Fusion protein

Abstract

The invention relates to fusion proteins comprising a membrane-targeting domain and an RNA-binding domain, linked by a cleavable peptide, which enable the packaging of an mRNA of interest into viral particles. The invention also relates to lentiviral vectors comprising said fusion protein, and cells and pharmaceutical compositions comprising said vectors, and their uses in therapy.

Description

[0001] FUSION PROTEIN

[0002] FIELD OF THE INVENTION

[0003] The invention relates to fusion proteins comprising a membrane-targeting domain and an RNA-binding domain, linked by a cleavable peptide, which enable the packaging of an mRNA of interest into viral particles. The invention also relates to vectors (e.g. lentiviral vectors) comprising said fusion protein, and cells and pharmaceutical compositions comprising said vectors, and their uses in therapy.

[0004] BACKGROUND TO THE INVENTION

[0005] Lentiviral vectors (LV), such as those derived from HIV-1, represent an efficient and versatile platform for gene therapy. LV can transfer genes to both dividing and non-dividing cells, can stably integrate in the genome of target cells and have relatively large cargo capacity. In addition, there is low prevalence of pre-existing anti-vector immunity in humans. LVs are being used in ex vivo gene therapy and have shown promising results in pre-clinical studies involving direct in vivo administration, such as in liver-directed gene therapy.

[0006] LVs and integrase deficient LVs have been employed to design vaccination strategies in pre-clinical studies. However, in this application long-term persistence of the transgene may not be desirable since this might cause dysfunctional activation of T cells.

[0007] Ling, et al. describes mRNA incorporation in lentiviral vectors to transiently express Cas9, together with a non-integrating viral genome encoding for the gRNA of interest (Ling, et al., 2021, Nat Biomed Eng 5, 144-156). However, there are no other technologies that enable the incorporation of exogenous RNAs into functional viral particles (i.e. viral vectors capable of carrying a functional genome).

[0008] There is thus a need for improved lentiviral vectors, for example that are capable of both transient and stable transgene expression.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention relates to a fusion protein, which may be incorporated into LVs, and which enables the packaging of an mRNA of interest into the viral particles (e.g. due to specific aptamer-aptamer binding protein interactions). The system couples the benefits of mRNA transient expression with LV’s potential for stable transduction and favourable safety profile. This approach offers a modular, customizable tool for cell engineering that can be especially useful in applications requiring joint short and long-term transgene expression. For instance, these engineered LVs could be employed for in vivo vaccination strategies that co-deliver antigens with immune-activating molecules, for genome editing strategies combining temporary expression of base / gene editors with corrective DNA templates, or for the transient expression of selectors from edited cells.

[0011] The fusion protein comprises a membrane-targeting domain, such as from Phospholipase C (PLC) (which colocalises in the lipid raft of the producer cell), enabling its incorporation in the viral envelope within the viral particle. The membrane-targeting domain is fused to an RNA-binding domain, such as MS2 phage capsid protein (MCP), which may specifically bind to MS2 RNA loops contained in the mRNA of interest. The two domains are linked by a peptide which is cleavable by a protease (e.g. a viral protease, such as a retroviral or lentiviral protease), which may enable the separation of the two proteins during viral particle maturation, making the mRNA complex available to ribosomes in the cytoplasm for translation. Through this system, the amount of co-packaged mRNA molecules per viral particle can be increased, while preserving LV structure and stable gene transfer capability.

[0012] The invention provides a fusion protein comprising a membrane-targeting domain and an RNA-binding domain, wherein the membrane-targeting domain and RNA-binding domain are linked by a peptide cleavable by a viral (e.g. retroviral or lentiviral) protease, and wherein the fusion protein is not operably linked to a viral (e.g. retroviral or lentiviral) structural protein. The invention provides a fusion protein comprising, in order from N-terminus to C-terminus, a membrane-targeting domain, a peptide cleavable by a viral (e.g. retroviral or lentiviral) protease, and an RNA-binding domain.

[0013] The invention provides a fusion protein comprising a membrane-targeting domain and an RNA-binding domain, wherein the membrane-targeting domain and RNA-binding domain are linked by a peptide cleavable by a lentiviral protease, and wherein the fusion protein is not operably linked to a lentiviral structural protein.

[0014] The invention provides a fusion protein comprising, in order from N-terminus to C-terminus, a membrane-targeting domain, a peptide cleavable by a lentiviral protease, and an RNA-binding domain.

[0015] The membrane-targeting domain may, for example, comprise a phospholipase C-δ1 pleckstrin homology domain or a membrane-targeting domain of a proto-oncogene tyrosine-protein kinase Src. In preferred embodiments, the membrane-targeting domain comprises a phospholipase C-δ1 pleckstrin homology domain. In preferred embodiments, the phospholipase C-δ1 pleckstrin homology domain comprises or consists of an amino acid sequence according to SEQ ID NO: 3, or a sequence having at least 80% sequence identity thereto.

[0016] The RNA-binding domain may comprise an aptamer-binding protein. In preferred embodiments, the RNA-binding domain comprises an MS2 phage capsid protein. In preferred embodiments, the MS2 phage capsid protein comprises or consists of an amino acid sequence according to SEQ ID NO: 5, or a sequence having at least 80% sequence identity thereto.

[0017] The peptide cleavable by a lentiviral protease may be an HIV protease cleavage sequence. In preferred embodiments, the peptide cleavable by a lentiviral protease may be an HIV protease cleavage sequence comprising or consisting of the amino acid sequence VSQNYPIVQ (SEQ ID NO: 6).

[0018] In some embodiments, the fusion protein according to the invention comprises or consists of an amino acid sequence according to SEQ ID NO: 1, or a sequence having at least 80% sequence identity thereto.

[0019] The invention provides a polynucleotide encoding a fusion protein according to the invention.

[0020] In some embodiments, the polynucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 11, or a sequence having at least 80% sequence identity thereto.

[0021] The invention provides a cell comprising the polynucleotide according to the invention, optionally wherein the cell is a viral packaging cell or a viral producer cell (e.g. a retroviral packaging cell or a retroviral producer cell, or a lentiviral packaging cell or a lentiviral producer cell). The invention provides a cell comprising the polynucleotide according to the invention, optionally wherein the cell is a lentiviral packaging cell or a lentiviral producer cell. The cell may be genetically engineered to decrease expression of CD47 on the surface of the cell. The cell may be genetically engineered to increase expression of CD47 on the surface of the cell. The cell may be genetically engineered to decrease expression of MHC on the surface of the cell.

[0022] The invention provides a viral (e.g. retroviral or lentiviral) vector comprising:

[0023] a) a fusion protein according to the invention; b) an mRNA comprising a first transgene and a target site for the RNA-binding domain; and

[0024] c) a viral genome, optionally wherein the viral genome comprises a second transgene.

[0025] The invention provides a lentiviral vector comprising:

[0026] a) a fusion protein according to the invention;

[0027] b) an mRNA comprising a first transgene and a target site for the RNA-binding domain;

[0028] and

[0029] c) a lentiviral genome, optionally wherein the lentiviral genome comprises a second transgene.

[0030] The target site for the RNA-binding domain may comprise an aptamer. In preferred embodiments, the target site for the RNA-binding domain comprises an MS2 stem-loop, preferably six MS2 stem-loops. In preferred embodiments, the mRNA comprises a nucleotide sequence according to SEQ ID NO: 41.

[0031] The mRNA may comprise a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). The WPRE element may enhance RNA stability and expression.

[0032] The mRNA may comprise one or more miRNA target sequences. The miRNA target sequence may prevent or reduce transgene expression in a cell that endogenously expresses the corresponding miRNA.

[0033] In some embodiments, the mRNA comprises an miR-122 target sequence, an miR-126 target sequence, and / or an miR-142 target sequence. In some embodiments, the miR-122 target sequence comprises or consists of SEQ ID NO: 53, the miR-126 target sequence comprises or consists of SEQ ID NO: 51, and / or the miR-142 target sequence comprises or consists of SEQ ID NO: 59.

[0034] The mRNA may comprise a first transgene which encodes a therapeutic polypeptide, an antigenic polypeptide, a cytokine, a chemokine receptor (such as CXCR4), a viral accessory protein (such as Vpx), a transduction enhancer, a gene editing enhancer (such as GSE56), a p53 inhibitor, a cell proliferation enhancer (such as HoxB4 or KrasQ61R), a cell cycle booster (such as Ad5-E4orf6 / 7) and / or an enzyme involved in dNTP pool enhancement.

[0035] The mRNA may comprise a first transgene which encodes a therapeutic polypeptide, an antigenic polypeptide, a cytokine, a chemokine receptor (such as CXCR4), a viral accessory protein (such as Vpx), a transduction enhancer, a p53 inhibitor, a cell cycle booster (such as Ad5-E4orf6 / 7) and / or an enzyme involved in dNTP pool enhancement.

[0036] The vector genome may comprise a second transgene which encodes a therapeutic polypeptide, an antigenic polypeptide, an immune activating molecule, a cytokine, a guide RNA and / or a corrective DNA template.

[0037] The lentiviral vector may be an integration defective lentiviral vector (IDLV).

[0038] The invention also provides a cell comprising a viral (e.g. retroviral or lentiviral) vector according to the invention. The invention also provides a cell comprising a lentiviral vector according to the invention. The cell may be a hematopoietic stem and progenitor cell (HSPC).

[0039] The invention also provides a pharmaceutical composition comprising a viral (e.g. retroviral or lentiviral, preferably lentiviral) vector according to the invention, or a cell according to the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0040] The invention provides a method for producing a viral (e.g. retroviral or lentiviral) vector according to the invention, comprising the steps of:

[0041] a) introducing into a cell:

[0042] a. a vector encoding a fusion protein according to the invention,

[0043] b. a vector encoding an mRNA comprising a first transgene and a target site for the RNA-binding domain; and

[0044] c. a transfer vector and optionally one or more helper vector,

[0045] b) culturing the cell under conditions suitable for the production of the viral vector.

[0046] The invention provides a method for producing a lentiviral vector according to the invention, comprising the steps of:

[0047] a) introducing into a cell:

[0048] a. a vector encoding a fusion protein according to the invention,

[0049] b. a vector encoding an mRNA comprising a first transgene and a target site for the RNA-binding domain; and

[0050] c. a transfer vector and optionally one or more helper vector,

[0051] b) culturing the cell under conditions suitable for the production of the lentiviral vector.

[0052] The invention provides a viral (e.g. retroviral or lentiviral) vector according to the invention for use in a method of treating and / or preventing a disease in a subject. The invention provides a lentiviral vector according to the invention for use in a method of treating and / or preventing a disease in a subject.

[0053] The invention provides a cell according to the invention for use in a method of treating and / or preventing a disease in a subject.

[0054] The invention provides a pharmaceutical composition according to the invention for use in a method of treating and / or preventing a disease in a subject.

[0055] The transgene encoded on the mRNA may be transiently expressed by a cell. The transgene encoded on the vector genome may be stable integrated into the genome of the cell.

[0056] The method may further comprise administering a Cas ribonucleoprotein, or a guide RNA and a polynucleotide encoding a Cas protein. In some embodiments, the Cas ribonucleoprotein is administered in a virus like particle (VLP).

[0057] The invention provides a kit of vectors, comprising:

[0058] a) a vector encoding a fusion protein according to the invention,

[0059] b) a vector encoding an mRNA comprising a first transgene and a target site for the RNA- binding domain; and

[0060] c) a transfer vector and optionally one or more helper vector.

[0061] DESCRIPTION OF THE FIGURES

[0062] Figure 1. (a) Schematics of the LV structure incorporating mRNA molecules, (b) Delivery of GFP mRNA to HEK 293T cells through LVs by employing different MCP fusion proteins. The graph depicts flow cytometry (FC) analysis of GFP (mRNA) and mCherry (genomic LV) expression in transduced cells. Dots represents samples transduced with different concentrations of LV. (c-g) Delivery of GFP mRNA incorporating or not Wpre sequence to HEK293T cells through LVs by employing either PLC. MCP or MCP. Gag. Pol. In c, FC analysis of transduced cells. Dots represents samples transduced with different concentrations of LV from n=2 / 3 distinct vector preps per construct. In d, LV titer estimated measuring transgene (mCherry) expression by FC in transduced HEK 293T cells. Dots represent distinct vector preps. In e, viral particle number measured by dynamic light scattering. Dots represent distinct vector preps, bar represents mean values ± SEM. In f, Infectivity calculated as the ratio between titer and particle number. Dots represent distinct vector preps, bar represents mean values ± SEM. In g, ratio between GFP and HIV copies measured by RT-ddPCR of viral RNA. Dots represent distinct vector preps, bar represents bar represents mean values ± SEM. Statistics by unpaired t test, (h-i) Comparison between PP7 and MS2 systems for the delivery of GFP mRNA within Comet LV. In h, schematics of the elements of MS2 or PP7 system incorporated within Comet LV. In i, FC analysis of transduced cells. Dots represent samples transduced with different concentrations of LV from n=2 / 3 distinct vector preps per MS2 system, and n=1 vector prep for PP7 system.

[0063] Figure 2. (a-d) Vector production optimization for vectors incorporating PLC. MCP and GFP. Wpre. MS2 mRNA. In a, table reporting PLC. MCP and GFP. Wpre. MS2 plasmids content per plate employed during vector production for each formulation. In b to d, FC analysis of HEK 293T transduced with the same volume of each LV formulation.

[0064] Figure 3. (a-d) Delivery of GFP mRNA incorporating or not miRT-142 sequences to HEK293T cells either expressing or not miR-142. In a, experimental setting and LV schematics. In b, FC plots showing expression of GFP and mCherry in HEK 293T cells transduced with different LVs. NGFR+ cells express miR-142-3p; LNGFR- cells do not express miR-142-3p. In c and d, quantification of FC analysis. Bar represents mean values of n=3 replicates ± SD.

[0065] Figure 4. (a-d) Transduction of cell lines of KCs (iKCs), LSECs (bEnd.3), hepatocytes (Hepa 1-6) with LVs incorporating GFP mRNA with or without miRT-126 and miRT-122 sequences, and containing or not PLC. MCP. In a, experimental setting and LV schematics. In b to d, FC analysis of transduced cells, respectively iKCs, bEnd.3 and Hepa1-6. Dots represents cells transduced with different concentrations of LV. (e-k) In vivo delivery of LVs for the expression of liOva (OVA LV) or IFNa (IFN LV), and Comet LVs for the delivery of liOva mRNA together with IFNa expression (OVAmIFN LV). In e, experimental setting. In f, measure of IFNa levels in the plasma of transduced mice 7 days post LV injection. Dots represent mice, bars represent mean, error bars represent SD. In g, FC analysis of blood of transduced mice 7 days post LV injection. Dots represent mice, lines represent mean values ± SD. In h, LV copies per genome in the liver measured by ddPCR. Dots represent mice, line represents mean values ± SD. In i-k, FC analysis of liver of transduced mice 10 days post LV injection. Dots represent mice, line represents mean values ± SD. Statistics by two-way ANOVA. (l-q) Prophylactic vaccination of mice with LVs for the expression of liOva (OVA LV) or GFP (GFP LV), and Comet LVs for the delivery of liOva mRNA together with GFP expression (OVAmGFP LV), followed by subcutaneous injection of MC38 OVA cells. In I, experimental setting. In m, tumor measure 10 days post tumor injection. Dots represent mice, bars represent mean, error bars represent SD. Statistics by Mann-Whitney test with Holm-Šídák correction method. In n, gene expression analysis by digital droplet PCR (ddPCR) of tumor samples collected at sacrifice. Dots represent mice, lines represent mean values ± SD. In o, FC analysis of blood of transduced mice 7 days post tumor injection. Dots represent mice, line represents mean values ± SD. Statistics by Kruskal-Wallis. In p, FC analysis of tumor of transduced mice at sacrifice. Dots represent mice, line represents mean values ± SD. Statistics by Mann-Whitney with Holm-Šídák correction method. In q, FC analysis of liver of transduced mice at sacrifice. Dots represent mice, line represents mean values ± SD. Statistics by Kruskal-Wallis. Cells were defined as terminally exhausted (Tex, EOMES+PD1high) and progenitor exhausted (Pex, Tbet+PD1int).

[0066] Figure 5. (a) Schematics of LV used in the following experiments, (b-e) Delivery of CXCR4 mRNA by Comet LVs or electroporation to K562 cells. In b, experimental setting. In c, cell growth kinetic of treated cells. In d and e, FC analysis of treated cells showing CXCR4 expression, (f-k) Comparison between different protocols for the transduction of HSPCs with Comet LVs. In f, experimental setting. In g, cell growth kinetic of transduced cells. In h to k, FC analysis, (l-p) Delivery of CXCR4 mRNA by Comet LVs or electroporation to HSPCs. In I, experimental setting. In m, cell growth kinetic of transduced cells. In n to p, FC analysis, (q-s) Comparison of the engraftment potential of human HSPCs transiently expressing CXCR4 either via mRNA electroporation or CXCR4mGFP transduction, when transplanted into mobilized humanized mice. In q, experimental setting. In r and s, FC analysis of peripheral blood 12 weeks after second transplant. Dots represent mice, line represents mean values ± SD.

[0067] Figure 6. (a-c) Transduction of human macrophages (hMac) by employing standard GFP LVs or Comet LVs delivering VPX mRNA (VPXmGFP LV). In a, experimental setting and schematics of employed LV. In b, light-field and fluorescent microscopy images of hMac 3 days after LV transduction. In c, FC analysis showing percentage of cells expressing GFP or Cherry. Dots represent hMas cultures transduced with different concentrations of LV.

[0068] Figure 7. (a-c) Editing of iKCs by employing VLPs for the delivery of CAS9 ribonucleoprotein conjugated to a guide targeting mouse Atp6C locus and either standard donor IDLVs or IDLVs co-packaging an editing enhancer (Ad5). In a, experimental setting and schematics of employed VLP / IDLVs. In b, FC analysis 7 days post transduction. Dots represent independently transduced samples. In c, titers measured by ddPCR quantification of viral copies.

[0069] Figure 8. (a-d) Delivery of proliferation enhancers to human HSPCs by Comet LV (n = 3 donor / group). In a, experimental setting. In b, FC analysis showing transduction efficiency. Dots represent mean values ± SD. In c, growth curve. Dots represent mean values ± SD. In d, stem cell composition of the sample, measured by FC and adjusted by overall cell count. Bar represent mean values ± SD.

[0070] Figure 9. (a) Kinetics of expression of the integrating transgene (mCherry) and co-packaged mRNA (GFP) following LV transduction of Nalm6 cells. Reporter expression was monitored by live-cell imaging (Incucyte) acquired hourly after transduction. Fluorescence intensity was normalized to cell confluency and is presented as the median ± SEM of three biological replicates per timepoint. Nalm6 cells were transduced with standard lentiviral vectors encoding mCherry (mCherry LV) or CometLVs (with or without PLC MCP, as indicated) encoding mCherry and co-packaging GFP mRNA (PLC MCP- GFPmmCherry LV or GFPmmCherry LV). (b-c). Kinetics of expression of the integrating transgene (Blue Fluorescent Protein, BFP) and co-packaged mRNA (GFP) following LV transduction of activated primary human T cells. T cells from healthy donors were transduced with either an LV encoding BFP (BFP LV) or Comet LVs co-packaging GFP mRNA (GFPmBFP LV). Reporter expression was analyzed by flow cytometry at the indicated timepoints. In b, representative flow cytometry plots showing BFP and GFP expression in live T cells at the indicated timepoints. In c, percentages of BFP+and GFP+cells over time.

[0071] Figure 10. (a) Experimental setting, (b-c) Flow cytometry analysis of wild type Jurkat cells following transduction with either a VSV-G pseudotyped or an aCD3 pseudotyped GFP LV. Bars represent independently transduced cell cultures, (d-f) Wild type (JCK WT) or CXCR4 knock out (JCK KO) Jurkat cells transduced with either a VSV-G pseudotyped GFP LV, an aCD3 pseudotyped GFP LV, or an aCD3 pseudotyped Comet LV delivering CXCR4 mRNA (CXCR4mGFP LV). In d, flow cytometry plots. In e and f, flow cytometry analysis. Bars represent independently transduced cell cultures.

