into double-stranded DNA, the expression cassette may be transcribed in the direction of from the 3’ LTR to the 5’ LTR. In preferred embodiments, the one or more antisense-oriented miRNA target sequences are upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the one or more sense-oriented miRNA target sequences are upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the one or more insulator elements are upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the one or more antisense-oriented miRNA target sequences are upstream from the expression cassette and / or downstream from the expression cassette; the one or more sense-oriented miRNA target sequences are upstream from the expression cassette and / or downstream from the expression cassette; and the one or more insulator elements are upstream from the expression cassette and / or downstream from the expression cassette. In some embodiments, the 3’ LTR does not comprise one or more antisense-oriented miRNA target sequences. In some embodiments, the 3’ LTR does not comprise one or more sense-oriented miRNA target sequences. In some embodiments, the 3’ LTR does not comprise one or more miRNA target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences are sense- oriented miR-142 target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences are sense-oriented miR-142-3p target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 1, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the one or more sense-oriented miRNA target sequences comprise two or more sense-oriented miRNA target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences comprises two sense-oriented miRNA target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences comprise two or more sense-oriented miR-142 target sequences. In some embodiments, the one or more sense-oriented miRNA target sequences two or more sense- oriented miR-142-3p target sequences. In some embodiments, the one or more sense- oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 21, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more sense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the one or more antisense-oriented miRNA target sequences are sense-oriented miR-223 target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences are sense-oriented miR-223-3p target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 12, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise two or more antisense-oriented miRNA target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences comprises two antisense- oriented miRNA target sequences. In some embodiments, the one or more antisense- oriented miRNA target sequences comprise two or more antisense-oriented miR-223 target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences two or more antisense-oriented miR-223-3p target sequences. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 22, or a variant thereof having one to four nucleotide variations. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 22, or a variant thereof having one or two nucleotide variations. In some embodiments, the one or more antisense-oriented miRNA target sequences comprise or consist of the nucleotide sequence of SEQ ID NO: 22. The miRNA target sequences and the one or more insulator elements may be in any configuration and may be any type described above in the section entitled “Polynucleotides”. Further, example configurations are shown in Figures 6D-G, Figures 7D-F, and Figures 7J-L. As described above, Figures 6 and 7 are schematics of the provirus. The 5’LTR of the retroviral vector may comprise none of the genetic elements present in the provirus (e.g. the 5’LTR of the retroviral vector may comprise only R and U5 regions). In some embodiments, the retroviral vector does not comprise an antisense-oriented miRNA target sequence or a sense-oriented miRNA target sequence upstream of a splice donor site or downstream of a splice acceptor site. Methods of improving the safety of a polynucleotide, vector, or retroviral vector In one aspect, the present invention provides a method of improving the safety of a polynucleotide or vector (e.g. by reducing genotoxicity), wherein the method comprises introducing into the polynucleotide or vector: (a) one or more miRNA target sequences having the same orientation as the expression cassette; (b) one or more miRNA target sequences having the opposite orientation as the expression cassette; and / or (c) one or more insulator elements. The methods be carried out using standard recombinant DNA techniques such as site- directed mutagenesis or gene-editing. Where insertions are to be made, synthetic DNA encoding the insertion together with 5’ and 3’ flanking regions corresponding to the naturally- occurring sequence either side of the insertion site may be made. The flanking regions may contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. Numerous other standard techniques are known in the art for manipulation of DNA sequences and other known techniques may also be used, such as methods using RNA-guided nucleases. The configurations and types of miRNA target sequences and insulator elements introduced into the polynucleotide or vector and their configurations may be any described above in the sections entitled “Polynucleotides” or “Vectors”. In another aspect, the present invention provides a method of improving the safety of a retroviral vector (e.g. by reducing genotoxicity) comprising a sense-oriented expression cassette, wherein the method comprises introducing into the retroviral vector (a) one or more sense-oriented miRNA target sequences upstream from the sense-oriented expression cassette. In preferred embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the method further