[0072] Figure 11. (a-c) Transduction of HEK 293T cells with Comet LVs carrying an integrating GFP payload and co-delivering murine IL12 mRNA (IL12mGFP LV) or respective LV control produced in absence of PLC. MCP accessory plasmid (PLC- LV). In a, experimental setting. In b, flow cytometry analysis of transduced cells. Dots represent independently transduced cell cultures, bars represent mean values ± SD. In c, IL12 ELISA on supernatants. Dots represent mean values ± SD.

[0073] Figure 12. (a) Schematics of Comet LV employed in following experiments, (b) Table reporting plasmids for the vector production of each LV employed in following experiments, (c-e) Evaluation of kinetics of induction of integrating transgene and co-packaged mRNA following LV transduction of HEK 293T cells. In c, FC plots of transduced HEK 293T cells at different timepoints. In d and e, transgene expression measured by FC over time (f) Evaluation of transgene persistence long-term following LV transduction of HEK 293T cells. The graph represents GFP and HIV quantification by ddPCR in genomic DNA extracted from HEK 293T cells 14 days after transduction.

[0074] Figure 13. (a) Schematics of Comet LV employed in following experiments, (b-g) Prophylactic vaccination of mice with LVs for the expression of liOva (OVA LV) or GFP (GFP LV), and Comet LVs for the delivery of liOVA mRNA alongside an integrating GFP payload (OVAmGFP LV), followed by intrahepatic injection of MC38 OVA cells (n=7 or 8). In b, experimental setting. In c, tumor weight at sacrifice. Dots represent mice, bars represent median with interquartile range. Statistics by Kruskal-Wallis test with Dunn’s correction. In d, Percentage of mice showing OVA clearance as measured by GEX by ddPCR of tumor samples collected at sacrifice (OVA expression = 0 for complete clearance, < 0.1 for partial clearance, > 0.1 for no clearance). In e and f, FC analysis of liver at sacrifice. Dots represent mice, bars represent median with interquartile range. Statistics by Kruskal-Wallis with Dunn’s correction. In g, LV copies per genome in the liver measured by ddPCR. Dots represent mice, lines represent mean ± SD. Statistics by Kruskal-Wallis with Dunn’s correction, (h-n) Therapeutic vaccination of mice challenged with intrahepatic MC38 OVA cell injection with an LV for the expression of IFNa (IFN LV), and Comet LVs for the delivery of liOVA mRNA alongside an integrating IFNa payload (OVAmIFN LV) (n=9). In h, experimental setting. In i, measure of IFNa levels in the plasma 14 days after tumor implant. Dots represent mice, bars represent mean ± SEM. In j, FC analysis of blood 14 days after tumor implant. Dots represent mice, bars represent mean ± SEM. Statistics by 2 way ANOVA with multiple corrections. In k, tumor weight at sacrifice. Dots represent mice, bars represent median with interquartile range. In I, GEX by ddPCR of sumor samples collected at sacrifice. Dots represent mice, bars represent mean ± SEM. In m and n, FC analysis of tumor and liver respectively. Dots represent mice, lines represent mean ± SD. Statistics by Kruskal-Wallis with Dunn’s correction.

[0075] Figure 14. (a-f) Comparison between CXCR4 mRNA delivery via Comet LV or electroporation in human HSPCs (n=3 distinct donors). In a, experimental setting. In b, cell growth kinetic of transduced cells. In c to f, FC analysis, (g-q) Comparison of the engraftment potential of human HSPCs transiently expressing CXCR4 either via mRNA electroporation or CXCR4mGFP transduction, when transplanted into mobilized humanized mice. In g, experimental setting. In h and i, FC analysis of peripheral blood 6, 9, and 12 weeks after transplant. Dots represent mean ± SEM. In j too, FC analysis of peripheral blood, bone marrow and spleen at sacrifice. Dots represent mice, lines or bars represent mean ± SEM. Statistics by Mann-Whitney test. In p, Shannon diversity index computed following ISA analysis on DNA extracted from human cells-enriched bone marrow samples. Statistics by Mann-Whitney test. In q, alluvial plots depicting IS abundance (>1%) in each sample. Each grayscale bar represents an individual IS with a relative abundance higher than 1%; bottom background areas include ISs that remained below the 1% threshold. Plots are stratified by experimental group.

[0076] DETAILED DESCRIPTION OF THE INVENTION

[0077] Fusion protein

[0078] The invention provides a fusion protein comprising a membrane-targeting domain and an RNA-binding domain, wherein the membrane-targeting domain and RNA-binding domain are linked by a cleavable peptide.

[0079] By “fusion protein” (or “chimeric protein”) is meant a protein created through the joining of two or more genes that originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins. For example, the fusion protein may be a single polypeptide chain, or comprise a single polypeptide chain comprising the membrane-targeting domain, RNA-binding domain, and cleavable peptide.

[0080] In some embodiments, the fusion protein is not operably linked (e.g. via a peptide bond) to a lentiviral structural protein. For example, the fusion protein is not operably linked to a protein encoded by gag, pol or env. In some embodiments, the fusion protein is not operably linked to a viral matrix, capsid or nucleocapsid protein.

[0081] In some embodiments, the fusion protein is not translated as a polypeptide comprising a lentiviral structural protein. For example, the fusion protein is not translated as a polypeptide comprising a protein encoded by gag, pol or env. In some embodiments, the fusion protein is not translated as a polypeptide comprising a viral matrix, capsid or nucleocapsid protein.

[0082] In some embodiments, the fusion protein comprises, in order from N-terminus to C-terminus, a membrane-targeting domain, a cleavable peptide, and an RNA-binding domain. In another aspect, the invention provides a fusion protein comprising, in order from N-terminus to C-terminus, a membrane-targeting domain, a cleavable peptide, and an RNA-binding domain.

[0083] In some embodiments, the cleavable peptide is a peptide cleavable by a viral protease. In some embodiments, the cleavable peptide is a peptide cleavable by a retroviral protease. In preferred embodiments, the cleavable peptide is a peptide cleavable by a lentiviral protease. In some embodiments, the fusion protein comprises, in order from N-terminus to C-terminus, a membrane-targeting domain, a peptide cleavable by a lentiviral protease, and an RNA-binding domain.

[0084] In some embodiments the fusion protein comprises or consists of an amino acid sequence according to SEQ ID NO: 1.

[0085] In some embodiments the fusion protein comprises or consists of an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to SEQ ID NO: 1.

[0086] Example PLC. PC. MCP construct (SEQ ID NO: 1)

[0087] MDSGRDFLTLHGLQDDEDLQALLKGSQLLKVKSSSWRRERFYKLQEDCKTIWQESRKVMRTPESQLFS IEDIQEVRMGHRTEGLEKFARDVPEDRCFSIVFKDQRNTLDLIAPSPADAQHWVLGLHKIIHHSGSMD QRQKLQHWIHSCLRKADKNKDNKMSFKELQNFLKELNIQVSQNYPIVQMASNFTQFVLVDNGGTGDVT VAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLN MELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

[0088] In some embodiments the fusion protein comprises or consists of an amino acid sequence according to SEQ ID NO: 2.

[0089] In some embodiments the fusion protein comprises or consists of an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to SEQ ID NO: 2.

[0090] Example SRC. PC. MCP construct (SEQ ID NO: 2).

[0091] MGSSKSKPKDPSQRRNNNNSQNYPIVQMASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQ AYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAM QGLLKDGNPIPSAIAANSGIY

[0092] In some embodiments the fusion protein comprises or consists of an amino acid sequence according to SEQ ID NO: 66.

[0093] In some embodiments the fusion protein comprises or consists of an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to SEQ ID NO: 66. Example PLC. PCP construct (SEQ ID NO: 66)

[0094] MDSGRDFLTLHGLQDDEDLQALLKGSQLLKVKSSSWRRERFYKLQEDCKTIWQESRKVMRTPESQLFS IEDIQEVRMGHRTEGLEKFARDVPEDRCFSIVFKDQRNTLDLIAPSPADAQHWVLGLHKIIHHSGSMD QRQKLQHWIHSCLRKADKNKDNKMSFKELQNFLKELNIQVSQNYPIVQMSKTIVLSVGEATRTLTEIQ STADRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLDQADVVDSGLPKVRYTQVWSHDVTIVAN STEASRKSLYDLTKSLVATSQVEDLVVNLVPLGR

[0095] Membrane-targeting domain

[0096] The fusion protein according to the invention comprises a membrane-targeting domain.

[0097] The membrane-targeting domain may be any peptide or protein that enables targeting of the fusion protein to the plasma membrane. Thus, the membrane-targeting domain may be able to associate with the host cell plasma membrane and / or otherwise be incorporated into the viral envelope within the viral particle. In this way, the membrane-targeting domain anchors the fusion protein to the viral envelope within the viral particle.

[0098] For example, the membrane-targeting domain may be a transmembrane domain. The membrane-targeting domain may temporarily attach to the plasma membrane through hydrophobic, electrostatic, and / or other non-covalent interactions. The membrane-targeting domain may be a membrane-integral signal peptide.

[0099] In some embodiments, the membrane-targeting domain comprises the pleckstrin homology domain from Phospholipase C-51.

[0100] PLC51 is a member of a family of inositol phospholipid-specific PLC isozymes involved in transducer-mediated intracellular responses. The ~120 aa pleckstrin homology domain can bind to phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2)], a phospholipid component of cell membranes, and thus localize to the plasma membrane with high affinity and specificity.

[0101] The membrane-targeting domain may further comprise an EF hand motif, or a fragment thereof.

[0102] In some embodiments, the membrane-targeting domain comprises or consists of an amino acid sequence according to SEQ ID NO: 3.

[0103] In some embodiments, the membrane-targeting domain comprises or consists of an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to SEQ ID NO: 3. Example phospholipase C-61 pleckstrin homology domain (SEQ ID NO: 3)

[0104] MDSGRDFLTLHGLQDDEDLQALLKGSQLLKVKSSSWRRERFYKLQEDCKTIWQESRKVMRTPESQLFS IEDIQEVRMGHRTEGLEKFARDVPEDRCFSIVFKDQRNTLDLIAPSPADAQHWVLGLHKIIHHSGSMD QRQKLQHWIHSCLRKADKNKDNKMSFKELQNFLKELNIQ

[0105] In some embodiments, the membrane-targeting domain comprises the membrane-targeting domain of a proto-oncogene tyrosine-protein kinase Src.

[0106] In some embodiments, the membrane-targeting domain comprises or consists of an amino acid sequence according to SEQ ID NO: 4.

[0107] In some embodiments, the membrane-targeting domain comprises or consists of an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to SEQ ID NO: 4.

[0108] Example membrane-targeting domain of a proto-oncogene tyrosine-protein kinase Src (SEQ ID NO: 4)

[0109] MGSSKSKPKDPSQRRNNNN

[0110] RNA-binding domain

[0111] The fusion protein according to the invention comprises a RNA-binding domain.

[0112] The RNA-binding domain may be any peptide or protein that is capable of binding an RNA molecule, in order to complex the fusion protein to an mRNA of interest. The mRNA may comprise a transgene and a target site corresponding to the RNA-binding domain. The RNA-binding domain may specifically bind to the target site on the mRNA molecule. Thus, the RNA-binding domain enables the formation of ribonucleoprotein complexes comprising the fusion protein and mRNA of interest, such that mRNA molecules are co-packaged in the viral particle.

[0113] In some embodiments, the RNA-binding domain comprises an aptamer-binding protein. An aptamer-binding protein may be a protein that is recognised by or bound by an aptamer.

[0114] Suitably, the aptamer-binding protein and / or RNA-binding domain is an MS2 phage capsid protein.

[0115] In some embodiments, the RNA-binding domain comprises an MS2 phage capsid protein. In some embodiments, the RNA-binding domain comprises or consists of an amino acid sequence according to SEQ ID NO: 5.

[0116] In some embodiments, the RNA-binding domain comprises or consists of an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to SEQ ID NO: 5.

[0117] Example MS2 phage capsid protein (SEQ ID NO: 5)

[0118] MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKV ATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

[0119] Suitably, the aptamer-binding protein and / or RNA-binding domain is a Pseudomonas phage PP7 capsid protein.

[0120] In some embodiments, the RNA-binding domain comprises a Pseudomonas phage PP7 capsid protein.

[0121] In some embodiments, the RNA-binding domain comprises or consists of an amino acid sequence according to SEQ ID NO: 67.

[0122] In some embodiments, the RNA-binding domain comprises or consists of an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to SEQ ID NO: 67.

[0123] Example Pseudomonas phage PP7 capsid protein (SEQ ID NO: 67)

[0124] MSKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLDQADVVDS GLPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQVEDLVVNLVPLGR

[0125] Cleavable peptides

[0126] The fusion protein comprises a cleavable peptide.

[0127] The cleavable peptide enables the separation of the RNA-binding domain and membranetargeting domain, thus making the RNA-binding domain-mRNA complex available to ribosomes in the cytoplasm for translation.

[0128] In some embodiments, the cleavable peptide is a peptide cleavable by a viral protease. In some embodiments, the cleavable peptide is a peptide cleavable by a retroviral protease. In preferred embodiments, the cleavable peptide is a peptide cleavable by a lentiviral protease. In preferred embodiments, the cleavable peptide is a peptide cleavable by an HIV protease.

[0129] In these embodiments, cleavage may occur during viral particle maturation.

[0130] In some embodiments, the peptide cleavable by a lentiviral protease is an HIV protease cleavage sequence. In some embodiments, the cleavable peptide comprises or consists of SEQ ID NO: 6.

[0131] VSQNYPIVQ (SEQ ID NO: 6)

[0132] In some embodiments, the cleavable peptide comprises or consists of SEQ ID NO: 7.

[0133] SQNYPIVQ (SEQ ID NO: 7)

[0134] Any suitable cleavable peptide may be used. Exemplary cleavable peptides are shown in SEQ ID NOs: 8, 9, 10, and 61.

[0135] ARVLAEAM (SEQ ID NO: 8)

[0136] ATIMMQKG (SEQ ID NO: 9)

[0137] RQANFLGK (SEQ ID NO: 10)

[0138] TAIMMQKG (SEQ ID NO: 61)

[0139] Polynucleotide

[0140] In a further aspect, the invention provides a polynucleotide encoding a fusion protein according to the invention.

[0141] Thus, the invention provides a polynucleotide encoding a fusion protein comprising a membrane-targeting domain and an RNA-binding domain, wherein the membrane-targeting domain and RNA-binding domain are linked by a cleavable peptide.

[0142] In some embodiments, the invention provides a polynucleotide encoding a fusion protein comprising or consisting of an amino acid sequence having SEQ ID NO: 1, or a sequence having at least 80% sequence identity thereto.

[0143] An exemplary polynucleotide sequence is set forth in SEQ ID NO: 11. In some embodiments, the polynucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 11.

[0144] In some embodiments, the polynucleotide comprises or consists of a nucleic acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to SEQ ID NO: 11.

[0145] PLC. PC. MCP nucleic acid sequence (SEQ ID NO: 11)

[0146] ATGGACTCGGGCCGGGACTTCCTGACCCTGCACGGCCTACAGGATGATGAGGATCTACAGGCGCTGCT GAAGGGCAGCCAGCTCCTGAAGGTGAAGTCCAGCTCATGGAGGAGAGAGCGCTTCTACAAGTTGCAGG AGGACTGCAAGACCATCTGGCAGGAGTCCCGCAAGGTCATGCGGACCCCGGAGTCCCAGCTGTTCTCC ATCGAGGACATTCAGGAGGTGCGAATGGGGCACCGCACGGAGGGTCTGGAGAAGTTCGCCCGTGATGT GCCCGAGGACCGCTGCTTCTCCATTGTCTTCAAGGACCAGCGCAATACACTAGACCTCATCGCCCCAT CGCCAGCTGATGCCCAGCACTGGGTGCTGGGGCTGCACAAGATCATCCACCACTCAGGCTCCATGGAC CAGCGTCAGAAGCTACAGCACTGGATTCACTCCTGCTTGCGAAAAGCTGACAAAAACAAGGACAACAA GATGAGCTTCAAGGAGCTGCAGAACTTCCTGAAGGAGCTCAACATCCAGGTCAGCCAAAATTACCCGA TTGTGCAGATGGCCAGCAACTTCACCCAGTTCGTGCTGGTGGATAATGGCGGCACAGGCGACGTGACA GTGGCCCCTAGCAATTTCGCTAATGGCGTGGCCGAGTGGATCAGCAGCAACAGCAGATCCCAGGCCTA CAAAGTGACCTGCAGCGTGCGACAGTCTAGCGCCCAGAAGCGGAAGTACACCATCAAGGTGGAAGTGC CCAAGGTGGCCACACAGACAGTTGGCGGAGTGGAACTGCCAGTGGCCGCTTGGAGAAGCTACCTGAAC ATGGAACTGACAATCCCCATCTTCGCCACCAACAGCGACTGCGAGCTGATCGTGAAAGCCATGCAGGG CCTGCTGAAGGACGGCAACCCTATTCCTTCTGCCATTGCCGCCAACAGCGGCATCTACTGA

[0147] In some embodiments, the polynucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 68.

[0148] In some embodiments, the polynucleotide comprises or consists of a nucleic acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to SEQ ID NO: 68.

[0149] PLC. PCP nucleic acid sequence (SEQ ID NO: 68)

[0150] ATGGACTCGGGCCGGGACTTCCTGACCCTGCACGGCCTACAGGATGATGAGGATCTACAGGCGCTGCT GAAGGGCAGCCAGCTCCTGAAGGTGAAGTCCAGCTCATGGAGGAGAGAGCGCTTCTACAAGTTGCAGG AGGACTGCAAGACCATCTGGCAGGAGTCCCGCAAGGTCATGCGGACCCCGGAGTCCCAGCTGTTCTCC ATCGAGGACATTCAGGAGGTGCGAATGGGGCACCGCACGGAGGGTCTGGAGAAGTTCGCCCGTGATGT GCCCGAGGACCGCTGCTTCTCCATTGTCTTCAAGGACCAGCGCAATACACTAGACCTCATCGCCCCAT CGCCAGCTGATGCCCAGCACTGGGTGCTGGGGCTGCACAAGATCATCCACCACTCAGGCTCCATGGAC CAGCGTCAGAAGCTACAGCACTGGATTCACTCCTGCTTGCGAAAAGCTGACAAAAACAAGGACAACAA GATGAGCTTCAAGGAGCTGCAGAACTTCCTGAAGGAGCTCAACATCCAGGTCAGCCAAAATTACCCGA TTGTGCAGATGTCCAAAACCATCGTTCTTAGCGTCGGCGAGGCTACTCGCACTCTGACTGAGATCCAG TCCACCGCAGACAGACAGATCTTCGAAGAGAAGGTCGGGCCTCTGGTGGGTCGGCTGCGCCTCACGGC CAGCCTCAGACAAAACGGAGCCAAGACCGCGTATCGCGTCAACCTGAAACTGGATCAGGCGGACGTCG TTGATAGCGGACTTCCGAAAGTGCGCTACACTCAGGTATGGTCCCACGACGTGACAATCGTTGCGAAT AGCACCGAGGCCAGCCGCAAATCTTTGTACGATTTGACCAAGTCCCTCGTCGCGACCAGCCAGGTCGA AGATCTTGTCGTCAACCTTGTGCCGCTGGGCCGTTAA

[0151] The terms “polynucleotide” and "nucleic acid sequence" as used herein are interchangeable. “Polynucleotide” generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. A polynucleotide in relation to the present invention can be a double stranded or single stranded molecule and includes genomic DNA, cDNA, synthetic DNA, RNA and a chimeric DNA / RNA molecule.

[0152] It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described here to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.

[0153] Typically, the nucleic acid sequence encompassed by the scope of the present invention is prepared using recombinant DNA techniques (i.e. recombinant DNA).

[0154] Variants, derivatives, analogues, and fragments

[0155] In addition to the specific proteins and nucleotides mentioned herein, the invention also encompasses variants, derivatives, and fragments thereof.

[0156] In the context of the 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 retains at least one of its endogenous functions. A variant 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.

[0157] 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 residue from or to the sequence, providing that the resultant protein or polypeptide retains at least one of its endogenous functions.

[0158] Typically, amino acid substitutions may be made, for example from 1, 2 or 3, to 10 or 20 substitutions, provided that the modified sequence retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues. Proteins used in the invention may also 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 endogenous 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.

[0159] Conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and in the same line in the third column may be substituted for each other:

[0160]

[0161] Typically, a variant may have a certain identity with the wild type amino acid sequence or the wild type nucleotide sequence.

[0162] In the present context, a variant sequence is taken to include an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, suitably at least 95%, 96% or 97% or 98% or 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.

[0163] In the present context, a variant sequence is taken to include a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, suitably at least 95%, 96% or 97% or 98% or 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. 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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 of course 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.

[0168] 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 (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid - Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410), EMBOSS Needle (Madeira, F., et al., 2019. Nucleic acids research, 47(W1), pp. W636-W641) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, BLAST 2 Sequences, is also available for comparing protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174(2):247-50; FEMS Microbiol. Lett. (1999) 177(1): 187-8).

[0169] 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 (the default matrix for the BLAST suite of programs). GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

[0170] 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.

[0171] “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.

[0172] Such variants, derivatives, 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 protein. 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.

[0173] Vectors

[0174] The invention provides a vector comprising a fusion protein according to the invention.

[0175] The invention provides a vector encoding a fusion protein according to the invention.

[0176] In some embodiments, the invention provides a vector comprising:

[0177] a) a fusion protein according to the invention;

[0178] b) an mRNA comprising a first transgene and a target site for the RNA-binding domain;

[0179] and

[0180] c) a lentiviral genome, optionally wherein the lentiviral genome comprises a second transgene.

[0181] 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 and / or facilitating the expression of the protein encoded by a segment of nucleic acid.

[0182] Vectors may include a promoter for the expression of a polynucleotide and optionally a regulator of the promoter.

[0183] Vectors comprising polynucleotides may be introduced into cells using a variety of techniques known in the art, such as transfection, transduction and transformation.

[0184] Transfection may refer to a general process of incorporating a nucleic acid into a cell and includes a process using a non-viral vector to deliver a polynucleotide to a cell. Transduction may refer to a process of incorporating a nucleic acid into a cell using a viral vector.

[0185] Suitably, the vector of the invention is a viral vector. The vector of the invention may be a lentiviral vector, although it is contemplated that other viral vectors may be used. Other suitable viral vectors include those described in Lundstrom, et al. (2018) Diseases 6: 42. For example, other suitable viral vectors include a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes simplex viral 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.

[0186] The vector of the invention may be in the form of a viral vector particle. Suitably, the viral vector of the invention is in the form of a lentiviral vector particle.

[0187] The vector may be an integrating viral vector or a non-integrating viral vector. An “integrating viral vector” is capable of integrating into the host cell genome following transduction into the host cell. A “non-integrating viral vector” is not capable of integrating into the host cell genome following transduction into the host cell or demonstrates very weak integration capability.

[0188] Methods of preparing and modifying viral vectors and viral vector particles, such as lentiviral vectors, are well known in the art.

[0189] Retroviral and lentiviral vectors

[0190] The vector of the invention may, for example, be a retroviral vector or a lentiviral vector. The vector of the invention may, for example, be a retroviral vector particle or a lentiviral vector particle.

[0191] 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 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).

[0192] Retroviruses may be broadly divided into two categories, “simple” and “complex”. Retroviruses may be even further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses.

[0193] The basic structure of retrovirus and lentivirus 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 - these are polypeptides required for the assembly of viral particles. Lentiviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell.

[0194] In the provirus, these genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for proviral integration and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes.

[0195] The LTRs themselves are identical sequences that can be divided into three elements: U3, R and U5. U3 is derived from the sequence unique to the 3’ end of the RNA. R is derived from a sequence repeated at both ends of the RNA. U5 is derived from the sequence unique to the 5’ end of the RNA. The sizes of the three elements can vary considerably among different retroviruses.

[0196] In a defective retroviral vector genome gag, pol and env may be absent or not functional.

[0197] 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.

[0198] Lentivirus vectors are part of the larger group of retroviral vectors. In brief, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include but are not limited to human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS); and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototype “slow virus” visna / maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[0199] In some embodiments, the lentiviral vector is derived from HIV, such as HIV-1 or HIV-2.

[0200] The lentivirus family differs from retroviruses in that lentiviruses have the capability to infect both dividing and non-dividing cells. In contrast, other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.

[0201] A “lentiviral vector”, as used herein, is a vector which comprises at least one component part derivable from a lentivirus. Suitably, that component part is involved in the biological mechanisms by which the vector infects cells, expresses genes or is replicated.

[0202] The lentiviral vector may be a “primate” vector. The lentiviral vector may be a “non-primate” vector (i.e. derived from a virus which does not primarily infect primates, especially humans). Examples of non-primate lentiviruses may be any member of the family of lentiviridae which does not naturally infect a primate.

[0203] As examples of lentivirus-based vectors, HIV-1- and HIV-2-based vectors are described below.

[0204] The HIV-1 vector contains cis-acting elements that are also found in simple retroviruses. It has been shown that sequences that extend into the gag open reading frame are 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, most HIV-1 vectors 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 RRE, full-length HIV-1 RNAs 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 RRE. Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.

[0205] Most HIV-2-based vectors are structurally very similar to HIV-1 vectors. Similar to HIV-1-based vectors, HIV-2 vectors also require RRE for efficient transport of the full-length or singly spliced viral RNAs.

[0206] Optionally, the viral vector used in the invention has a minimal viral genome.

[0207] 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. Further details of this strategy can be found in WO 1998 / 017815.

[0208] Optionally, the plasmid vector used to produce the viral genome within a host cell / packaging cell will have sufficient lentiviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle which is capable of infecting a target cell, but is incapable of independent replication to produce infectious viral particles within the final target cell. Optionally, the vector lacks a functional gag-pol and / or env gene and / or other genes essential for replication.

[0209] However, the plasmid vector used to produce the viral genome within a host cell / packaging cell will also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a host cell / packaging cell. These regulatory sequences may be the natural sequences associated with the transcribed viral sequence (i.e. the 5’ U3 region), or they may be a heterologous promoter, such as another viral promoter (e.g. the CMV promoter).

[0210] The vectors may be self-inactivating (SIN) vectors in which the viral enhancer and promoter sequences have been deleted. SIN 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 should prevent mobilisation by replication-competent virus. This should also enable the regulated expression of genes from internal promoters by eliminating any cis-acting effects of the LTR.

[0211] The vector may be integration-defective. Integration defective lentiviral vectors can be produced, for example, either by packaging the 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 vector LTR, or by a combination of the above.

[0212] In some embodiments, the vector is an integration-defective lentiviral vector (IDLV). In some embodiments, the vector is an integration-proficient lentiviral vector.

[0213] The vector may be pseudotyped vector. The vector may be a pseudotyped lentiviral vector.

[0214] As used herein, the term “pseudotyped” refers to a vector that has been engineered to comprise a non-endogenous envelope protein, such as an envelope protein derived from another virus or viral vector, for example, in order to alter host cell tropism. The skilled person would be aware of suitable envelope proteins.

[0215] Suitably, the vector is VSV-G-pseudotyped. In some embodiments, the vector is a VSV-G-pseudotyped lentiviral vector particle. Suitably, the vector is aCD3-pseudotyped. In some embodiments, the vector is a aCD3-pseudotyped lentiviral vector particle. In some embodiments, the lentiviral vector comprises aCD3.

[0216] CD47highlentiviral particles

[0217] The lentiviral particle of the present invention may be a CD47highlentiviral particle. As used herein, a “CD47highlentiviral particle” may refer to a lentiviral particle with increased levels of CD47 (or a fragment thereof) on its surface. A CD47highlentiviral particle may have reduced uptake by professional phagocytes. In some embodiments the surface of a CD47highlentiviral particle comprises a higher level of CD47 protein than a control lentiviral particle produced in HEK293T cells (ATCC® CRL-1 1268™).

[0218] CD47 (Cluster of Differentiation 47) also known as integrin associated protein (IAP) is a transmembrane protein that in humans is encoded by the CD47 gene. Phagocytosis is physiologically inhibited by CD47, which is a ubiquitously expressed ligand of signal regulatory protein a (SIRP-a) receptor, that is expressed by professional phagocytes. CD47 may be incorporated into lentiviral particles when they bud from producer cells.

[0219] The lentiviral particle of the present invention may comprise one or more CD47 polypeptides (or a fragment thereof) on its surface. The amount of CD47 (or a fragment thereof) on the surface may be enough to reduce uptake by professional phagocytes. Any suitable assay to quantify the amount of CD47 polypeptides (or fragments thereof) present on the surface of the lentiviral particle may be used.

[0220] In some embodiments, the density of CD47 polypeptides (or fragments thereof) may be determined by immunostaining for CD47 and total internal reflection fluorescence microscopy, for example as described in US20100316570A1. The CD47 polypeptides (or fragments thereof) may be present in a density of at least about 20 molecules / μm2, at least about 25 molecules / μm2, at least about 30 molecules / μm2, at least about 35 molecules / μm2, at least about 40 molecules / μm2, at least about 45 molecules / μm2, at least about 50 molecules / μm2, at least about 60 molecules / μm2, at least about 70 molecules / μm2, at least about 80 molecules / μm2, at least about 90 molecules / μm2, at least about 100 molecules / μm2, at least about 150 molecules / μm2, at least about 200 molecules / μm2, at least about 250 molecules / μm2, at least about 300 molecules / μm2, at least about 350 molecules / μm2, at least about 400 molecules / μm2, at least about 450 molecules / μm2, at least about 500 molecules / μm2, at least about 600 molecules / μm2, at least about 700 molecules / μm2, at least about 800 molecules / μm2, at least about 900 molecules / μm2or at least about 1000 molecules / μm2. The CD47 polypeptides (or fragments thereof) may be present in a density of about 1000 molecules / μm2or less, about 500 molecules / μm2or less or about 250 molecules / μm2or less. The CD47 polypeptides (or fragments thereof) may be present in a density of from about 20 molecules / μm2to about 1000 molecules / μm2, from about 20 molecules / μm2to about 500 molecules / μm2or from about 20 molecules / μm2to about 250 molecules / μm2.

[0221] In some embodiments, the amount of CD47 polypeptides (or fragments thereof) may be determined by immunostaining for CD47 and electron microscopy, as described in Milani et al. (2019) Science Translational Medicine 11: eaav7325. The CD47 polypeptides (or fragments thereof) may be detected in an amount of at least about 10 gold particles / lentiviral particle, at least about 15 gold particles / lentiviral particle or at least about 20 gold particles / lentiviral particle. The CD47 polypeptides (or fragments thereof) may be detected in an amount of about 100 gold particles / lentiviral particle or less, about 80 gold particles / lentiviral particle or less or about 60 gold particles / lentiviral particle or less. The CD47 polypeptides (or fragments thereof) may be detected in an amount of from about 10 to about 100 gold particles / lentiviral particle, from about 15 to about 80 gold particles / lentiviral particle or from about 20 to about 60 gold particles / lentiviral particle.

[0222] CD47 is a member of the immunoglobulin (Ig) superfamily of membrane proteins, with a single IgV-like domain at its N-terminus, a highly hydrophobic stretch with five membrane-spanning segments and an alternatively spliced cytoplasmic C-terminus ranging in length from 3 to 36 amino acids. Mouse, rat, bovine and human CD47 molecules have been cloned and show about 70% overall amino acid identity (see, for example, Brown et al. (2001) Trends Cell Biology 11: 130-135).

[0223] The CD47 polypeptide (or a fragment thereof) may be a human CD47 polypeptide (or a fragment thereof). A CD47 polypeptide may have an amino acid sequence of UniProtKB Q08722.

[0224] Exemplary CD47 polypeptides are provided by SEQ ID NOs: 12-14. Suitably, a CD47 polypeptide comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to any of SEQ ID NOs: 12-14. Suitably, a CD47 polypeptide comprises or consists of the amino acid sequence of any of SEQ ID NOs: 12-14.

[0225] Example CD47 amino acid sequences: MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTF DGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSW FSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSL KNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLI S GL S I LALAQLLGL VYMKF VASNQ KT I Q P P RNN

[0226] (SEQ ID NO: 12)

[0227] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTF DGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSW FSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSL KNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLI SGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE

[0228] (SEQ ID NO: 13)

[0229] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTF DGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSW FSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSL KNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLI SGL S I LALAQLLGLVYMKFV

[0230] (SEQ ID NO: 14)

[0231]

[0232] The lentiviral particle of the present invention may be a CD47freelentiviral particle.

[0233] As used herein, a “CD47|OWlentiviral particle” may refer to a lentiviral particle with reduced levels of CD47 molecules on its surface (i.e. reduced levels of surface-exposed CD47 molecules).

[0234] As used herein, a “CD47freelentiviral particle” may refer to a lentiviral particle which is substantially devoid of (or free of) CD47 molecules on its surface (i.e. substantially devoid of (or free of) surface-exposed CD47 molecules). Specifically, the surface of the lentiviral particle may, for example, not comprise CD47.

[0235] In one embodiment, the viral particles each comprise less than 10, 5, 4, 3, 2 or 1 surface-exposed CD47 molecules.

[0236] In one embodiment, the viral particles each comprise less than 10 surface-exposed CD47 molecules. In one embodiment, the viral particles each comprise less than 5 surface-exposed CD47 molecules. In one embodiment, the viral particles each comprise less than 2 surface-exposed CD47 molecules. In one embodiment, the viral particles do not comprise any surface-exposed CD47 molecules.

[0237] MHC-Ilowor MHC-Ifreelentiviral particles

[0238] The lentiviral particle of the present invention may be a MHC-Ilowlentiviral particle or a M H C-lfreelentiviral particle. In preferred embodiments, the lentiviral particle of the present invention is a MHC-Ifreelentiviral particle.

[0239] As used herein, a “MHC-Ilowlentiviral particle” may refer to a lentiviral particle with reduced levels of one or more MHC-I molecules on its surface (i.e. reduced levels of surface-exposed MHC-I molecules). The number of surface-exposed MHC-I molecules may be reduced such that the immune response to the MHC-I is decreased to a therapeutically relevant degree.

[0240] As used herein, a “MHC-Ifreelentiviral particle” may refer to a lentiviral particle which is substantially devoid of (or free of) one or more MHC-I molecules on its surface (i.e. substantially devoid of (or free of) surface-exposed MHC-I molecules). Specifically, the surface of the lentiviral particle may not comprise MHC-I.

[0241] The major histocompatibility complex class I (MHC-I) is a heterodimeric membrane protein that is displayed on the outer leaflet of the cell membrane (see, for example, Penn et al. (2005) Major histocompatibility complex (MHC). eLS). MHC-I functions to bind and display peptide fragments of proteins to the extracellular environment where they may be recognised by CD8+ cytotoxic T cells. Peptide fragments generated from normal cellular proteins will not activate cytotoxic T cells due to central and peripheral tolerance mechanisms. However, foreign peptides (e.g. those originating from viral proteins) will cause activation of an immune response to destroy the cell. An allogeneic MHC-I protein itself may be recognised by the immune system. For example, antibodies may bind MHC-I epitopes directly. As a result, lentiviral particles that comprise MHC-I molecules originating from an allogeneic source may be targeted and neutralised by the immune system.

[0242] The term “MHC-I molecules” may refer to human MHC-I molecules. Human MHC-I is also referred to as human leukocyte antigen class I (HLA-I) and is expressed on almost all nucleated cells. HLA-I consists of two polypeptide chains, an HLA-I heavy chain (α chain) and β2 microglobulin (β2M or β chain). The HLA-I α chain and β2M are linked non-covalently. The HLA-I α chain is polymorphic. Six HLA-I α chains have been identified to date, including three classical, highly polymorphic α chains (HLA-A, HLA-B and HLA-C) and three non-classical, less polymorphic (HLA-E, HLA-F and HLA-G) α chains. The MHC-I molecules may comprise or consist of HLA-A, HLA-B and HLA-C molecules, which comprise an invariant β2M sequence. Any suitable assay to quantify the amount of MHC-I molecules present on the surface of the lentiviral particle may be used.

[0243] In some embodiments, the amount of MHC-I molecules may be determined by immunostaining for MHC-I and electron microscopy, for example as described in Milani et al. (2017) EMBO Molecular Medicine 9: 1558-1573. The MHC-I molecules may be detected in an amount of less than about 10 gold particles / lentiviral particle, less than about 9 gold particles / lentiviral particle, less than about 8 gold particles / lentiviral particle, less than about 7 gold particles / lentiviral particle, less than about 6 gold particles / lentiviral particle, less than about 5 gold particles / lentiviral particle, less than about 4 gold particles / lentiviral particle, less than about 3 gold particles / lentiviral particle, less than about 2 gold particles / lentiviral particle, less than about 1 gold particle / lentiviral particle or about 0 gold particles / lentiviral particle. The MHC-I molecules may be undetectable (e.g. the amount of gold particles detected may not be significantly higher than background levels).

[0244] The lentiviral particle of the present invention may be a CD47high / MHC-lfreelentiviral particle or a CD47high / MHC-ll0Wlentiviral particle. In preferred embodiments, the lentiviral particle of the present invention is a CD47high / MHC-lfreelentiviral particle.

[0245] Transgene

[0246] The vector of the present invention may comprise one or more transgene.

[0247] The transgene may be encoded on the vector genome. Such transgene may be stably integrated into a host cell genome in the methods according to the invention.

[0248] The transgene may be encoded on an mRNA incorporated into the vector particle. Such transgene may be transiently expressed in a host cell in the methods according to the invention.

[0249] The transgene is not particularly limited and any suitable transgene may be used. The transgene may encode a naturally-occurring human gene, or a variant and / or fragment thereof.

[0250] In some embodiments, the vector genome encodes a therapeutic polypeptide, an antigenic polypeptide, an immune activating molecule, or a cytokine. In some embodiments, the mRNA encodes a therapeutic polypeptide, an antigenic polypeptide, a cytokine, a chemokine receptor, a viral accessory protein, a transduction enhancer, or an enzyme involved in dNTP pool enhancement.

[0251] The transgene may be a therapeutic transgene.

[0252] The transgene may encode a therapeutic polypeptide and / or an antigenic polypeptide.

[0253] Suitably, the transgene encodes a therapeutic polypeptide.

[0254] As used herein, a “therapeutic polypeptide” is any polypeptide which can be used for therapy. For example, therapeutic polypeptides include therapeutic cytokines that can activate immune responses. Therapeutic polypeptides may include, for example, antibodies or antigen-binding proteins.

[0255] In some embodiments, the transgene encodes a cytokine, for example a cytokine that can activate immune responses, particularly anti-tumour responses.

[0256] Cytokines are molecular messengers that allow the cells of the immune system to communicate with one another to generate a coordinated, robust, but self-limited response to a target antigen. Cytokines directly stimulate immune effector cells and stromal cells at the tumour site, enhance tumour cell recognition by cytotoxic effector cells. Cytokines may have broad anti-tumour activity (Lee, S. and Margolin, K., 2011. Cancers, 3(4), pp.3856-3893).

[0257] For example, any cytokine which can activate immune responses, particularly anti-tumour responses can be used. Exemplary cytokines include IFN (e.g. a, p, and y), IL-2, IL-12, TNFa, CXCL9, and IL-1β. Further exemplary cytokines include I L10, IL15 or IL18. Further exemplary cytokines include GMCSF, FLT3, IL7 or IL21.

[0258] In some embodiments, the transgene encodes an antigen-binding protein. The antigenbinding protein may be, for example, a antibody, bispecific engager, antibody-cytokine fusion protein (immunocytokine), or chimeric antigen receptor (CAR).

[0259] In some embodiments, the transgene encodes a chimeric antigen receptor.

[0260] In some embodiments, the chimeric antigen receptor is encoded on the vector genome.