comprises introducing into the retroviral vector (b) one or more antisense-oriented miRNA target sequences upstream from the sense-oriented expression cassette and / or downstream from the sense-oriented expression cassette. In some embodiments, the method further comprises introducing into the retroviral vector (c) one or more insulator elements upstream from the sense-oriented expression cassette and / or downstream from the sense-oriented expression cassette. In some embodiments, the method further comprises introducing into the retroviral vector (b) one or more antisense- oriented miRNA target sequences upstream from the sense-oriented expression cassette and / or downstream from the sense-oriented expression cassette and (c) one or more insulator elements upstream from the sense-oriented expression cassette and / or downstream from the sense-oriented expression cassette. The configurations and types of miRNA target sequences and insulator elements introduced into the retroviral vector may be any described above in the section entitled “Retroviral vector (sense-oriented expression cassette)”. In another aspect, the present invention provides a method of improving the safety of a retroviral vector (e.g. by reducing genotoxicity) comprising an antisense-oriented expression cassette, wherein the method comprises introducing into the retroviral vector: (a) one or more antisense-oriented miRNA target sequences upstream from the antisense-oriented expression cassette and / or downstream from the antisense-oriented expression cassette; (b) one or more sense-oriented miRNA target sequences upstream from the antisense-oriented expression cassette and / or downstream from the antisense-oriented expression cassette; and (c) one or more insulator elements upstream from the antisense-oriented expression cassette and / or downstream from the antisense-oriented expression cassette. In preferred embodiments, the retroviral vector is a lentiviral vector. The configurations and types of miRNA target sequences and insulator elements introduced into the retroviral vector may be any described above in the section entitled “Retroviral vector (antisense-oriented expression cassette)”. Safety and reduced genotoxicity of polynucleotides, vectors, or retroviral vectors The polynucleotides, vectors, or retroviral vectors of the present invention may have improved safety. miRNA target sequences upstream from the expression cassette and / or downstream from the expression cassette may improve safety of the polynucleotides, vectors, or retroviral vectors by avoiding and / or reducing aberrant transcription, aberrant splicing, and / or read- through events. The miRNA target sequences may specifically degrade aberrant transcripts. Insulator elements may improve safety of the polynucleotides, vectors, or retroviral vectors by avoiding and / or reducing enhancer-mediated gene activation. The polynucleotides, vectors, or retroviral vectors of the present invention may be substantially non-genotoxic and / or have reduced genotoxicity. As used herein, “genotoxicity” may refer to damage to the genetic information within a cell causing mutations, which may lead to cancer. The reduction in genotoxicity may be determined by any method known in the art (see e.g. Cesana, D., et al.2014. Mol Ther 22, 774-785). In some embodiments, the polynucleotide, vector, or retroviral vector is substantially non- genotoxic compared to a mock control. A “mock control” may refer to a control setting where the specimen is treated the same way as the specimen administered the polynucleotide, vector, or retroviral vector, except no polynucleotide, vector, retroviral vector is administered. In some embodiments, the polynucleotide, vector, or retroviral vector has reduced genotoxicity compared to a control polynucleotide, vector, or retroviral vector. A “control polynucleotide, vector, or retroviral vector” may refer to a polynucleotide, vector, or retroviral vector in which one or more of the safety genetic elements are absent or in which all of the safety genetic elements are absent, but in which all other genetic elements are present. In some embodiments, a control polynucleotide, vector, or retroviral vector is identical to the polynucleotide, vector, or retroviral vector of the present invention but does not comprise any of: (a) a miRNA target sequence having the same orientation as the expression cassette, upstream from the expression cassette and / or downstream from the transcription termination site; (b) a miRNA target sequence having the opposite orientation as the expression cassette, upstream from the expression cassette and / or downstream from the expression cassette; or (c) an insulator element, upstream from the expression cassette and / or downstream from the expression cassette. The genotoxicity of the polynucleotides, vectors, or retroviral vectors may be determined by an in vivo genotoxicity assay based on a tumor prone mouse model. A tumor-prone mouse model based on a Cdkn2a+ / -mice has previously been described (see e.g. Cesana, D., et al. 2014. Mol Ther 22, 774-785). Cdkn2a+ / -mice are significantly more sensitive than wild type mice since they develop early tumors when treated with genotoxic retroviral vectors. In the Cdkn2a+ / -mouse model, the survival kinetics may be used to determine the genotoxicity of the polynucleotides, vectors, or retroviral vectors. In some embodiments, Cdkn2a+ / -mice which are administered the polynucleotides, vectors, or retroviral vectors of the present invention do not have a significantly reduced survival compared to Cdkn2a+ / -mice which are administered a mock control. In some embodiments, Cdkn2a+ / -mice which are administered the polynucleotides, vectors, or retroviral vectors