[0261] Suitably, the transgene encodes an antigenic polypeptide. As used herein, an “antigenic polypeptide” is any polypeptide which can induce an immune response. In particular, an antigenic polypeptide may be internalized and presented by an antigen-presenting cell (APC). Antigen presentation allows for specificity of adaptive immunity and can contribute to immune responses against both intracellular and extracellular pathogens. APCs also naturally have a role in fighting tumours, via stimulation of B and cytotoxic T cells to respectively produce antibodies against tumour-related antigens and kill malignant cells.

[0262] The antigen may be patient-specific.

[0263] In some embodiments, the transgene encodes a tumour antigen, for example a tumourspecific antigen or a tumour-associated antigen.

[0264] As used herein, a “tumour antigen” is an antigenic substance (e.g. antigenic polypeptide) produced in tumour cells. A “tumour-specific antigen” is present only on tumours cells and not on any other cell. A “tumour-associated antigen” is present on some tumour cells and also some normal cells.

[0265] Any suitable tumour antigen can be used. Suitable tumour antigens will be well known to those of skill in the art, for example tumour antigens are recorded in the Cancer Antigenic Peptide Database.

[0266] Suitably, the transgene encodes a chemokine receptor.

[0267] In some embodiments, the transgene encodes C-X-C chemokine receptor type 4 (CXCR4). CXCR4 (or a fragment or variant thereof) may be used for increasing engraftment by haematopoietic stem and / or progenitor cells (HSPCs).

[0268] C-X-C chemokine receptor type 4 (CXCR4) is a receptor expressed on the surface of HSPCs. The interaction of CXCR4 with CXCL12 is one of the major mechanisms that directs migration to the bone marrow. CXCR4 may also known as fusin or CD184. Mouse and human CXCR4 have been cloned and show about 91% overall amino acid identity (see e.g. Heesen, M., et al., 1996. The Journal of Immunology, 157(12), pp.5455-5460).

[0269] In a preferred embodiment, the CXCR4 is human CXCR4. A human CXCR4 may have an amino acid sequence of UniProtKB P61073. In some embodiments, the CXCR4 is isoform I.

[0270] Exemplary CXCR4 polypeptides are provided by SEQ ID NOs: 15 and 16. In one embodiment, the CXCR4 comprises or consists of an amino acid sequence that has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 15 or 16, preferably wherein the amino acid sequence substantially retains the engraftment enhancing activity of the protein represented by SEQ ID NO: 15 or 16, respectively.

[0271] In one embodiment, the CXCR4 comprises or consists of an amino acid sequence that has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 16, preferably wherein the amino acid sequence substantially retains the engraftment enhancing activity of the protein represented by SEQ ID NO: 16.

[0272] In one embodiment, the amino acid sequence of CXCR4 isoform II is:

[0273] MSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGL VILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNL YSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADD RYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVIL ILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLG AKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS

[0274] Exemplary CXCR4 isoform II (SEQ ID NO: 15)

[0275] In one embodiment, the amino acid sequence of CXCR4 isoform I is:

[0276] MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILV MGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSV LILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYIC DRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAF FACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFK TSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS

[0277] Exemplary CXCR4 isoform I (SEQ ID NO: 16)

[0278] Suitably, the transgene encodes a transduction enhancer.

[0279] Suitably, the transgene encodes a viral accessory protein.

[0280] In some embodiments, the transgene encodes Vpx. Vpx is a virion-associated protein encoded by human immunodeficiency virus type 2 and most simian immunodeficiency virus (SIV) strains. Vpx enhances HIV-2 replication in humans by counteracting the host factor SAMHD1. SAMHD1 is found in human myeloid cells, such as dendritic cells and macrophages, that restricts HIV-1 replication by depleting the cytoplasmic pool of deoxynucleoside triphosphates needed for viral DNA production. Vpx counteracts restriction by inducing the ubiquitin-proteasome-dependent degradation of SAMHD1. Vpx-mediated degradation of SAMHD1 therefore decreases deoxynucleoside triphosphate hydrolysis, thereby increasing the availability of dNTPs for viral reverse transcription in the cytoplasm.

[0281] In some embodiments, the transgene encodes a gene editing enhancer.

[0282] The term “gene editing enhancer” refers to an agent that increases the efficiency of gene editing.

[0283] Increasing the efficiency of gene editing may refer to an increase in the gene editing of the cells using a gene editing enhancer, in comparison to the gene editing achieved in the absence of the gene editing enhancer but under otherwise substantially identical conditions. Where a viral vector is used to introduce gene editing machinery, an increased efficiency may therefore allow the multiplicity of infection (MOI) and / or the time required to achieve effective transduction to be reduced.

[0284] In one embodiment, the percentage of cells which have been edited is increased. Methods for determining the percentage of cells which have been edited are known in the art. Suitable methods include flow cytometry, fluorescence-activated cell sorting (FACS) and fluorescence microscopy. The technique employed is preferably one which is amenable to automation and / or high throughput screening.

[0285] For example, a population of cells may be edited with a vector which harbours a reporter gene. Suitably, the reporter gene may be expressed when the cell has been edited. Suitable reporter genes include genes encoding fluorescent proteins, for example green, yellow, cherry, cyan or orange fluorescent proteins. Once the population of cells has been edited, both the number of cells expressing and not-expressing the reporter gene may be quantified using a suitable technique, such as FACS. The percentage of cells which have been edited may then be calculated.

[0286] Alternatively, quantitative PCR (qPCR) may be used to determine the percentage of cells which have been gene edited without the use of a reporter gene. For example, single colonies of cells (e.g. CD34+ cells) may be picked from a semi-solid culture and qPCR may be performed on each colony separately to determine the percentage of gene-edited-positive colonies among those analysed.

[0287] Methods for determining vector copy number are also known in the art. The technique employed is preferably one which is amenable to automation and / or high throughput screening. Suitable techniques include quantitative PCR (qPCR) and Southern blot-based approaches.

[0288] In one embodiment, gene editing enhancer for use according to the present invention improves gene editing efficiency compared with gene editing without use of the agent (i.e. standard gene editing). Suitably, gene editing efficiency may be improved by at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 1.8 fold, 1.9 fold, 2.0 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 3 fold or more.

[0289] In one embodiment, the gene editing enhancer for use according to the present invention improves gene editing efficiency compared with gene editing without use of the agent (i.e. standard gene editing). Suitably, the percentage of cells which have been edited is increased. Suitably, the percentage of cells which have been edited may be increased by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300% or more. Suitably, the percentage of the cells which have been edited may be 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

[0290] The gene editing enhancer may be a p53 inhibitor, such as i53, an inhibitor of 53BP1 which impairs non-homologous end joining (NHEJ) DNA repair mechanism, and its gain of function variants (e.g. L67R or T12V. T14H. L67H); or proteins actively involved in the HDR process, such as RAD51 and RAD52.

[0291] In some embodiments, the transgene encodes a p53 inhibitor.

[0292] The term “p53 inhibitor” refers to an inhibitor of p53 activation, the terms may be used interchangeably.

[0293] The term “p53 activation” refers to an increase in the activity of p53, for example through a post-translational modification of the p53 protein. Example post-translational modifications include phosphorylation, acetylation and methylation, and are described in Kruse, J. P. et al. (2008) Snapshot: p53 Posttranslational Modifications Cell 133: 930-931. In the context of the invention, the p53 activation preferably results from phosphorylation of p53, particularly preferably at amino acid Serine 15.

[0294] Methods for analysing such post-translational modifications are known in the art (example methods for analysing kinase activity are disclosed herein, further methods include, for example, mass spectrometry- and antibody recognition-based methods). In a preferred embodiment, the inhibitor of p53 activation is a dominant negative peptide (e.g. a dominant negative p53 peptide).

[0295] Suitably, a dominant negative peptide may comprise mutations in the homo-oligomerisation domain. Suitably, dominant negative peptides comprising mutations in the homooligomerisation domain may dimerise with wild-type p53 and prevent wild-type p53 from activating transcription.

[0296] In a preferred embodiment, the dominant negative peptide is GSE56 or a variant thereof.

[0297] In one embodiment, the amino acid sequence of GSE56 is set forth in SEQ ID NO: 17:

[0298] MDGWCPGRDRRTEEENFRKKEEHCPELPPGSAKRALPTSTSSSPQQKKKPLDGEYFTLKIRGRERFEM FRELNEALELKDARAAEESGDSRAHSSYPKIVS

[0299] In some embodiments, the transgene encodes GSE56 or a variant thereof, i53 or a gain of function variant thereof (for example, L67R or T12V. T 14H. L67H), RAD51 or RAD52.

[0300] In some embodiments, the transgene encodes GSE56, i53, i53 L67R, i53 T12V. T14H. L67H, RAD51 or RAD52.

[0301] In some embodiments, the transgene encodes GSE56, i53, i53 L67R, i53 T12V. T14H. L67H, RAD51 or RAD52, or a variant thereof.

[0302] Suitably, the transgene encodes cell cycle booster.

[0303] Suitably, the transgene encodes an adenoviral protein.

[0304] Adenoviruses are natural co-helpers of AAV infection and provide a set of genes: E1a, E1b, E2a and E4 which optimize AAV infection. The delivery of adenoviral proteins during gene editing may improve the efficiency of gene editing.

[0305] In one embodiment, the adenoviral protein is from an Adenovirus of serotype 5.

[0306] In one embodiment, the adenoviral protein is selected from the group comprising E1a, E1b, E2a and E4.

[0307] In a preferred embodiment, the adenoviral protein is an open reading frame of the E4 gene.

[0308] In a one embodiment, the adenoviral protein is E4orf1 or a variant thereof. In a one embodiment, the adenoviral protein is Ad5-E4orf1 or a variant thereof. An example of an amino acid sequence of Ad5-E4orf1 is set forth in SEQ ID No. 18. Suitably, the at least one adenoviral protein may comprise an amino acid sequence as set forth in SEQ ID No. 18 or a variant thereof.

[0309] MAAAVEALYVVLEREGAILPRQEGFSGVYVFFSPINFVIPPMGAVMLSLRLRVCIPPGYFGRFLALTD VNQPDVFTESYIMTPDMTEELSVVLFNHGDQFFYGHAGMAVVRLMLIRVVFPVVRQASNV

[0310] (SEQ ID NO 18)

[0311] Other examples of an amino acid sequence of E4orf1 are set forth in SEQ ID NO. 19 to SEQ ID NO. 38. Suitably, the at least one adenoviral protein may comprise an amino acid sequence as set forth in SEQ ID No. 19 to SEQ ID No. 38 or a variant thereof.

[0312]

[0313]

[0314] In a preferred embodiment, the adenoviral protein is E4orf6 / 7 or a variant thereof. In a preferred embodiment, the adenoviral protein is Ad5-E4orf6 / 7 or a variant thereof.

[0315] An example of an amino acid sequence of Ad5-E4orf6 / 7 is set forth in SEQ ID No. 39. Suitably, the at least one adenoviral protein may comprise an amino acid sequence as set forth in SEQ ID No. 39 or a variant thereof.

[0316] MTTSGVPFGMTLRPTRSRLSRRTPYSRDRLPPFETETRATILEDHPLLPECNTLTMHNAWTSPSPPVK QPQVGQQPVAQQLDSDMNLSELPGEFINITDERLARQETVWNITPKNMSVTHDMMLFKASRGERTVYS VCWEGGGRLNTRVL

[0317] (SEQ ID NO: 39)

[0318] Suitably, the transgene encodes a cell proliferation enhancer.

[0319] The term “cell proliferation enhancer” refers to an agent that can promote cell proliferation and expansion, particularly cells which have been transduced by the vector encoding the cell proliferation enhancer.

[0320] Promoting cell proliferation and expansion may refer to an increase in the proliferation and expansion of cells using an enhancer, in comparison to the proliferation and expansion achieved in the absence of the enhancer but under otherwise substantially identical conditions. Where a viral vector is used to introduce a transgene, increased proliferation and expansion of the fraction of transduced cells may therefore provide a selective advantage to transduced cells, thereby allowing to expand ex vivo engineered cells (e.g. HSPCs) before transplantation, or to enhance the efficacy of in vivo cells (e.g. HSPCs) gene therapy approaches. This approach may be particularly beneficial for cells having low transduction efficiency. The cell proliferation enhancer may be transiently expressed, and thereby provide a transient advantage to transduced cells.

[0321] Promotion of cell proliferation and expansion may be measured using techniques known in the art. Suitably, promotion of cell proliferation and expansion may be measured as described herein (see Example).

[0322] In one embodiment, the cell proliferation enhancer for use according to the present invention increases the transduced cell number compared with the transduced cell number obtained without use of the agent (i.e. standard proliferation and expansion). Suitably, cell proliferation and expansion may be improved by at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 1.8 fold, 1.9 fold, 2.0 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 3 fold or more. In one embodiment, the cell proliferation enhancer for use according to the present invention increases the transduced cell number compared with the transduced cell number without use of the agent (i.e. standard proliferation and expansion). Suitably, the number of transduced cells is increased following transduction. Suitably, the number of cells following transduction may be increased by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300% or more. Suitably, the number of cells may be 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

[0323] Suitably, agents that could be used to enhance cell proliferation are HoxB4, a transcription factor involved in HSPCs self-renewal; KrasQ61R, a gain-of-function variant of small GTPase KRAS involved in the signalling pathways regulating cell growth and differentiation; MYC, which promotes cell cycle entry and metabolic activation; CCND1 (Cyclin D1) and CCNE1 (Cyclin E1), which drive G1 / S transition; EVI1, a transcriptional regulator implicated in HSC expansion and survival; BMI1, which supports long-term maintenance by repressing senescence pathways; GATA2, TAL1, and RUNX1, key hematopoietic transcription factors essential for stem cell identity and differentiation balance; HLF, which preserves dormancy and functional potential of human HSCs; FOXO3, which maintains quiescence and oxidative stress resistance; and growth factor receptors or their downstream mediators such as FLT3, c-KIT (KIT), and STAT5A, which enhance proliferative responses to hematopoietic cytokines.

[0324] In some embodiments, the transgene encodes HoxB4, KrasQ61R, MYC, CCND1 (Cyclin D1), CCNE1 (Cyclin E1), EVI1, BMI1, GATA2, TAL1, RUNX1, HLF, FOXO3, FLT3, c-KIT (KIT), or STAT5A.

[0325] In some embodiments, the transgene encodes HoxB4, KrasQ61R, MYC, CCND1 (Cyclin D1), CCNE1 (Cyclin E1), EVI1, BMI1, GATA2, TAL1, RUNX1, HLF, FOXO3, FLT3, c-KIT (KIT), or STAT5A, or a variant thereof.

[0326] In some embodiments, the transgene encodes HoxB4 or KrasQ61R.

[0327] In one embodiment, the transgene encodes HoxB4.

[0328] In one embodiment, the transgene encodes KrasQ61R. In one embodiment, the transgene encodes KrasQ61Ror a variant thereof (e.g. a gain-of-function variant of small GTPase KRAS, such as wherein the gain-of-function mutation is at position Gin61).

[0329] In one embodiment, the amino acid sequence of HoxB4 is: MAMSSFLINSNYVDPKFPPCEEYSQSDYLPSDHSPGYYAGGQRRESGFQPEAAFGRRAPCTVQRYAAC RDPGPPPPPPPPPPPPPPGLSPRAPVQPTAGALLPEPGQRSEAVSSSPPPPPCAQNPLHPSPSHSACK EPVVYPWMRKVHVSTVNPNYAGGEPKRSRTAYTRQQVLELEKEFHYNRYLTRRRRVEIAHALCLSERQ IKI FQNRRMKWKKDHKLPNTKIRSGGTAGAAGGPPGRPNGGPPAL

[0330] (SEQ ID NO: 62)

[0331] In one embodiment, the amino acid sequence of KrasQ61Ris:

[0332] MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGREEYSAMR DQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQELARS YGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKKKKKKSRTRCTVM

[0333] (SEQ ID NO: 63)

[0334] In one embodiment, the amino acid sequence of HoxB4 is:

[0335] MAMSSFLINSNYVDPKFPPCEEYSQSDYLPSDHSPGYYAGGQRRESSFQPEAGFGRRAACTVQRYAAC RDPGPPPPPPPPPPPPPPPGLSPRAPAPPPAGALLPEPGQRCEAVSSSPPPPPCAQNPLHPSPSHSAC KEPVVYPWMRKVHVSTVNPNYAGGEPKRSRTAYTRQQVLELEKEFHYNRYLTRRRRVEIAHALCLSER QIKIWFQNRRMKWKKDHKLPNTKIRSGGAAGSAGGPPGRPNGGPRAL

[0336] (SEQ ID NO: 64)

[0337] In one embodiment, the amino acid sequence of KrasQ61Ris:

[0338] MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGREEYSAMR DQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARS YGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKKKKKKSKTKCVIM

[0339] (SEQ ID NO: 65)

[0340] In a preferred embodiment, the HoxB4 and / or KrasQ61Ris human HoxB4 and / or KrasQ61R. In one embodiment, the transgene encodes human KrasQ61Ror a variant thereof (e.g. a gain-of-function variant of small GTPase KRAS, such as wherein the gain-of-function mutation is at position Gin61).

[0341] Exemplary cell proliferation enhancers are provided by SEQ ID NOs: 62 and 63. In one embodiment, the cell proliferation enhancer comprises or consists of an amino acid sequence that has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 62 or 63, preferably wherein the amino acid sequence substantially retains the cell proliferation enhancing activity of the protein represented by SEQ ID NO: 62 or 63, respectively.

[0342] In one embodiment, the cell proliferation enhancer comprises or consists of an amino acid sequence that has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 64 or 65, preferably wherein the amino acid sequence substantially retains the cell proliferation enhancing activity of the protein represented by SEQ ID NO: 64 or 65, respectively.

[0343] Suitably, the transgene encodes an enzyme involved in dNTP pool enhancement.

[0344] Suitably, the enzyme is a ribonucleotide reductase.

[0345] Suitably, the enzyme is a ribonucleotide reductase, thymidylate synthase, dihydrofolate reductase, nucleoside diphosphate kinase, deoxyuridine triphosphate nucleotidohydrolase, or a viral protein involved in de novo nucleotide biosynthesis (e.g. AAV E4ORF1).

[0346] Suitably, the transgene encodes a nuclease, for example a Cas protein, transcription activatorlike effector nuclease (TALEN) or zinc finger nuclease.

[0347] In some embodiments, the transgene encodes a Cas protein, such as a Cas9 protein.

[0348] In some embodiments, the Cas protein is encoded on the mRNA incorporated into the vector particle.

[0349] A “nuclease” is an enzyme that can cleave the phosphodiester bond present within a polynucleotide chain. Suitably, the nuclease is an endonuclease. Endonucleases are capable of breaking the bond from the middle of a chain.

[0350] An “RNA-guided nuclease” is a nuclease which can be directed to a specific site by a guide RNA. The present invention can be implemented using any suitable RNA-guided nuclease, for example any RNA-guided nuclease described in Murugan, K., et al., 2017. Molecular cell, 68(1), pp.15-25. RNA-guided nucleases include, but are not limited to, Type II CRISPR nucleases such as Cas9, and Type V CRISPR nucleases such as Cas12a and Cas12b, as well as other nucleases derived therefrom. RNA-guided nucleases can be defined, in broad terms, by their PAM specificity and cleavage activity.

[0351] Suitably, the RNA-guided nuclease is a Type II CRISPR nuclease, for example a Cas9 nuclease. Cas9 is a dual RNA-guided endonuclease enzyme associated with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) adaptive immune system. Cas9 nucleases include the well-characterized ortholog from Streptococcus pyogenes (SpCas9). SpCas9 and other orthologs (including SaCas9, FnCa9, and AnaCas9) have been reviewed by Jiang, F. and Doudna, J. A., 2017. Annual review of biophysics, 46, pp.505-529.

[0352] Suitably, the transgene encodes a guide RNA.

[0353] In some embodiments, the guide RNA is encoded on the vector genome.