of the present invention have a significantly increased survival compared to Cdkn2a+ / -mice which are administered a control polynucleotide, vector, or retroviral vector. In the Cdkn2a+ / -mouse model, the time to tumor onset may be used to determine the genotoxicity of the polynucleotides, vectors, or retroviral vectors. In some embodiments, Cdkn2a+ / -mice which are administered the polynucleotides, vectors, or retroviral vectors of the present invention do not have a significantly increased time to tumor onset compared to Cdkn2a+ / -mice which are administered a mock control. In some embodiments, Cdkn2a+ / -mice which are administered the polynucleotides, vectors, or retroviral vectors of the present invention have a significantly reduced time to tumor onset compared to Cdkn2a+ / -mice which are administered a control polynucleotide, vector, or retroviral vector. Alternatively or additionally, in the Cdkn2a+ / -mouse model, histopathological analysis may be used to determine the genotoxicity of the polynucleotides, vectors, or retroviral vectors. The histopathological analysis may determine the incidence of tumors (e.g. liver tumors) and / or tissue abnormalities (e.g. focal cell alterations). In some embodiments, Cdkn2a+ / -mice which are administered the polynucleotides, vectors, or retroviral vectors of the present invention do not develop tumours and / or tissue abnormalities. In some embodiments, Cdkn2a+ / -mice which are administered the polynucleotides, vectors, or retroviral vectors of the present invention do not develop significantly more tumours and / or tissue abnormalities compared to Cdkn2a+ / -mice which are administered a mock control. In some embodiments, Cdkn2a+ / -mice which are administered the polynucleotides, vectors, or retroviral vectors of the present invention develop significantly fewer tumours and / or tissue abnormalities compared to Cdkn2a+ / -mice which are administered a control polynucleotide, vector, or retroviral vector. Alternatively or additionally, in the Cdkn2a+ / -mouse model, the frequency of insertions targeting common integration sites (CIS) genes may be used to determine the genotoxicity of the polynucleotides, vectors, or retroviral vectors. The CIS genes may be cancer-causing CIS genes. In some embodiments, two or less, one or less, or no CIS genes are identified in Cdkn2a+ / -mice which are administered the polynucleotides, vectors, or retroviral vectors of the present invention. In some embodiments, significantly fewer CIS genes are identified in Cdkn2a+ / -mice which are administered the polynucleotides, vectors, or retroviral vectors of the present invention compared to Cdkn2a+ / -mice which are administered a control polynucleotide, vector, or retroviral vector. In some embodiments, the one or more sense-oriented miRNA target sequences and the one or more antisense-oriented miRNA target sequences avoid and / or reduce aberrant splicing and / or read-through events, and / or wherein the one or more insulator elements avoid and / or reduce enhancer-mediated gene activation compared to a polynucleotide, vector, or retroviral vector lacking the one or more sense-oriented miRNA target sequences and the one or more antisense-oriented miRNA target sequences and / or the one or more insulator elements. In some embodiments, the one or more sense-oriented miRNA target sequences and the one or more antisense-oriented miRNA target sequences avoid and / or reduce aberrant splicing and / or read-through events compared to a polynucleotide, vector, or retroviral vector lacking the one or more sense-oriented miRNA target sequences and the one or more antisense-oriented miRNA target sequences. For example, the number of aberrant transcripts may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a polynucleotide, vector, or retroviral vector lacking the one or more sense-oriented miRNA target sequences and the one or more antisense-oriented miRNA target sequences. A suitable assay to determine the number of aberrant transcripts is described in Cesana, D., et al., 2012. The Journal of clinical investigation, 122(5), pp.1667- 1676. In some embodiments, the one or more insulator elements avoid and / or reduce enhancer- mediated gene activation compared to a polynucleotide, vector, or retroviral vector lacking the one or more insulator elements. For example, differential expression of endogenous genes following genomic insertion of the polynucleotide, vector, or retroviral vector may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a polynucleotide, vector, or retroviral vector lacking the one or more insulator elements. A suitable assay to determine the differential expression of endogenous genes is described in Ribeiro-dos-Santos, A.M., et al., 2022. Genome Research, 32(3), pp.425-436. Variants, derivatives, analogues, and fragments In addition to the specific polypeptides and polynucleotides mentioned herein, the invention also encompasses variants, derivatives, analogues, and fragments thereof. In the context of the present invention, a “variant” of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question substantially retains at least one of its functions. Suitably, for example, any amino acid changes in the TCR sequences should maintain the capacity of the TCR to bind the peptide presented by MHC molecules. A variant amino acid or nucleic acid sequence can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the naturally-occurring polypeptide or polynucleotide. A variant amino acid sequence of the invention referred to as having up to three amino acid substitutions, additions or deletions may have, for example, one, two or three amino acid substitutions, additions or deletions. Suitably, for example, the variation may be concentrated in one or more regions, such as the constant regions, the linker, or the framework regions of the α or β chains, or they may be spread throughout the TCR. The term “derivative” as used herein, in relation to proteins or polypeptides of the invention includes any substitution of, variation of, modification of, replacement of, deletion of and / or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide substantially retains at least one of its functions. The term “analogue” as used herein, in relation to polypeptides or polynucleotides includes any mimetic, that is, a chemical compound that possesses at least one of the functions of the polypeptides or polynucleotides which it mimics. Polypeptides of the present invention may have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues as long as the function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine. Typically, amino acid substitutions may be made, for example from 1, 2 or 3 to 10 or 20 substitutions provided that the modified sequence substantially retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues. A substitution may involve replacement of an amino acid for a similar amino acid (a conservative substitution). A similar amino acid is one which has a side chain moiety with related properties as grouped together, for example as shown below: (i) basic side chains: lysine (K), arginine (R), histidine (H); (ii) acidic side chains: aspartic acid (D) and glutamic acid (E); (iii) uncharged polar side chains: asparagine (N), glutamine (Q), serine (S), threonine (T) and tyrosine (Y); or (iv) non-polar side chains: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W) and cysteine (C). Conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar - charged D E K R H AROMATIC F W Y The effect of additions, deletions, substitutions, modifications, replacements and / or variations may be predicted using any suitable prediction tool e.g. SIFT (Vaser, R., et al., 2016. Nature protocols, 11(1), pp.1-9), PolyPhen-2 (Adzhubei, I., et al., 2013. Current protocols in human genetics, 76(1), pp.7-20), CADD (Rentzsch, P., et al., 2021. Genome medicine, 13(1), pp.1-12), REVEL (Ioannidis, N.M., et al., 2016. The American Journal of Human Genetics, 99(4), pp.877-885), MetaLR (Dong, C., et al., 2015. Human molecular genetics, 24(8), pp.2125-2137), and / or MutationAssessor (Reva, B., et al., 2011. Nucleic acids research, 39(17), pp.e118-e118). Typically, a variant may have a certain identity with the subject amino acid sequence or the subject nucleotide sequence. Suitably, reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to. In the present context, a variant amino acid sequence is taken to include an amino acid sequence which may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence. Although a variant can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties / functions), in the context of the present invention it is preferred to express in terms of sequence identity. In the present context, a variant nucleotide sequence is taken to include a nucleotide sequence which may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence. Although a variant can also be considered in terms of similarity, in the context of the present invention it is preferred to express it in terms of sequence identity. Sequence identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent identity between two or more sequences. Percent identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues. Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the amino acid or nucleotide sequence may cause the following residues or codons to be put out of alignment, thus potentially resulting in a large reduction in percent identity when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall identity score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local identity. However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum percent identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (see e.g. Devereux, J., et al., 1984. Nucleic acids research, 12(1), pp.387-395). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see e.g. Altschul, S.F., et al., 1990. Journal of molecular biology, 215(3), pp.403-410), BLAST 2 (see e.g. Tatusova, T.A. and Madden, T.L., 1999. FEMS microbiology letters, 174(2), pp.247-250), FASTA (see e.g. Pearson, W.R. and Lipman, D.J., 1988. PNAS, 85(8), pp.2444-2448.), EMBOSS Needle (Madeira, F., et al., 2019. Nucleic acids research, 47(W1), pp.W636-W641) and the GENEWORKS suite of comparison tools. For some applications, it is preferred to use EMBOSS Needle. Although the final percent identity can be measured, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix. Once the software has produced an optimal alignment, it is possible to calculate percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The percent sequence identity may be calculated as the number of identical residues as a percentage of the total residues in the SEQ ID NO referred to. “Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide. Such variants, derivatives, analogues and fragments may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5’ and 3’ flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded polypeptide. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used. Expression vectors, kits and systems In one aspect, the present invention provides a vector encoding the polynucleotide, vector, or retroviral vector of the present invention. In another aspect, the present invention provides a vector encoding the virus genome (e.g. retrovirus genome) of the present invention. The vector may be a transfer vector. For example, the vector may be a plasmid and / or the retroviral genome may be operably linked to a promoter (e.g. a viral pro...