[0354] A “guide RNA” (gRNA) confers target sequence specificity to a RNA-guided nuclease. Guide RNAs are non-coding short RNA sequences which bind to the complementary target DNA sequences. For example, in the CRISPR / Cas9 system, guide RNA first binds to the Cas9 enzyme and the gRNA sequence guides the resulting complex via base-pairing to a specific location on the DNA, where Cas9 performs its nuclease activity by cutting the target DNA strand.

[0355] The term “guide RNA” encompasses any suitable gRNA that can be used with any RNA-guided nuclease, and not only those gRNAs that are compatible with a particular nuclease such as Cas9.

[0356] The guide RNA may comprise a trans-activating CRISPR RNA (tracrRNA) that provides the stem loop structure and a target-specific CRISPR RNA (crRNA) designed to cleave the gene target site of interest. The tracrRNA and crRNA may be annealed, for example by heating them at 95°C for 5 minutes and letting them slowly cool down to room temperature for 10 minutes. Alternatively, the guide RNA may be a single guide RNA (sgRNA) that consists of both the crRNA and tracrRNA as a single construct.

[0357] The guide RNA may comprise of a 3’-end, which forms a scaffold for nuclease binding, and a 5'-end which is programmable to target different DNA sites. For example, the targeting specificity of CRISPR-Cas9 may be determined by the 15-25 bp sequence at the 5' end of the guide RNA. The desired target sequence typically precedes a protospacer adjacent motif (PAM) which is a short DNA sequence usually 2-6 bp in length that follows the DNA region targeted for cleavage by the CRISPR system, such as CRISPR-Cas9. The PAM is required for a Cas nuclease to cut and is typically found 3-4 bp downstream from the cut site. After base pairing of the guide RNA to the target, Cas9 mediates a double strand break about 3-nt upstream of PAM. Numerous tools exist for designing guide RNAs (e.g. Cui, Y., et al., 2018. Interdisciplinary Sciences: Computational Life Sciences, 10(2), pp.455-465). For example, COSMID is a webbased tool for identifying and validating guide RNAs (Cradick TJ, et al. Mol Ther - Nucleic Acids. 2014;3(12):e214).

[0358] Suitably, the transgene encodes a corrective DNA template or repair template.

[0359] The corrective DNA template may comprise a gene of interest, or suitably nucleotide sequence and 5’- and 3’-homology arms, which are homologous to a sequence either side of a target sequence. The corrective DNA template thus enables the insertion or correction of a target sequence by homology directed repair of a double strand break caused by a nuclease, such as an RNA-guided nuclease.

[0360] Lentiviral vectors according to the present invention may be employed to improve T cell-based therapies, including TCR-engineered T cell products, CAR T cell products and NK-based products.

[0361] Integrated lentiviral genome may provide the possibility to stably express a CAR, a TCR or another chimeric receptor. While transient mRNA expression may be employed to transiently confer a series of functionalities that could improve their transduction, efficacy, migration and activation.

[0362] For example, mRNA co-packaged in the lentiviral vector according to the present invention could encode for one or more selected from:

[0363] 1. Chemokine receptors, growth factor receptors, integrins, cell adhesion molecules, or tropic receptors, with the final goal of promoting T cell migration to the tissue of interest (e.g. CXCR3, CXCR4, CXCR6, CCR2, CCR5, CCR6, CCR7, CCR9, CCR10, ITGA4, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ICAM1, ICAM2, VCAM1, SELP, SELE, MET, EGFR, FGFR1, PDGFRA, PDGFRB, IGF1R).

[0364] 2. Therapeutic antibodies, including agonistic, blocking or depleting monoclonal antibodies, bispecific engagers, and antibody-cytokine fusion proteins (immunocytokines), chimeric antigen receptors (CARs), and nanobodies engineered for targeted immune modulation or tumor cell killing.

[0365] 3. Cell fitness enhancers that enhance proliferation and activation (e.g. CD28, ICOS, 4- 1BB (TNFRSF9), 0X40 (TNFRSF4), GITR (TNFRSF18), CD27, CD40, CD2, LCK, ZAP70, AKT1, AKT2, MTOR, KRAS, NRAS, HRAS, MYC, JUN, FOS, NFATC1, RELA, STAT3, STAT5A, STAT5B, IL2RA, IL2RB, IL7R, PIK3CA, PIK3CD, MAPK1, MAPK3) 4. Tumor antigens that promote the expansion and activation of preexisting T cells with superior effector capacity (e.g. MARTI (MLANA), PMEL (gp100), TYR, MAGEA1, MAGEA3, NY-ESO-1 (CTAG1B), PRAME, GAGE1, SSX2, WT1, TP53, KRAS(G12D), BRAF(V600E), EGFRvlll, HER2 (ERBB2), HPV16E6, HPV16E7, CMV pp65 (UL83), EBV LMP1, EBV EBNA1, Influenza HA, SARS-CoV-2 Spike (S), PSA (KLK3), CEA (CEACAM5), and MUC1).

[0366] 5. Cytokines and growth factors that promote T cell activation, migration and / or effector function, and / or immune activation, tumor microenvironment remodeling, and / or antigen presentation (e.g. IL2, IL7, IL12A, IL12B, IL15, IL18, IL21, IL23A, IFNG, TNF, LTA, LTB, CSF2 (GM-CSF), CSF1, FLT3LG, TGFB1, VEGFA, CXCL9, CXCL10, CXCL11, CCL2, CCL3, CCL4, CCL5, CCL19, CCL21, IFNA1, IFNB1).

[0367] Target site

[0368] Described herein is an mRNA comprising a transgene and a target site for a RNA-binding domain. The RNA-binding domain is present on the fusion protein, such that interaction between the RNA-binding domain and the target site on the mRNA results in a fusion protein-mRNA complex that may be incorporated into vector particles.

[0369] The target site may be an aptamer. An aptamer is a single-stranded oligonucleotide sequence that can fold into a defined architecture and bind to a specific target such as a protein. Most aptamers are based on a specific oligomer sequence of 20-100 bases.

[0370] In some embodiments the RNA-binding domain is an MS2 phage capsid protein, and the target site is an aptamer comprising an MS2 stem-loop nucleotide sequence or MS2 operator hairpin nucleotide sequence.

[0371] The MS2 stem-loop may have the nucleotide sequence of SEQ ID NO: 40:

[0372] ACATGAGGATCACCCATGT

[0373] In some embodiments, the mRNA comprises one, two, three, four, five, six, seven or more MS2 stem-loop sequences. In some embodiments, the mRNA comprises six MS2 stem-loop sequences.

[0374] In some embodiments, the mRNA described herein comprises the sequence SEQ ID NO: 41:

[0375] ACATGAGGATCACCCATGTCTGCAGGTCGACTCTAGAAAACATGAGGATCACCCATGTCTGCAGTATT CCCGGGTTCATTAGATCCTAAGGTACCTAATTGCCTAGAAAACATGAGGATCACCCATGTCTGCAGGT CGACTCTAGAAAACATGAGGATCACCCATGTCTGCAGTATTCCCGGGTTCATTAGATCCTAAGGTACC TAATTGCCTAGAAAACATGAGGATCACCCATGTCTGCAGGTCGACTCTAGAAAACATGAGGATCACCC ATGT

[0376] mRNA

[0377] The mRNA described herein may comprise one or more structural elements for improving stability and translation efficiency.

[0378] Any suitable structural elements may be used. Modifying mRNA structural elements, particularly the 5' cap, 5'-and 3'-untranslated regions (UTRs), the coding region, and polyadenylation tail, may help improve its intracellular stability and translational efficiency.

[0379] The structural elements may be operably linked to the transgene, when appropriate. The term “operably linked” may mean that the components described are in a relationship permitting them to function in their intended manner.

[0380] Expression control sequences

[0381] The vector of the invention may further comprise one or more regulatory elements which may act pre- or post-transcriptionally. Suitably, the transgene is operably linked to one or more regulatory elements which may act pre- or post-transcriptionally.

[0382] A “regulatory element” is any nucleotide sequence which facilitates expression of a polypeptide, e.g. acts to increase expression of a transcript or to enhance mRNA stability. Suitable regulatory elements include for example promoters, enhancer elements, post-transcriptional regulatory elements and polyadenylation sites.

[0383] Post-transcriptional regulatory elements

[0384] The vector of the invention may comprise one or more post-transcriptional regulatory element. Suitably, the transgene is operably linked to one or more post-transcriptional regulatory element. The post-transcriptional regulatory element may improve gene expression.

[0385] The vector of the invention may comprise a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE). Suitably, the transgene is operably linked to a WPRE.

[0386] In some embodiments, the mRNA described herein comprises a WPRE.

[0387] Suitable WPRE sequences will be well known to those of skill in the art (see, for example, Zufferey et al. (1999) Journal of Virology 73: 2886-2892; Zanta-Boussif et al. (2009) Gene Therapy 16: 605-619). Suitably, the WPRE is a wild-type WPRE or is a mutant WPRE. For example, the WPRE may be mutated to abrogate translation of the woodchuck hepatitis virus X protein (WHX), for example by mutating the WHX ORF translation start codon.

[0388] In some embodiments, the WPRE comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 42 or a fragment thereof. Suitably, the WPRE comprises or consists of a nucleotide sequence that has 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% sequence identity to SEQ ID NO: 42 or a fragment thereof.

[0389] In some embodiments, the WPRE comprises or consists of the nucleotide sequence SEQ ID NO: 42 or a fragment thereof.

[0390] AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTAC GCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCT CCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGC GTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCT TTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCT GCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTT CCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGC CCTCAATCCAGCGGACCTTCCTTCCCGC

[0391] (SEQ ID NO: 42)

[0392] Polyadenylation sequence

[0393] The vector of the invention may comprise a polyadenylation sequence. Suitably, the transgene is operably linked to a polyadenylation sequence. A polyadenylation sequence may be inserted after the transgene to improve transgene expression.

[0394] In some embodiments, the mRNA described herein comprises a polyadenylation sequence.

[0395] A polyadenylation sequence typically comprises a polyadenylation signal, a polyadenylation site and a downstream element: the polyadenylation signal comprises the sequence motif recognised by the RNA cleavage complex; the polyadenylation site is the site of cleavage at which a poly-A tail is added to the mRNA; the downstream element is a GT-rich region which usually lies just downstream of the polyadenylation site, which is important for efficient processing.

[0396] Suitable polyadenylation sequences will be well known to those of skill in the art (see, for example, Schambach et al. (2007) Molecular Therapy 15: 1167-1173; Choi et al. (2014) Molecular Brain 7: 1-10). Exemplary polyadenylation sequences include the bGH poly(A) signal sequence and SV40pA signal sequence. Kozak sequence

[0397] The vector of the invention may comprise a Kozak sequence. Suitably, the transgene is operably linked to a Kozak sequence. A Kozak sequence may be inserted before the start codon to improve the initiation of translation.

[0398] In some embodiments, the mRNA described herein comprises a Kozak sequence.

[0399] Suitable Kozak sequences will be well known to the skilled person (see, for example, Kozak (1987) Nucleic Acids Research 15: 8125-8148).

[0400] In some embodiments, the Kozak sequence comprises or consists of a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 43 or a fragment thereof.

[0401] In some embodiments, the Kozak sequence comprises or consists of the nucleotide sequence SEQ ID NO: 43 or a fragment thereof.

[0402] GCCACC

[0403] (SEQ ID NO: 43)

[0404] Other cis-acting elements

[0405] The vector (e.g. lentiviral vector) of the invention may comprise any other suitable cis-acting elements, such as one or more of a rev response element (RRE); a retroviral psi packaging element; a primer binding site (PBS); a TAT activation region (TAR); splice donor and acceptor sites; and central and terminal polypurine tracts.

[0406] Long terminal repeats (LTRs)

[0407] The vector (e.g. lentiviral vector) of the invention may comprise one or more long terminal repeat (LTR). LTRs are responsible for proviral integration and transcription. Typically, a naturally occurring LTR comprises U3, R, and U5 regions.

[0408] The vector (e.g. lentiviral vector) may comprise a 5’ LTR and / or a 3’ LTR. The vector (e.g. lentiviral vector) may comprise a 5’ LTR and a 3’ LTR. Suitably, a 5’ LTR comprises R and U5 regions, and optionally comprises a U3 region. Suitably, a 3’ LTR comprises U3, R and U5 regions.

[0409] Suitable LTR sequences will be well known to the skilled person (see, for example, Freeh et al. (1996) Virology 224: 256-267). In some embodiments, a LTR comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 44 or a fragment thereof. Suitably, a LTR comprises or consists of a nucleotide sequence that has 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% sequence identity to SEQ ID NO: 44 or a fragment thereof.

[0410] In some embodiments, a LTR comprises or consists of the nucleotide sequence SEQ ID NO: 44 or a fragment thereof.

[0411] TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTA GACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTT GCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGAC CCTTTTAGTCAGTGTGGAAAATCTCTAGCAG

[0412] (SEQ ID NO: 44)

[0413] The vector (e.g. lentiviral vector) of the invention may comprise 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, for example, Zufferey et al. (1998) Journal of Virology 72: 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.

[0414] Suitable SIN-LTR sequences will be well known to the skilled person (see, for example, Zufferey et al. (1998) Journal of Virology 72: 9873-9880; Miyoshi et al. (1998) Journal of Virology 72: 8150-8157).

[0415] In some embodiments, the 5’ LTR comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 45 or a fragment thereof. Suitably, the 5’ LTR comprises or consists of a nucleotide sequence that has 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% sequence identity to SEQ ID NO: 45 or a fragment thereof.

[0416] In some embodiments, the 5’ LTR comprises or consists of the nucleotide sequence SEQ ID NO: 45 or a fragment thereof.

[0417] GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAA GCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACT AGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG (SEQ ID NO: 45)

[0418] In some embodiments, the 5’ LTR and / or the 3’ LTR comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 44 or a fragment thereof. Suitably, the 5’ LTR and / or the 3’ LTR comprises or consists of a nucleotide sequence that has 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% sequence identity to SEQ ID NO: 44 or a fragment thereof.

[0419] In some embodiments, the 5’ LTR and / or the 3’ LTR comprises or consists of the nucleotide sequence SEQ ID NO: 44 or a fragment thereof.

[0420] In some embodiments, the 5’ LTR and the 3’ LTR comprise or consist of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 44 or a fragment thereof. Suitably, the 5’ LTR and the 3’ LTR comprise or consist of a nucleotide sequence that has 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% sequence identity to SEQ ID NO: 44 or a fragment thereof.

[0421] In some embodiments, the 5’ LTR and the 3’ LTR comprise or consist of the nucleotide sequence SEQ ID NO: 44 or a fragment thereof.

[0422] Primer binding site (PBS)

[0423] The vector (e.g. lentiviral vector) of the invention may comprise a primer binding site (PBS). A PBS is a cis-acting element where a primer may bind to initiate reverse transcription of the RNA genome (see, for example, Lanchy et al. (1998) Journal of Biological Chemistry 273: 24425-24432).

[0424] Suitable retroviral PBSs will be well known to the skilled person.

[0425] In some embodiments, a PBS comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 46 or a fragment thereof. Suitably, a PBS comprises or consists of a nucleotide sequence that has 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: 46 or a fragment thereof.

[0426] In some embodiments, a PBS comprises or consists of the nucleotide sequence SEQ ID NO: 46 or a fragment thereof. TGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCT TGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCG GAGGCTAGAAGGAGAGAG

[0427] (SEQ ID NO: 46)

[0428] Retroviral psi packaging element

[0429] The vector (e.g. lentiviral vector) of the invention may comprise a retroviral psi packaging element. A retroviral psi packaging element is a cis-acting element which is involved in regulating the process of packaging the retroviral RNA genome into the viral capsid during replication (see, for example, McBride et al. (1997) Journal of Virology 71: 4544-4554). A retroviral psi packaging element may form part of the 5’ region of the gag gene.

[0430] Suitable retroviral psi packaging elements will be well known to the skilled person.

[0431] In some embodiments, a retroviral psi packaging element comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 47 or a fragment thereof. Suitably, a retroviral psi packaging element comprises or consists of a nucleotide sequence that has 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% sequence identity to SEQ ID NO: 47 or a fragment thereof.

[0432] In some embodiments, a retroviral psi packaging element comprises or consists of the nucleotide sequence SEQ ID NO: 47 or a fragment thereof.

[0433] ATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGG CCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGC AGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCC TTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAA AGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGAC CACCGCACAGCAAGCGGCCGCTGAT

[0434] (SEQ ID NO: 47)

[0435] Rev response element (RRE)

[0436] The vector (e.g. lentiviral vector) of the invention may comprise a rev response element (RRE). A RRE is a cis-acting element that enables the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell (see, for example, Pollard et al. (1998) Annual Review of Microbiology 52: 491-532).

[0437] Suitable RRE sequences will be well known to the skilled person. In some embodiments, a RRE comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 48 or a fragment thereof. Suitably, a RRE comprises or consists of a nucleotide sequence that has 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: 48 or a fragment thereof.

[0438] In some embodiments, a RRE comprises or consists of the nucleotide sequence SEQ ID NO: 48 or a fragment thereof.

[0439] GGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGAC GGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGG CGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTG GAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTT

[0440] (SEQ ID NO: 48)

[0441] Central polypurine tract (cPPT)

[0442] The vector (e.g. lentiviral vector) of the invention may comprise a central polypurine tract (cPPT). A cPPT may allow initiation of plus-strand synthesis (see, for example, Follenzi et al. (2000) Nature Genetics 25: 217-222).

[0443] Suitable cPPT sequences will be well known to the skilled person.

[0444] In some embodiments, a cPPT comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 49 or a fragment thereof. Suitably, a cPPT comprises or consists of a nucleotide sequence that has 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% sequence identity to SEQ ID NO: 49 or a fragment thereof.

[0445] In some embodiments, a cPPT comprises or consists of the nucleotide sequence SEQ ID NO: 49 or a fragment thereof.

[0446] AACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAA C AG AC AT AC AAAC T AAAGAAT T AC AAAAAC AAAT T AC AAAAAT T C AAAAT T T T AT C

[0447] (SEQ ID NO: 49)

[0448] miRNA target sequence

[0449] The vector of the present invention may comprise one or more miRNA target sequence. Suitably, the transgene is operably linked to one or more miRNA target sequence. In some embodiments, the mRNA described herein comprises one or more miRNA target sequence(s).

[0450] MicroRNA (miRNA) genes are scattered across all human chromosomes, except for the Y chromosome. They can be either located in non-coding regions of the genome or within introns of protein-coding genes. Around 50% of miRNAs appear in clusters which are transcribed as polycistronic primary transcripts. Similar to protein-coding genes, miRNAs are usually transcribed from polymerase-ll promoters, generating a so-called primary miRNA transcript (pri-miRNA). This pri-miRNA is then processed through a series of endonucleolytic cleavage steps, performed by two enzymes belonging to the RNAse Type III family, Drosha and Dicer. From the pri-miRNA, a stem loop of about 60 nucleotides in length, called miRNA precursor (pre-miRNA), is excised by a specific nuclear complex, composed of Drosha and DiGeorge syndrome critical region gene (DGCR8), which crops both strands near the base of the primary stem loop and leaves a 5’ phosphate and a 2 bp long, 3’ overhang. The pre-miRNA is then actively transported from the nucleus to the cytoplasm by RAN-GTP and Exportin. Then, Dicer performs a double strand cut at the end of the stem loop not defined by the Drosha cut, generating a 19-24 bp duplex, which is composed of the mature miRNA and the opposite strand of the duplex, called miRNA*. In agreement with the thermodynamic asymmetry rule, only 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, as was demonstrated by single-nucleotide mismatches introduced into the 5’ end of each strand of siRNA duplexes. However, there are some miRNAs that support accumulation of both duplex strands to similar extent.