Claims

CLAIMS 1. A polynucleotide comprising a nucleotide sequence comprising an expression cassette and a transcription termination site, wherein the nucleotide sequence further comprises: (a) one or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette and / or downstream from the transcription termination site; (b) one or more miRNA target sequences having the opposite orientation as the expression cassette; and (c) one or more insulator elements.

2. The polynucleotide of claim 1, wherein the one or more miRNA target sequences having the same orientation as the expression cassette are selected from a miR223 target sequence, a miR142 target sequence, a miR22 target sequence, a miR122 target sequence, a miR126 target sequence, a miR124 target sequence, a miR133a target sequence, a miR133b target sequence, a let7f target sequence, a miR7 target sequence, or a combination thereof, preferably wherein the one or more miRNA target sequences having the same orientation as the expression cassette are miR142-3p target sequences; and / or wherein the one or more miRNA target sequences having the opposite orientation as the expression cassette are selected from a miR223 target sequence, a miR142 target sequence, a miR22 target sequence, a miR122 target sequence, a miR126 target sequence, a miR124 target sequence, a miR133a target sequence, a miR133b target sequence, a let7f target sequence, a miR7 target sequence, or a combination thereof, preferably wherein the one or more miRNA target sequences having the opposite orientation as the expression cassette are miR223-3p target sequences.

3. The polynucleotide of claim 1 or 2, wherein the nucleotide sequence comprises a pair of insulator elements flanking the expression cassette, optionally wherein the pair of insulator elements is in a divergent orientation, a convergent orientation or a parallel orientation and / or wherein the one or more insulator elements are CTCF-based insulator elements.

4. A vector comprising the polynucleotide of any of claims 1 to 3, optionally wherein the vector is a viral vector selected from the group consisting of a lentiviral vector, a retroviral vector, and an adeno-associated viral (AAV) vector, preferably wherein the vector is a retroviral vector, more preferably wherein the vector is a lentiviral vector.

5. A viral particle comprising the polynucleotide of any of claims 1 to 3, optionally wherein the viral particle is selected from the group consisting of a lentiviral particle, a retroviral particle, and an adeno-associated viral (AAV) particle, preferably wherein the viral particle is a retroviral particle, more preferably wherein the viral particle is a lentiviral particle.

6. A retroviral vector comprising from 5’ to 3’ direction: a 5’ LTR; one or more sense- oriented miRNA target sequences; a sense-oriented expression cassette; and a 3’ LTR, optionally wherein the retroviral vector further comprises one or more antisense-oriented miRNA target sequences upstream from the expression cassette and / or downstream from the expression cassette; and / or one or more insulator elements upstream from the expression cassette and / or downstream from the expression cassette.

7. A retroviral vector comprising from 5’ to 3’ direction: a 5’ LTR; an antisense-oriented expression cassette; and a 3’ LTR, wherein the retroviral vector further comprises: (a) one or more antisense-oriented miRNA target sequences, upstream from the expression cassette and / or downstream from the expression cassette; (b) one or more sense-oriented miRNA target sequences, optionally upstream from the expression cassette and / or downstream from the expression cassette; and (c) one or more insulator elements, optionally upstream from the expression cassette and / or downstream from the expression cassette.

8. The retroviral vector of claim 6 or 7, wherein the retroviral vector is substantially non- genotoxic, wherein the one or more sense-oriented miRNA target sequences and the one or more antisense-oriented miRNA target sequences avoid and / or reduce aberrant splicing and / or read-through events, and / or wherein the one or more insulator elements avoid and / or reduce enhancer-mediated gene activation.

9. An isolated cell comprising the polynucleotide of any of claims 1 to 3, the vector of claim 4, the viral particle of claim 5, or the retroviral vector of any of claims 6 to 8, optionally wherein the cell is a hematopoietic stem and / or progenitor cell (HSPC) or a non-stem cell, such as a liver cell or a T cell.

10. A pharmaceutical composition comprising the polynucleotide of any of claims 1 to 3, the vector of claim 4, the viral particle of claim 5, the retroviral vector of any of claims 6 to 8, or the isolated cell of claim 9, in combination with a pharmaceutically acceptable carrier, diluent or excipient.

11. The polynucleotide of any of claims 1 to 3, the vector of any of claim 4, the viral particle of claim 5, the retroviral vector of any of claims 6 to 8, the isolated cell of claim 9, or the pharmaceutical composition of claim 10, for use as a medicament.

12. A method of reducing the genotoxicity of a polynucleotide or vector comprising a nucleotide sequence comprising an expression cassette and a transcription termination site, wherein the method comprises introducing into the nucleotide sequence: (a) one or more miRNA target sequences having the same orientation as the expression cassette, upstream from the expression cassette and / or downstream from the transcription termination site; (b) one or more miRNA target sequences having the opposite orientation as the expression cassette; and (c) one or more insulator elements.

13. A method of reducing the genotoxicity of a retroviral vector comprising a sense- oriented expression cassette, wherein the method comprises introducing into the retroviral vector: (a) one or more sense-oriented miRNA target sequences upstream from the sense- oriented expression cassette, optionally wherein the method further comprises introducing into the retroviral vector: (b) one or more antisense-oriented miRNA target sequences upstream from the sense-oriented expression cassette and / or downstream from the sense-oriented expression cassette; and / or (c) one or more insulator elements upstream from the sense-oriented expression cassette and / or downstream from the sense-oriented expression cassette.

14. A method of reducing the genotoxicity of a retroviral vector comprising an antisense- oriented expression cassette, wherein the method comprises introducing into the retroviral vector: (a) one or more antisense-oriented miRNA target sequences upstream from the antisense-oriented expression cassette and / or downstream from the antisense- oriented expression cassette;(b) one or more sense-oriented miRNA target sequences, optionally upstream from the antisense-oriented expression cassette and / or downstream from the antisense- oriented expression cassette; and (c) one or more insulator elements, optionally upstream from the antisense-oriented expression cassette and / or downstream from the antisense-oriented expression cassette.

15. An insulator element comprising or consisting of: (a) a sequence having at least 95% sequence identity to SEQ ID NO: 103, wherein the insulator element comprises the nucleotides G and C at positions corresponding to nucleotides 45 and 46, respectively, wherein the nucleotides are numbered with reference to SEQ ID NO: 103; or (b) a sequence having at least 95% sequence identity to SEQ ID NO: 102, wherein the insulator element comprises the nucleotides G and C at positions corresponding to nucleotides 221 and 222, respectively, wherein the nucleotides are numbered with reference to SEQ ID NO:

102.

16. A self-inactivating (SIN) 3’ LTR comprising one or more antisense-oriented miRNA target sequences and one or more insulator elements; optionally wherein the SIN 3’ LTR comprises or consists of a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 104 to 112.

17. A polynucleotide comprising a nucleotide sequence comprising the insulator element or claim 15 or the SIN 3’ LTR of claim 16.

18. A vector comprising the polynucleotide of claim 17.

19. An isolated cell comprising the polynucleotide of claim 17 or the vector of claim 18.

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