[0451] MicroRNAs trigger RNAi, very much like small interfering RNAs (siRNA) which are extensively used for experimental gene knockdown. The main difference between miRNA and siRNA is their biogenesis. Once loaded into RISC, the guide strand of the small RNA molecule interacts with mRNA target sequences preferentially found in the 3' untranslated region (3'UTR) of protein-coding genes. It has been shown that nucleotides 2-8 counted from the 5' end of the miRNA, the so-called seed sequence, are essential for triggering RNAi. If the whole guide strand sequence is perfectly complementary to the mRNA target, as is usually the case for siRNAs and plant miRNAs, the mRNA is endonucleolytically cleaved by involvement of the Argonaute (Ago) protein, also called “slicer” of the small RNA duplex into the RNA-induced silencing complex (RISC). DGRC (DiGeorge syndrome critical region gene 8) and TRBP (TAR (HIV) RNA binding protein 2) are double-stranded RNA-binding proteins that facilitate mature miRNA biogenesis by Drosha and Dicer RNase III enzymes, respectively. The guide strand of the miRNA duplex gets incorporated into the effector complex RISC, which recognises specific targets through imperfect base-pairing and induces post-transcriptional gene silencing. Several mechanisms have been proposed for this mode of regulation: miRNAs can induce the repression of translation initiation, mark target mRNAs for degradation by deadenylation, or sequester targets into the cytoplasmic P-body.

[0452] On the other hand, if only the seed is perfectly complementary to the target mRNA but the remaining bases show incomplete pairing, RNAi acts through multiple mechanisms leading to translational repression. Eukaryotic mRNA degradation mainly occurs through the shortening of the polyA tail at the 3’ end of the mRNA, and de-capping at the 5’ end, followed by 5’-3’ exonuclease digestion and accumulation of the miRNA in discrete cytoplasmic areas, the so called P-bodies, enriched in components of the mRNA decay pathway.

[0453] Expression of the transgene may be regulated by one or more endogenous miRNAs using one or more corresponding miRNA target sequences. Using this method, one or more miRNAs endogenously expressed in a cell prevent or reduce transgene expression in that cell by binding to its corresponding miRNA target sequence positioned in the vector or polynucleotide (Brown, B. D. et al. (2007) Nat Biotechnol 25: 1457-1467).

[0454] Suitable miRNA target sequences which suppress transgene expression in specific cells will be known to the skilled person. Any suitable method can be used to identify suitable miRNA target sequences, for example by performing microarrays containing known miRNAs, for example from miRbase.

[0455] More than one copy of a miRNA target sequence included in the vector may increase the effectiveness of the system. Also it is envisaged that different miRNA target sequences could be included. For example, the transgene may be operably linked to more than one miRNA target sequence, which may or may not be different. The miRNA target sequences may be in tandem, but other arrangements are envisaged. The vector may, for example, comprise 1, 2, 3, 4, 5, 6, 7 or 8 copies of the same or different miRNA target sequence. Suitably, the vector comprises 4 miRNA target sequences of each miRNA target sequence.

[0456] The target sequence may be fully or partially complementary to the 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 and effecting prevention or reduction of transgene expression in cells expressing that miRNA.

[0457] Copies of miRNA target sequences may be separated by a spacer sequence. The spacer sequence may comprise, for example, at least one, at least two, at least three, at least four or at least five nucleotide bases.

[0458] A vector driving transgene expression from a M2-like macrophage-specific promoter (e.g. the MRC1 promoter) can be used to drive selective transgene expression in Kupffer cells (KCs), and to a lesser extent in MRC1+ splenic macrophages and liver sinusoidal endothelial cells (LSECs). miRNA target sequences can be used to further increase the specificity of the vector.

[0459] The one or more miRNA target sequence may suppress transgene expression in some liver cell populations and / or some spleen cell populations. The one or more miRNA target sequence may suppress transgene expression in some liver macrophages and / or some spleen macrophages. For example, expression may be targeted to LSECs.

[0460] The term “suppress expression” as used herein may refer to a reduction of expression in the relevant cell type(s) of a transgene to which the one or more miRNA target sequence is operably linked as compared to transgene expression in the absence of the one or more miRNA target sequence, but under otherwise substantially identical conditions. In some embodiments, transgene expression is suppressed by at least 50%. In some embodiments, transgene expression is suppressed by at least 60%, 70%, 80%, 90% or 95%. In some embodiments, transgene expression is substantially prevented.

[0461] Suitably, the one or more miRNA target sequence suppresses transgene expression in liver sinusoidal endothelial cells (LSECs) and / or hepatocytes.

[0462] In some embodiments, the one or more miRNA target sequence suppresses transgene expression in hepatocytes and / or LSECS. For example, the vector may comprise (i) one or more copies of a miRNA target sequence that suppresses transgene expression in LSECs; and / or (ii) one or more copies of a miRNA target sequence that suppresses transgene expression in hepatocytes.

[0463] Suitably, the one or more miRNA target sequence comprises: (i) one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8) miR-126 target sequence; and / or (ii) one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8) miR-122 target sequence. The miR-126 target sequence is an exemplary miRNA target sequence that suppresses transgene expression in LSECs. miR-126 is a microRNA that is expressed in endothelial cells (e.g. LSEC), and when it binds to its target sequence it reduces the expression of the target gene.

[0464] In some embodiments of the invention, the miR-126 target sequence comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 50 or a fragment thereof. Suitably, the miR-126 target sequence comprises or consists of a nucleotide sequence which is at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 50 or a fragment thereof.

[0465] In some embodiments of the invention, the miR-126 target sequence comprises or consists of the nucleotide sequence SEQ ID NO: 50 or a fragment thereof.

[0466] Exemplary miRT-126

[0467] CGCATTATTACTCACGGTACGA

[0468] (SEQ ID NO: 50)

[0469] In some embodiments of the invention, the miR-126 target sequence comprises or consists of the nucleotide sequence SEQ ID NO: 51 or a fragment thereof.

[0470] CGCATTATTACTCACGGTACGACCATCGCATTATTACTCACGGTACGAACTTCGCATTATTACTCACG GTACGACGAACGCATTATTACTCACGGTACGA

[0471] (SEQ ID NO: 51)

[0472] The miR-122 target sequence is an exemplary miRNA target sequence that suppresses transgene expression in hepatocytes. miR-122 is the most abundant microRNA in hepatocytes, and when it binds to its target sequence it reduces the expression of the target gene.

[0473] In some embodiments of the invention, the miR-122 target sequence comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 52 or a fragment thereof. Suitably, the miR-122 target sequence comprises or consists of a nucleotide sequence which is at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 52 or a fragment thereof.

[0474] In some embodiments of the invention, the miR-122 target sequence comprises or consists of the nucleotide sequence SEQ ID NO: 52 or a fragment thereof.

[0475] Exemplary miRT-122 ACAAACACC AT T GT CACACT CC A

[0476] (SEQ ID NO: 52)

[0477] In some embodiments of the invention, the miR-122 target sequence comprises or consists of the nucleotide sequence SEQ ID NO: 53 or a fragment thereof.

[0478] ACAAACACCATTGTCACACTCCAACATACAAACACCATTGTCACACTCCAGATTACAAACACCATTGT C AC AC T C C AC AGAAC AAAC AC CAT T GT CACACT CCA

[0479] (SEQ ID NO: 53)

[0480] Further miRNA target sequences that suppresses transgene expression in LSECs and / or hepatocytes can be identified by any suitable method, for example miRNA expression analysis as described in Oda, S., et al., 2018. The American journal of pathology, 188(4), pp.916-928.

[0481] In some embodiments, the one or more miRNA target sequence comprises: (i) two or more miR-126 target sequences; and / or (ii) two or more miR-122 target sequences. In some embodiments, the one or more miRNA target sequence comprises: (i) four miR-126 target sequences; and / or (ii) four miR-122 target sequences. Suitably, the target sequences are separated by spacer sequences.

[0482] In some embodiments of the invention, the one or more miRNA target sequence comprises or consists of a nucleotide sequence which is at least 70% identical to one or more of SEQ ID NOs: 54-56 or a fragment thereof. Suitably, the one or more miRNA target sequence comprises or consists of a nucleotide sequence which is at least 80%, at least 90% or at least 95% identical to one or more of SEQ ID NOs: 54-56 or a fragment thereof.

[0483] In some embodiments of the invention, the one or more miRNA target sequence comprises or consists of the nucleotide sequence of one or more of SEQ ID NOs: 54-56 or a fragment thereof.

[0484] Exemplary miRT-1224 x miRT

[0485] TCTAGATAAACAAACACCATTGTCACACTCCATTCGAAACAAACACCATTGTCACACTCCAACGCGTA CAAACACCATTGTCACACTCCAATGCATACAAACACCATTGTCACACTCCACCCGGGTCGAGCTCGGT ACC

[0486] (SEQ ID NO: 54)

[0487] Exemplary miRT-1264 x miRT GGTACCAGCAAACGCATTATTACTCACGGTACGACCATCGCATTATTACTCACGGTACGAACTTCGCA TTATTACTCACGGTACGACGAACGCATTATTACTCACGGTACGACACGTGTCGGTACC

[0488] (SEQ ID NO: 55)

[0489] Exemplary miRT-122 and miR126 4 x miRT

[0490] GGTACCAGCGCTACAAACACCATTGTCACACTCCAACATACAAACACCATTGTCACACTCCAGATTAC AAACACCATTGTCACACTCCACAGAACAAACACCATTGTCACACTCCAGTTTAAACGCATTATTACTC ACGGTACGACCATCGCATTATTACTCACGGTACGAACTTCGCATTATTACTCACGGTACGACGAACGC ATTATTACTCACGGTACGACACGTGTCGGTACC

[0491] (SEQ ID NO: 56)

[0492] In some embodiments of the invention, the one or more miRNA target sequence comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 57 or a fragment thereof. Suitably, the one or more miRNA target sequence comprises or consists of a nucleotide sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 57 or a fragment thereof.

[0493] In some embodiments of the invention, the one or more miRNA target sequence comprises or consists of the nucleotide sequence SEQ ID NO: 57 or a fragment thereof.

[0494] Exemplary Afel-4xmiRT122-4xmiRT126-Pmll

[0495] AGCGCTACAAACACCATTGTCACACTCCAACATACAAACACCATTGTCACACTCCAGATTACAAACAC CATTGTCACACTCCACAGAACAAACACCATTGTCACACTCCAGTTTAAACGCATTATTACTCACGGTA CGACCATCGCATTATTACTCACGGTACGAACTTCGCATTATTACTCACGGTACGACGAACGCATTATT ACT CACGGT ACGAC ACGT GT C

[0496] (SEQ ID NO: 57)

[0497] In some embodiments of the invention, the miR-142 target sequence comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 58 or a fragment thereof. Suitably, the miR-142 target sequence comprises or consists of a nucleotide sequence which is at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 58 or a fragment thereof.

[0498] In some embodiments of the invention, the miR-142 target sequence comprises or consists of the nucleotide sequence SEQ ID NO: 58 or a fragment thereof.

[0499] Exemplary miR-142

[0500] TCCATAAAGTAGGAAACACTACA (SEQ ID NO: 58)

[0501] In some embodiments of the invention, the miR-142 target sequence comprises or consists of the nucleotide sequence SEQ ID NO: 59 or a fragment thereof.

[0502] TCCATAAAGTAGGAAACACTACACGATTCCATAAAGTAGGAAACACTACAACCGGTTCCATAAAGTAG G AAAC AC TAG AT C AC T C C AT AAAGT AGGAAAC ACT AC A

[0503] (SEQ ID NO: 59)

[0504] In some embodiments, the one or more miRNA target sequence suppresses transgene expression in some liver and / or some splenic macrophages. For example, the one or more miRNA target sequence may suppress transgene expression in M2-like macrophages. For example, the one or more miRNA target sequence may suppress transgene expression in Kupffer cells and / or MRC1+ splenic macrophages.

[0505] In some embodiments, the one or more miRNA target sequence suppresses transgene expression in splenic phagocytes (e.g. splenic macrophages).

[0506] miRNA target sequences that suppresses transgene expression in some liver and / or some splenic macrophages can be identified by any suitable method, for example miRNA expression analysis as described in Zhang, Y., et al., 2013. International journal of molecular medicine, 31(4), pp.797-802.

[0507] Exemplary mRNA

[0508] The mRNA as described herein may comprise from 5’ to 3’: a transgene, a target site for the RNA-binding domain and a poly A tail.

[0509] The mRNA as described herein may comprise from 5’ to 3’: a transgene, a target site for the RNA-binding domain, a WPRE element and a poly A tail.

[0510] The mRNA as described herein may comprise from 5’ to 3’: a transgene, a target site for the RNA-binding domain, one or more miRNA target sequences and a poly A tail.

[0511] The mRNA as described herein may comprise from 5’ to 3’: a transgene, a target site for the RNA-binding domain, a WPRE element, one or more miRNA target sequences, and a poly A tail.

[0512] The mRNA as described herein may comprise from 5’ to 3’: a transgene, MS2-stem loops and a poly A tail. The mRNA as described herein may comprise from 5’ to 3’: a transgene, MS2-stem loops, a WPRE element and a poly A tail.

[0513] The mRNA as described herein may comprise from 5’ to 3’: a transgene, MS2-stem loops, one or more miRNA target sequences and a poly A tail.

[0514] The mRNA as described herein may comprise from 5’ to 3’: a transgene, MS2-stem loops, a WPRE element, one or more miRNA target sequences, and a poly A tail.

[0515] An exemplary mRNA sequences is provided below in SEQ ID NO 60. In some embodiments, the RNA polynucleotide comprises or consists of a nucleotide sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 60. In some embodiments, the RNA polynucleotide comprises or consists of the nucleotide sequence of SEQ ID NO: 60.

[0516] Exemplary MCS. MS2. Wpre.miRT cloning site (SEQ ID NO: 60)

[0517] GAATTCACCGGTGCGGCCGCCTAGGATCCGACGAGCTGTACAAGTAAGAGCTCAATTACATGAGGATC ACCCATGTCTGCAGGTCGACTCTAGAAAACATGAGGATCACCCATGTCTGCAGTATTCCCGGGTTCAT TAGATCCTAAGGTACCTAATTGCCTAGAAAACATGAGGATCACCCATGTCTGCAGGTCGACTCTAGAA AACATGAGGATCACCCATGTCTGCAGTATTCCCGGGTTCATTAGATCCTAAGGTACCTAATTGCCTAG AAAACATGAGGATCACCCATGTCTGCAGGTCGACTCTAGAAAACATGAGGATCACCCATGTCTCGACA ATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACG CTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTC CTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCG TGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTT TCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTG CTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTC CATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCC CTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTAGAGCCTCTTCCGCGTCTTCGCCT TCCCTCGAG

[0518] Restriction sites for cloning of transgenes (at 5’) or miRNA targeting sequences (3’); Bacteriophages MS2 loops; Mutated Wpre

[0519] Method of production

[0520] In one aspect, the present invention provides a method of producing enveloped viral particles (e.g. lentiviral particles).

[0521] The method of production may comprise:

[0522] a) introducing into a cell (e.g. a host cell):

[0523] a. a vector encoding a fusion protein according to the invention,

[0524] b. a vector encoding an mRNA as described herein, and c. a transfer vector and optionally one or more helper vector

[0525] b) culturing the cell under conditions suitable to produce enveloped viral particles (e.g.

[0526] lentiviral particles) according to the present invention.

[0527] The enveloped viral particles (e.g. lentiviral particles) may then be obtained from the cell. The invention provides a vector obtained or obtainable by the method of production according to the invention.

[0528] As used herein, a “transfer vector” may encode the viral (e.g. lentiviral) genome of the present invention. Suitably, the transfer vector used to produce the lentiviral genome within a host cell / packaging cell will have sufficient lentiviral genetic information to allow packaging of an RNA genome, in the presence of packaging components (e.g. gag-pol, rev, env), into a viral particle which is capable of infecting a target cell, but is incapable of independent replication to produce infectious viral particles within the final target cell.

[0529] The transfer vector used to produce the viral genome within a host cell / packaging cell may include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a cell. These regulatory sequences may be the natural sequences associated with the transcribed viral sequence (i.e. the 5’ U3 region), or they may be a heterologous promoter, such as another viral promoter (e.g. the CMV promoter). The transfer vector may be a plasmid.

[0530] As used herein, a “helper vector” may encode one or more packaging components (e.g. gag-pol, rev, env). The nucleotide sequence encoding the packaging component(s) may be operably linked to a promoter (e.g. a CMV promoter or a RSV promoter) and / or a polyadenylation signal. The term “helper vector” may include “packaging vectors” (e.g. encoding gag-pol or rev) and “envelope vectors” (e.g. encoding an env gene, such as VSV-g). The helper vectors, packaging vectors and / or envelope vectors may be plasmids.

[0531] The transfer vector and / or one or more helper vector may be introduced into the host cell by any suitable technique known in the art, such as transfection, transduction and / or transformation. Suitably, the helper vectors may be transiently transfected or transduced into the host cell or may be stably maintained (e.g. stably integrated into the cell genome) within the host cell. Alternatively, a combination of transient transfection or transduction and stable maintenance may be used to introduce the helper vectors into the host cell.

[0532] Suitably, the transfer vector and / or the helper vectors may be plasmids and introduced by transfection. Suitably, a four plasmid system may be used consisting of a transfer plasmid and three helper plasmids. The three helper plasmids may consist of: a first helper plasmid encoding a gag-pol gene; a second helper plasmid encoding a rev gene; and a third helper plasmid encoding an env gene. Alternatively, a three plasmid system may be used which consists of a transfer plasmid, one helper plasmid encoding a gag-pol gene and a rev gene; and one helper plasmid encoding an env gene. Alternatively, a two plasmid system may be used in which all helper functions (e.g. gag-pol, rev and env) are encoded by one helper plasmid.

[0533] Any suitable host cell may be used to produce the enveloped viral particles (e.g. lentiviral particles). Suitable cells include producer cells and packaging cells, such as those described herein (e.g. HEK 293 or derivatives thereof). Suitable conditions for culturing the cell will be well known to the skilled person. For example, the cells may be incubated in culture medium (e.g. chemically defined medium) for from about 1 day to about 5 days (e.g. about 48 hours, about 54 hours or about 72 hours). For example, the cells may be incubated in culture medium (e.g. chemically defined medium) for from about 2 days to about 3 days.

[0534] The enveloped viral particles (e.g. lentiviral particles) may be obtained using any suitable methods known in the art. For example, the culture supernatant may be harvested and enveloped viral particles (e.g. lentiviral particles) subsequently purified from the culture supernatant (e.g. by centrifugation, membrane filtration and / or chromatography). The method of production may further comprise any other suitable process steps, for example DNA reduction, concentration, formulation and / or sterilisation.

[0535] Packaging / producer cell

[0536] The invention provides a cell comprising a polynucleotide encoding a fusion protein according to the invention.

[0537] The cell may be an isolated cell. Suitably, the cell is a mammalian cell, for example a human cell. The cell may be an isolated human cell.

[0538] Suitably, the cell may be a producer cell. The term “producer cell” may refer to a cell that produces viral particles, for example comprises (e.g. has been transiently transfected, stably transfected and / or transduced with) all the elements necessary to produce the viral particles. Suitable producer cells will be well known to the skilled person and may include HEK293, COS-1, COS-7, CV-1, HeLa, CHO and A549 cell lines. In some embodiments, the producer cell is a HEK293 cell, or a derivative thereof (e.g. a HEK293T cell, a HEK293T Lenti-X, a HEK293T-Rex cell, a HEK293FT cell, a HEK293SF-3F6 cell, a HEK293SF-3F9 cell, a HEK293-EBNA1 cell or a SJ293TS cell). Suitably, the cell may be a packaging cell. The term “packaging cell” may refer to a cell which contains some or all of the elements necessary for packaging a recombinant virus genome. Typically, such packaging cells contain one or more vectors which are capable of expressing viral structural proteins (e.g. gag-pol, rev, env) and / or one or more genes encoding the viral structural proteins have been integrated into the genome of the packaging cell. Cells comprising only some of the elements required for the production of enveloped viral particles are useful as intermediate reagents in the generation of viral particle producer cell lines, through subsequent steps of transient transfection, transduction or stable integration of each additional required element. These intermediate reagents are encompassed by the term “packaging cell”.

[0539] Packaging cells lacking a transfer vector may be used for the production of enveloped VLPs as they do not comprise a sequence encoding a viral genome.

[0540] The vector particle (e.g. lentiviral vector particle) of the present invention may be obtained from a cell that is genetically engineered to decrease expression of CD47 on the surface of the cell.

[0541] Decreased expression of CD47 on the surface of the cell refers to a decrease in the number of CD47 molecules that are expressed on the surface of the cell that has been genetically engineered, in comparison to the number of CD47 molecules that are expressed on the surface of a cell lacking the genetic engineering, but under otherwise substantially identical conditions.

[0542] The expression of CD47 on the surface of the cell may be decreased such that the number of surface-exposed CD47 molecules is, for example, less than about 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the number of surface-exposed CD47 molecules that are displayed in the absence of the genetic engineering. In one embodiment, the expression of CD47 on the surface of the cell is decreased such that the number of surface-exposed CD47 molecules is 0% of the number of surface-exposed CD47 molecules that are displayed in the absence of the genetic engineering.

[0543] The expression of CD47 on the surface of the cell is preferably decreased such that the cell is substantially devoid of surface-exposed CD47 molecules.

[0544] The term “substantially devoid” as used herein means that there is a substantial decrease in the number of CD47 molecules that are expressed on the surface of the cell that has been genetically engineered, in comparison to the number of CD47 molecules that are expressed on the surface of a cell lacking the genetic engineering (but under otherwise substantially identical conditions), such that enveloped viral particles produced by the cell exhibit a therapeutically useful increase in ability to transduce macrophages, phagocytes, antigen-presenting cells and / or monocytes, and / or induce a cytokine response upon systemic administration.

[0545] The vector particle (e.g. lentiviral vector particle) of the present invention may be obtained from a MHC-Ilowproducer cell or a MHC-Ifreeproducer cell. In preferred embodiments, the lentiviral particle of the present invention is obtained from a MHC-Ifreeproducer cell. As used herein, a “MHC-Ilowproducer cell” may refer to a producer cell with reduced levels of one or more MHC-I molecule on its surface. As used herein, a “MHC-Ifreeproducer cell” may refer to a producer cell which is substantially devoid of or free of one or more MHC-I molecule on its surface. Specifically, the surface of the lentiviral particle may, for example, not comprise MHC-I.

[0546] A MHC-Ilowor MHC-Ifreeproducer cell may be genetically engineered to decrease expression of MHC-I on the cell surface. For example, the cell may comprise a genetically engineered disruption of a gene encoding β2-microglobulin and / or a genetically engineered disruption of a gene encoding an MHC-I a chain.

[0547] Methods for genetic engineering to decrease protein expression are known in the art. For example, this may be achieved by targeted gene knockout. To decrease protein expression, the gene encoding the protein itself or its regulatory sequence (e.g. its promoter) may be knocked out. Knockout may be achieved by deletion of a section of the coding nucleic acid sequence, which may delete a section of the protein essential for expression or stability, or alter the reading frame of the coding sequence or by base-editing. Suitable methods for targeted gene knockout include use of zinc finger nucleases (ZFNs), transcription activatorlike effector nucleases (TALENs) and CRISPR / Cas-based RNA-guided nucleases (see e.g. Gaj et al. (2013) Trends Biotechnol 31: 397-405). For example, the CRISPR / Cas9 RNA-guided nuclease may be used to catalyse a double strand break at a specific locus in the genome if provided with appropriate RNA guides designed to bind that locus. Cas9 and the guide RNA may be delivered to a target cell by transfection of vectors encoding the protein and RNA. Cells attempt to repair any double strand breaks in their DNA using the non-homologous end joining (NHEJ) pathway. This is an error-prone mechanism which inserts random nucleotides and often disrupts the reading frame of the targeted gene. Alternatively, the genetic engineering to decrease protein expression may be accomplished using RNAi techniques, microRNA or antisense RNA to suppress expression of the target gene. Once the targeted gene knockout or suppression of expression approach has been carried out, the resulting population of cells may be screened to select and enrich for those cells exhibiting the phenotype of interest, for example decreased expression of surface-exposed MHC-I. Suitable techniques for screening and enrichment are known in the art and include flow cytometry and fluorescence-activated cell sorting (FACS).

[0548] The vector particle (e.g. lentiviral vector particle) of the present invention may be obtained from a CD47highproducer cell. As used herein, a “CD47highproducer cell” may refer to a producer cell with increased levels of CD47 (or a fragment thereof) on its surface.

[0549] A CD47highproducer cell may be genetically engineered to increase expression of CD47 (or a fragment thereof) on the cell surface. For example, the producer cell may comprise a vector encoding CD47 (or a fragment thereof) or may be edited to introduce a nucleotide sequence encoding CD47 (or a fragment thereof) into its genome. Suitably, the producer cell is transduced with a viral vector encoding a CD47 polypeptide (or a fragment thereof).

[0550] A CD47highproducer cell may have a higher concentration of CD47 (or a fragment thereof) on its surface than an unmodified producer cell. Suitably, the producer cell has at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold or at least about 30-fold more CD47 on its cell surface than an unmodified producer cell. Suitably, the producer cell has from about 5-fold to about 30-fold more CD47 (or a fragment thereof) on its cell surface than an unmodified producer cell.

[0551] Kits and systems

[0552] In one aspect, the present invention provides a kit or system for producing enveloped viral particles (e.g. lentiviral particles) of the present invention.

[0553] In one aspect, the present invention provides a kit of vectors comprising:

[0554] a) a vector encoding a fusion protein according to the invention,

[0555] b) a vector encoding an mRNA comprising a first transgene and a target site for the RNA- binding domain; and

[0556] c) a transfer vector and optionally one or more helper vector.

[0557] In one aspect, the invention provides a vector system, such as for use in producing enveloped viral particles (e.g. lentiviral particles) according to the invention. The vector system may comprise: a) a vector encoding a fusion protein according to the invention,

[0558] b) a vector encoding an mRNA comprising a first transgene and a target site for the RNA- binding domain; and

[0559] c) a transfer vector and optionally one or more helper vector.

[0560] The kit or system may further comprise an enveloped viral particle (e.g. lentiviral particle) producer or packaging cell of the invention. The kit or system may further comprise other reagents (e.g. transfection reagent, culture medium, etc.). The kit or system may further comprise any other suitable components, and optionally instructions for producing the enveloped viral particles (e.g. lentiviral particles) of the present invention.

[0561] Cell

[0562] In one aspect, the present invention provides a cell comprising the vector (e.g. lentiviral vector particle) of the invention.

[0563] In some embodiments, the vector(s) is comprised in a cell.

[0564] The cell may be an isolated cell. The cell may be a human cell, suitably an isolated human cell. The cell may be any cell type known in the art.

[0565] Pharmaceutical composition

[0566] The invention provides a pharmaceutical composition comprising a vector according to the invention.

[0567] The invention provides a pharmaceutical composition comprising a cell according to the invention.

[0568] A “pharmaceutical composition” may refer to a preparation which is stable and in a form which is acceptable to the patient.

[0569] The medicaments, for example vectors and vector particles, of the invention may be formulated into pharmaceutical compositions. These compositions may comprise, in addition to the medicament, a pharmaceutically acceptable carrier, diluent, excipient, buffer, stabiliser or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may be determined by the skilled person according to the route of administration, e.g. intravenous or intra-arterial. The pharmaceutical composition is typically in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, a surfactant, such as pluronic acid (PF68) 0.001% may be used. In some cases, serum albumin may be used in the composition.

[0570] For injection, the active ingredient may be in the form of an aqueous solution which is pyrogen-free, and has suitable pH, isotonicity and stability. The skilled person is well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection or Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and / or other additives may be included as required.

[0571] For delayed release, the medicament may be included in a pharmaceutical composition which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.

[0572] Handling of the cell therapy products is preferably performed in compliance with FACT-JACIE International Standards for cellular therapy.

[0573] Method of treatment

[0574] The invention provides a method of treating and / or preventing a disease comprising administering the vector, cell or pharmaceutical composition of the invention to a subject in need thereof.

[0575] In a related aspect, the present invention provides use of the vector, cell or pharmaceutical composition according to the invention in the manufacture of a medicament.

[0576] The invention provides a vector, a cell or a pharmaceutical composition according to the invention for use in a method of treating and / or preventing a disease in a subject.

[0577] The invention provides a vector, a cell or a pharmaceutical composition according to the invention for use in a method of therapy, for example gene therapy.

[0578] In another aspect, the invention provides a vector according to the invention for use in a method of therapy in vivo. The therapy may be, for example, gene therapy or CAR T cell therapy. The invention provides a vector, a cell or a pharmaceutical composition according to the invention for use in a method of vaccinating a subject.

[0579] In another aspect, the invention provides a method of transducing a cell, comprising contacting the cell with the vector of the invention. The method may be, for example, an in vivo, in vitro or ex vivo method.

[0580] In some embodiments, the method of transducing a cell, such as a T cell, comprises contacting the cell with the vector of the invention in vivo.

[0581] The vector of the invention may be used to transduce cells ex vivo. The transduced cells may subsequently be administered to a subject.

[0582] The invention provides a cell transduced by a vector of the invention. The invention provides a cell obtained or obtainable by the method according to the invention.

[0583] The cell may be a T cell. In some embodiments, the transgene encodes a chimeric antigen receptor.

[0584] The cell may be a hematopoietic stem and progenitor cell (HSPC). In some embodiments, the transgene encodes an engraftment enhancer, e.g. a CXCR4 protein.

[0585] In some embodiments, the transgene encodes a cell proliferation enhancer, e.g. HoxB4 or KrasQ61R. Suitably, the first transgene encodes a cell proliferation enhancer, e.g. HoxB4 or KrasQ61R. The increased proliferation and expansion of the fraction of transduced cells provided by the expression of the proliferation enhancer may therefore provide a selective advantage to transduced cells, thereby allowing to expand ex vivo engineered cells (e.g. HSPCs) before transplantation, or to enhance the efficacy of in vivo cells (e.g. HSPCs) gene therapy approaches. This approach may be particularly beneficial for cells having low transduction efficiency. The cell proliferation enhancer may be transiently expressed, and thereby provide a transient advantage to transduced cells.

[0586] The transgene encoded on the mRNA may be transiently expressed by the cell. The transgene encoded on the vector genome may be stably integrated into the genome of the cell.

[0587] In some embodiments, the vector is an integration-deficient lentiviral vector.

[0588] The integration-deficient lentiviral vector may comprise a genome which encodes for a guide RNA, and an mRNA which encodes for a Cas protein. The integration-deficient lentiviral vector may comprise a genome which encodes for a corrective DNA template, and an mRNA which encodes for a gene editing enhancer, a p53 inhibitor, a cell proliferation enhancer, a transduction enhancer, and / or an enzyme involved in dNTP pool enhancement. Suitably, the integration-deficient lentiviral vector may comprise a genome which encodes for a corrective DNA template, and an mRNA which encodes for a p53 inhibitor, a transduction enhancer, and / or an enzyme involved in dNTP pool enhancement.

[0589] The integration-deficient lentiviral vector may comprise a first transgene which encodes for a gene editing enhancer, a p53 inhibitor, a cell proliferation enhancer, a transduction enhancer, and / or an enzyme involved in dNTP pool enhancement, and a second transgene which encodes for a corrective DNA template.

[0590] The method may further comprise administering a Cas ribonucleoprotein, or a guide RNA and a polynucleotide encoding a Cas protein. In some embodiments, the method comprises administering a virus like particle (VLP) comprising a Cas ribonucleoprotein.

[0591] Thus, the combined transduction of Cas ribonucleoprotein, or a guide RNA and a polynucleotide encoding a Cas protein, and a vector according to the invention would provide all necessary elements for gene editing by RNA-guided gene editing system.

[0592] EXAMPLES

[0593] Results

[0594] PLC. MCP fusion protein and MS2 mRNA engineering allow increased transgene output in transduced cells

[0595] LVs were engineered to incorporate additional mRNA molecules exploiting the aptameraptamer binding protein system derived from bacteriophage MS2, in which secondary structures on RNA, termed MS2 loops, are specifically bound by MS2 capsid protein (MCP). To this aim, MCP was fused to either domains phospholipase C (PLC) or Proto-Oncogene Tyrosine-Protein Kinase Src (SRC), which are commonly found associated to the viral envelope within LVs, and expressed them during vector production together with an MS2 loopcontaining mRNA (Figure 1a). Between SRC and MCP, or PLC and MCP was incorporate a protein cleavage (PC) domain found in HIV, which is recognize and cleaved by HIV protease, resulting in SRC. PC. MCP and PLC. MCP constructs respectively. A version of SRC fused to MCP lacking the PC domain (SRC. MCP) was also included. The mRNA packaging capability of different MCP fusion proteins was tested in vectors incorporating GFP mRNA through MS2 aptamer sequences and encoding mCherry in the viral genome. An MCP fusion protein-free LV produced in presence of a plasmid encoding GFP. MS2 mRNA was employed as control to assess carryover of GFP protein / RNA. Different concentrations of the LVs were used to transduce HEK 293T cells and transduction efficiency was evaluated by measuring mCherry expression and MS2 mRNA delivery by GFP expression.

[0596] It was found that PLC. MCP promoted the highest expression of GFP in transduced cells (Figure 1b). Interestingly, PLC. MCP outperformed both the other tested constructs and the construct previously described by Ling and colleagues fusing MCP to the N terminal of viral Gag protein (MCP. Gag. Pol) (Ling et al., 2021, Nat Biomed Eng 5, 144-156). MS2 mRNA was also engineered to incorporate a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (Wpre) to enhance RNA stability and expression. It was found that MS2 mRNA incorporating Wpre promoted enhanced GFP expression within transduced cells independently of MCP fusion protein (Figure 1c).

[0597] LVs engineered to co-package MS2. Wpre-containing mRNA through PLC. MCP incorporation, hereon “co-packaged mRNA transferred from lentiviral vectors” (Comet LV), showed a reduction in titers and particle numbers compared to standard LVs (Figure 1d,e). Furthermore, infectivity of Comet LV, standard LVs and the previously described LVs were comparable (Figure 1f). GFP mRNA content within LV particles was checked by performing a reverse-transcription digital droplet PCR (RT-ddPCR) assay on viral RNA. It was found that Comet LV had a higher content of GFP. MS2 mRNA per viral particle compared to Ling et al technology, and that GFP. MS2 packaging was appreciable only in vectors containing both an MCP fusion protein and MS2 loops within GFP mRNA (Figure 1g). Interestingly, LV particles incorporating Wpre in the mRNA design did not show increased MS2 mRNA content compared to the Wpre deficient counterparts, suggesting that their advantage may reside in extended half-life and expression of MS2 mRNA molecules within transduced cells.

[0598] Alternative aptamer-aptamer binding protein systems for mRNA co-packaging within Comet LVs were also tested. The mRNA packaging capability of a PP7 bacteriophage-based system employing a PLC. PCP fusion protein in vectors incorporating GFP mRNA through PP7 aptamer sequences and encoding mCherry in the viral genome was tested. As controls, a Comet LV co-packaging GFP mRNA through MS2-based systems (as previously described) and MCP / PCP fusion protein-free LV produced in presence of a plasmid encoding GFP. MS2. Wpre mRNA (No MCP) were included. Compared to MS2 based system, PP7 system was less efficient in mediating GFP mRNA co-packaging but still showed enhanced GFP delivery compared to No MCP controls. These data support the feasibility of employing other aptamer-based systems for mRNA co-packaging within Comet LVs apart from MS2-derived systems (Figure 1 h, i).

[0599] Optimization of Comet LV production yields higher titers and more efficient mRNA transfer Subsequently, plasmid ratio in vector production was optimised to enhance both titers and MS2 mRNA packaging. Four different concentrations of PLC. MCP plasmid and 3 different concentrations of GFP. MS2. Wpre plasmid were tested, using a range of quantities spanning around those employed initially (Figure 2a). Furthermore, pAdVAntage plasmid was included among packaging plasmids during vector production to increase vector production yields. Comet LVs were once again produced employing GFP as MS2 mRNA transgene and mCherry as LV-genome encoded transgene, and subsequently used to transduce HEK 293T cells at standard concentration for all formulations. This allowed simultaneous evaluation of both titer / infectivity of the vector (through mCherry expression) and mRNA transfer capability (through GFP expression). As expected, higher concentrations of either PLC. MCP or GFP. MS2. Wpre plasmid during vector production impaired titers. Moreover, MS2 mRNA copackaging in LV particles increased proportionally with GFP. MS2. Wpre plasmid quantity (Figure 1b-d). Nevertheless, formulation 8 outperformed all the others in terms of both titer and mRNA delivery, and was thus employed for following applications.

[0600] Co-packaged mRNA expression can be post-transcriptionally regulated by miRNAs

[0601] The possibility to post-transcriptionally regulate co-packaged mRNA expression through the incorporation of miRNA target sites (miRT) downstream of Wpre was then tested. This strategy would allow to restrict mRNA transgene expression to specific cell types that do not express a miRNA of interest, and thus would prove particularly useful to abrogate transgene expression in unwanted cell populations in the context of in vivo gene therapy.

[0602] A cell line was generated in which half of the cells co-expressed miR-142 and LNGFR. This cell line was transduced with Comet LVs encoding mCherry in the viral genome and incorporating GFP mRNA molecules, which either contained or not miRT-142-3p sequences (Figure 3a). PLC. MCP-deficient LVs produced by including the GFP. MS2. Wpre. miRT plasmid during LV production were included as negative controls.

[0603] It was found that GFP expression was completely and specifically abrogated in LNGFR+ cells when miRT sequences were included in the transferred GFP mRNA. GFP expression in these cells reached levels comparable to those of PLC. MCP- deficient controls (Figure 3b-d). Of note, mCherry expression was similar in all conditions. Conversely, the presence of miRT sequences did not impact GFP expression in LNGFR-negative cells, due to absence of miR- 142-3p expression. Indeed, GFP expression in the LNGFR-negative fraction was not impacted by the presence or absence of the miRT sequences within the transferred GFP mRNA.

[0604] Interestingly, this observation further underlines how the viral RNA genome and the copackaged mRNA are differentially localized, and thus undergo distinct processing. Of note, the viral genome is resistant to mi RNA post-transcriptional regulation, and the incorporation of miRT sequences within the LV transfer genome doesn’t prevent its retrotranscription or integration. Inversely, co-packaged mRNA is readily available in the cytoplasm of transduced cells and thus is susceptible to post-transcriptional regulation by miRNAs.

[0605] Comet LV can deliver mRNA antigens for in vivo vaccination while inducing long-lasting immune-activating cytokine production

[0606] As proof-of-concept of Comet LV technology, it was investigated whether this platform could be employed to activate immune cells in a vaccination setting. To achieve this goal, systemically delivered Comet LV was employed to transiently convey antigens to liver antigen presenting cells (APCs). Importantly, it has been previously observed that upon intravenous administration, LVs efficiently target several splenic and hepatic cell populations. Among them, liver resident macrophages, termed Kupffer cells (KCs), represent the most abundant cell population. These macrophages, together with some subsets of splenic resident macrophages and hepatic dendritic cells, can act as professional APCs to trigger immune responses against antigens. However, other transduced cell populations, including liver sinusoidal endothelial cells (LSECs) and hepatocytes, are described to exert pro-tolerogenic effects when presenting antigens in the immune-suppressive hepatic environment.

[0607] To prevent antigen presentation in tolerogenic cell populations such as LSECs and hepatocytes, miRT against miR-126a-3p and miR-122-5p was incorporated in the copackaged mRNA to abrogate antigen expression in LSECs and hepatocytes, respectively. The expression of co-packaged mRNAs was measured in different murine cell lines of KCs (immortalized KCs, iKCs), LSECs (bEnd.3) or hepatocytes (Hepa 1-6) to investigate the selective silencing function of miRT sequences (Figure 4a). GFP expression was comparable among cell lines in Comet LVs incorporating GFP mRNAs without miRT sequences. On the other hand, the incorporation of miRT sequences in the co-packaged GFP mRNA prevented GFP expression selectively in bEnd.3 and Hepa 1-6, but not in iKCs (Figure 4b-d).

[0608] Subsequently, Comet LVs were tested in the context of in vivo vaccination. Comet LV employing interferon alpha (IFN) as integrating transgene and a version of chicken ovalbumin fused to CD74 invariant chain (OVA) as the transiently expressed mRNA incorporated in the LV was produced, hereon OVAmIFN LV. miRT sequences against miR-126a and miR-122 were included in the OVA mRNA to enable selective OVA expression and presentation in liver macrophages. LVs encoding either IFN or OVA, but not incorporating transiently expressed mRNAs, were included as controls, referred as IFN LV and OVA LV respectively. Notably, all vectors employed a KC-specific expression cassette for the expression of the integrating transgene (Figure 4e). KC-specific expression cassette was built upon a murine mannose receptor C-type 1 (Mrc1) promoter, as previously described.

[0609] Either OVAmIFN LV, IFNa LV, or IiOVA LV was injected intravenously (n = 5 mice / group) to immunocompetent C57BI6 mice at the dose of 1E+08 TU / mouse. Blood collection was performed 7 days after LV injection, and the experiment subsequently terminated at 10 days post LV injection. Circulating levels of IFNa 7 days post LV injection were comparable among IFN LV and OVAmIFN LV groups, as were IFNa effects on immune cell phenotype (Figure 4f,g). Similarly, vector copy number (VCN) in the liver of IFN LV and OVAmIFN LV mice was comparable, while it was reduced in OVA LV group. VCN reduction in the OVA LV mice might be a result of immune-driven clearance of cells, which were stably expressing OVA (Figure 4h). OVA-reactive CD8 T cells were detected in the liver of mice belonging both to OVA and OVAmIFN LV groups, although at lower levels in the latter, potentially due to the transient nature of antigen expression (Figure 4i). Interestingly, OVA-reactive T cells in OVAmIFN LV group displayed different phenotypical features compared to OVA group, with enhanced Ly6C expression, potentially linked to IFNa expression, and reduced exhaustion markers such as PD1 (Figure 4j). A similar phenotype was observed for the remaining CD8+ T cells, suggesting that transient antigen delivery might provide a more physiological activation and thus prevent exhaustion of the T cell compartment compared to chronic antigen presentation (Figure 4k).

[0610] The ability of Comet LV primed antigen-specific T cells to effectively clear antigen-expressing tumor cells was then tested. To this aim, a Comet LV encoding an integrating PGK. GFP expression cassette, to track transduced cells, and incorporating OVA as co-packaged mRNA (OVAmGFP LV) was produced. Intravenous injections (n = 10 mice / group) either with OVAmGFP LV, GFP LV, or OVA LV were given to immunocompetent C57BI6 mice at the dose of 1E+08 TU / mouse. 10 days post LV injection, the animals were challenged with a subcutaneous tumor injection of colorectal cancer cell line MC38 expressing chicken ovalbumin (MC38 OVA). Of note, MC38 OVA cell line is polyclonal as it contains both OVA-positive and OVA-negative tumor cells, and thus can be employed to monitor the selective clearance of antigen-expressing tumor clones. Blood collection was performed 7 days after tumor injection, and tumor growth was monitored from day 10 after tumor injection. The experiment was subsequently terminated 21 days after tumor injection (Figure 4I). Both OVA-vaccinated groups showed reduced tumor engraftment at day 10 after tumor implant (Figure 4m), however only OVAmGFP LVs induced the complete clearance of OVA-expressing clones from the tumor, while OVA LV seemed to induce tolerance against the antigen. This might be related to the distinct trafficking and phenotype of OVA-reactive T cells primed by integrating versus Comet LVs. OVAmGFP group showed increased Ly6C expression in circulating OVA-specific T cells, and enhanced OVA-specific T cells tumor infiltration compared to OVA LV group (Figure 4o-p). On the other hand, OVA-reactive T cells primed by OVA LV remained confined in the liver, where they acquired a terminally exhausted (Tex, EOMES+PD1high) phenotype (Figure 4q).

[0611] The ability of Comet LV to transfer antigens for T cell priming and activation in the context of oncology in both prophylactic and therapeutic settings was further validated.

[0612] Either OVAmGFP LV, GFP LV, or OVA LV were given to immunocompetent C57B / 6 mice (as described for Figure 4l-q). 10 days post LV injection, the animals were challenged with intrahepatic injection of MC38 OVA cells to obtain a murine model of CRC liver metastasis. Similar results in term of clearance of antigen-expressing tumor clones and phenotype of LV-primed OVA-reactive T cells were observed (Figure 13b-g). Taken together, these results support the capacity of Comet LV to deliver tumor antigens for the design of innovative vaccines.

[0613] The therapeutic potential of Comet LVs mediated vaccination in association to IFNa treatment in mice bearing established MC38-OVA liver metastases was evaluated (Figure 13h). One week after LV injection, IFNa was detected in the plasma of mice receiving OVAmIFN LV (Figure 13i). Moreover, the fraction of OVA-reactive T cells (i.e. tetramer+) showed a positive trend in mice treated with OVAmIFN LV compared to IFN LV and UT groups (Figure 13j).

[0614] OVA-reactive T cells in the OVAmIFN LV groups showed increased expression of activation markers such as Ly6C and Pd1 compared to UT. Tumor growth was reduced in IFN LV and OVAmIFN LV groups compared to UT mice, but only OVAmIFN LV induced a strong counterselection of OVA-expressing clones due to the activation of OVA-reactive T cells (Figure 13k, I). Tumor-infiltrating OVA-reactive T cells were virtually absent in the OVAmIFN LV group, in agreement with the loss of OVA expressing clones likely due to OVA-specific CD8 T cell activation (Figure 13m). Conversely, OVA-reactive T cells persisted in the liver in all groups (Figure 13n). Taken together, these results further support the use of Comet LVs as therapeutic vaccines that enable concurrent transient expression of antigens and sustained cytokine release. This dual activity promotes the activation of antigen-reactive T cells and remodelling of the immunosuppressive tumor microenvironment, ultimately leading to a therapeutic effect.

[0615] Taken together, these observations sustain the use of Comet LVs for mRNA vaccination applications, as these LVs retain infectivity in vivo and are able to efficiently prime antigenspecific T cells which are, in turn, able to effectively clear antigen-expressing tumor cells. Furthermore, Comet LVs enable combination with an immune-stimulating cytokine for immunotherapy, as transduced cells can transiently present the antigen of interest without being counter-selected by subsequently generated CD8 T cells.

[0616] Comet LV to transiently enforce the expression engrafting enhancers.

[0617] Hematopoietic stem and progenitor cell (HSPC) transplantation has proven useful in treating a number of human genetic pathologies through the mobilization, harvest, and ex vivo transduction of HSPCs. For transduced donor HSPCs to engraft, the bone marrow niche needs to be depleted through the use of chemotherapy-based conditioning regimens. In a recent study, Omer and colleagues propose the use of mobilization as a conditioning regimen to clear the bone marrow niche while sparing non-hematopoietic cells (Omer-Javed, et al., 2022, Cell 185, 2248-2264). In this approach, CXCR4 is transiently overexpressed through mRNA electroporation of ex vivo edited HSPCs to provide an engraftment advantage compared to mobilized host HSPCs.

[0618] Building on this approach, the possibility of using Comet LVs for transducing HSPCs through LV-based gene transfer while simultaneously delivering CXCR4 mRNA to provide a transient engraftment advantage to the transduced cells was tested. A Comet LV encoding a PGK. GFP expression cassette as integrating transgene and co-packaging CXCR4 mRNA was produced, hereon referred as CXCR4mGFP LV (Figure 5a). As control for this experiments a CXCR4mGFP LV produced in absence of PLC. MCP was employed, hereon referred as PLC-LV. CXCR4mGFP LV and PLC- LV was tested in K562 hematopoietic human cell line evaluating CXCR4 kinetics of expression and cell growth and viability in comparison to mRNA electroporation (Figure 5b). As expected, electroporation, although very efficient in delivering CXCR4 mRNA, severely impaired cell viability and slowed cell growth in comparison to transduction with Comet LVs (Figure 5c). Both CXCR4 mRNA electroporation and CXCR4mGFP LV transduction proved efficient in promoting CXCR4 expression, however while electroporation produced higher MFIs, CXCR4mGFP LV showed a more prolonged kinetic of expression, potentially due to different half-life of cellular mRNAs compared to in vitro transcribed RNAs employed in electroporation (Figure 5d). It’s important to notice that being CXCR4 a transmembrane protein, and being CXCR4 overexpressed during vector production, both CXCR4mGFP LV and PLC- LV envelopes would naturally be enriched in CXCR4 protein. Nevertheless, CXCR4mGFP LV provided an advantage in CXCR4 expression compared to pseudotransduction by protein carryover alone, as attested by the superior performance of CXCR4mGFP LV constructs compared to PLC- LV even at comparable transduction levels (Figure 5e).

[0619] Infectivity of Comet LVs on human HSPCs was tested. HSPCs were transduced with either a standard GFP LV or a CXCR4mGFP LV at MOI 100, testing a range of different transduction enhancers (Figure 5f). Transduction enhancers ciclosporin H (CsH), Lentibus (LB), both CsH + LB, prostaglandin E2 (PGE2), retronectin retroviral binding vector (Retro_RBV) or retronectin supernatant (Retro_SN) were employed, while no transduction enhancers were used as controls. Cell growth and viability was monitored on all conditions and found that they were all comparable except for cells transduced in Retro_RBV, in which cell growth was strongly inhibited (Figure 5g). Viability of the most primitive fraction of CD34+ CD90+ cells was preserved in all conditions except for cells transduced in presence of PGE2 (Figure 5h). In CD34+ CD90+ cells CXCR4 expression was high and sustained upon CXCR4mGFP LV transduction, and significantly increased compared to untransduced cells (Figure 5i). Only Retro_RBV condition seemed to display a more prolonged CXCR4 expression kinetic, probably related to reduced mRNA / protein dilution following the stall in cell growth. Interestingly, while CXCR4 expression was high and comparable among CXCR4mGFP LV-transduced cells, integrating GFP output widely varied depending on the employed transduction protocol (Figure 5 j,k). Interestingly, only transduction enhancers acting on mechanisms downstream of viral entry seemed to significantly improve GFP output, while CXCR4 expression was similar in all conditions.

[0620] Subsequently, CXCR4 mRNA delivery in HSPCs by CXCR4mGFP LV or CXCR4 mRNA electroporation was compared. HSPCs were transduced with CXCR4mGFP LV employing LB as transduction enhancer, and CXCR4 and GFP expression was monitored throughout the following 72h (Figure 5l). As expected, electroporation impacted negatively both cell growth kinetics and overall fraction of most primitive HSPCs, while CXCR4mGFP LV transduced cells showed no alterations compared to untransduced HSPCs (Figure 5m, n). CXCR4 electroporation in HSPCs showed both a higher and more sustained CXCR4 expression compared to Comet LVs delivery, potentially due to reduced mRNA / protein dilution following the stall in cell growth (Figure 5o). Nevertheless, Comet LV-treated cells showed a supra-physiological expression of CXCR4 compared to untransduced cells, even in presence of a relatively low overall transduction level (Figure 5p). Finally, it was tested whether Comet LV-mediated CXCR4 transient expression would grant an engraftment advantage in transduced HSPCs in agreement with previous observations using electroporated CXCR4. To this aim, hematochimeric NOD. Cg-KitW41JPrkdcscidH2rgtm1Wjl / WaskJ (NSGW41) mice engrafted with mobilized human CD34+ HSPCs were employed. Hematochimeric mice were then conditioned employing a previously described mobilization regiment (Omer-Javed et al. (2022). Cell 185, 2248-2264 e2221), and then transplanted with an equal dose of 3E+06 human CD34+ cells either (1) transduced with MOI 100 of GFP LV and then electroporated with CXCR4 mRNA (n = 4), or (2) transduced with MOI 100 of CXCR4mGFP LV in presence of LB (n = 6). Long term engraftment efficiency was verified by analyzing the peripheral blood of treated mice 12 weeks after the second transplant. Human chimerism was uneven between the two groups, potentially due to differences related to the route of administration for the mobilization agents employed in the experiment (Figure 5r). Nevertheless, looking at the GFP+ fraction within human CD45+ cells it can be appreciated how CXCR4mGFP LV promotes at least a comparable engraftment to electroporated CXCR4 mRNA, which could be further enhanced by transplanting a TO equivalent of CD34+ cells, considering the higher proliferation rate and viability of Comet LV-treated cells (Figure 5s).

[0621] The capacity of Comet LV to induce CXCR4 transient overexpression in human HSPCs, thus improving their in vivo engraftment potential, was further validated.

[0622] In a similar experiment to the one shown in Figure 5 l-p, hCD34+ cells deriving from n=3 distinct human donors (rather than only one as described previously) were transduced with CXCR4mGFP LV or electroporated with CXCR4 mRNA. This experiment replicated data previously shown both in terms of capacity of CXCR4 mRNA delivery and preservation of cell growth and HSC primitive fraction viability, while showcasing how our findings can be translated to several different human donors (Figure 14a-f).

[0623] The experiment previously described in Figure 5 q-s was repeated with the following differences: 1. only IP injection of mobilization reagents was employed; 2. at peak mobilization, mice received a graft composed of a TO equivalent of 5E+05 PB-derived mobilized hCD34+ cells.

[0624] As done previously, hCD34+ cells were treated with either (1) GFP LV transduction and then CXCR4 mRNA electroporation (n = 7), or (2) CXCR4mGFP LV transduction in presence of LB with no electroporation (n = 6) (Figure 14g). Of note, due to the enhanced proliferation and viability of Comet LV-treated cells, 4.77E+05 cells / mouse were transplanted in the GFP LV + Electro CXCR4 group, and 6.81 E+05 cells / mouse were transplanted in the CXCR4mGFP LV group.

[0625] Engraftment efficiency was monitored by analyzing the peripheral blood of treated mice 6, 9, and 12 weeks after transplantation. Human chimerism slowly increased over time and was similar between the two groups (Figure 14h). However, the GFP+ fraction within hCD45+ cells was enhanced in the CXCR4mGFP LV group, indicating a superior exchange rate between mobilized host cells and GFP+ donor cells when employing Comet LVs (Figure 14i). This finding was confirmed at termination of the experiment 16 weeks after transplantation, as flow cytometry analysis revealed comparable human chimerism, but enhanced GFP+ fraction within hCD45+ cells in peripheral blood, bone marrow, and spleen (Figure 14d-f). Importantly, GFP+ fraction, although more abundant in the CXCR4mGFP LV group, showed a similar composition to that in the GFP LV + electro CXCR4 group, confirming a balanced multilineage composition (Figure 14j-o). To evaluate clonal composition within the graft, we performed an integration site (IS) analysis of DNA extracted from human cells-enriched murine bone marrow. Clonal diversity, evaluated using the Shannon Diversity Index (H Index), was significantly higher for CXCR4mGFP LV group (3.85 ± 0.68) than for GFP LV + electro CXCR4 group (2.55 ± 0.38; p = 0.0047, Mann- Whitney test), reflecting a broader and more balanced clonal distribution (Figure 14p-q). Analysis of clonal abundance further confirmed groupspecific differences. In GFP LV + electro CXCR4 group, high-abundance ISs (>1%) accounted for 83-100% of total reads per sample, whereas in CXCR4mGFP LV group they represented 59-90% (Figure 14k). These results thus indicate that Comet LVs supports the engraftment of a richer and more polyclonal graft compared to CXCR4 electroporation treatment.

[0626] Taken together, these observations support the feasibility of employing Comet LVs to simultaneously transduce and provide an engraftment advantage to HSPCs, while preserving their viability and oligoclonality.

[0627] Comet L V can be employed to overcome SAMHD 1 -mediated transduction restriction in human macrophages

[0628] Conversely to murine macrophages, human macrophages restrict HIV-1 infection thanks to the action of the enzyme SAMHD1, which depletes the intracellular pool of deoxynucleotides required for viral reverse transcription. This mechanism effectively limits the ability of the virus to synthesize DNA from its RNA genome, and thus renders human macrophages restrictive to infection by HIV-1-based LVs. Certain lentiviruses, such as HIV-2 and specific strains of simian immunodeficiency virus (SIV), have evolved to express the accessory protein VPX, which circumvents SAMHD1 restriction by promoting its proteasomal degradation. Thus, HIV- 1 based LVs incorporating VPX have been used to transduce human myeloid cells, including monocytes, dendritic cells and macrophages in vitro.

[0629] The possibility to employ Comet LVs for the delivery of VPX mRNA in order to circumvent SAMHDI-mediated transduction restriction in human macrophages (hMac) was tested. A Comet LV encoding a PGK. GFP expression cassette as integrating transgene and incorporating VPX mRNA was produced, hereon referred as VPXmGFP LV. hMac or HEK 293T cells were transduced with either VPXmGFP LV or standard GFP LV at MOIs ranging from 1 to 5 (Figure 6a). At 96 h post transduction, only hMac transduced with VPXmGFP LVs expressed GFP (Figure 6b). Of note, while VPXmGFP LVs equally transduced hMAc and HEK 293T, standard GFP LV only transduced HEK 293T and failed to transduced hMac, as observed by GFP expression (Figure 6c). These observations show how co-packaging of VPX mRNA within LV particles by Comet LV technology offers a feasible way of overcoming SAMHDI-mediated infection restriction in human macrophages.

[0630] Comet IDLVs incorporating editing enhancers can be employed to enhance HDR efficiency in gene editing

[0631] State-of-the-art ex vivo gene editing takes advantage of editing enhancers in order to improve HDR efficiency in the cell populations of interest. However, such approaches require procedures (e.g. electroporation) that might reduce cell viability or limit their applicability. A study by Ferrari et al. described how the transient expression of a p53 inhibitor, GSE56, and a cell cycle booster, Ad5-E4orf6 / 7, obtained by mRNA delivery via electroporation resulted in increased homology directed repair (HDR) editing efficiency in HSPCs (Ferrari, et al., 2020, Nature biotechnology 38, 1298-1308). Thus, the possibility to incorporate mRNA molecules encoding either GSE56, Ad5-E4orf6 / 7 or both into IDLVs used for donor template delivery in the context of gene editing was evaluated.

[0632] Virus-like particles (VLPs) incorporating CAS9 as protein, hereon referred as CAS9 VLP, were used in order to develop an electroporation-free approach. In this way, the combined transduction by VLPs and Comet IDLVs would provide all necessary elements for gene editing while being compatible with in vivo applications.

[0633] As proof of concept, a highly expressed gene in macrophages, Atp6C, was selected, and a donor DNA cassette that enables the insertion of GFP within the first intron of Atp6C genomic locus via homology-directed repair (HDR) was designed. Of note, this donor template cassette did not contain a promoter, and GFP expression would be driven by the endogenous Atp6C promoter thanks to the addition of a splicing acceptor (SA) and a ribosome skipping sequence (2A) upstream of the GFP coding sequence. Standard IDLVs encoding the donor template cassette and Comet IDLVs that would additionally incorporate mRNAs for the editing enhancer Ad5-E4orf6 / 7 (Ad5), hereon referred as Ad5mGFP IDLV, were produced. Comet IDLV donor was used to co-transduce iKCs in combination with CAS9 VLPs incorporating a guide RNA targeting murine Atp6C (Figure 7a). Although all IDLV donors were employed at MO1 1, Comet IDLV incorporating Ad5 editor enhancer produced a higher fraction of edited cells compared to standard IDLVs, detected by GFP expression (Figure 7b). Conversely, none of the control conditions in which the IDLV donor was administered in absence of VLP give rise to GFP+ cells, confirming the specificity of the assay. Overall, Comet IDLVs incorporating Ad5 seemed to yield better results than standard IDLVs in absence of editor enhancers (Figure 7c, d). Other editor enhancers could be incorporated to increase editing efficiency, as GSE56.

[0634] These observations thus constitute a proof of concept for the use of Comet IDLVs incorporating editing enhancers mRNAs together with a donor template cassette for HDR editing of a cell population of interest.

[0635] Comet LVs provide transient proliferation advantage to engineered HSPCs

[0636] Ex vivo HSPCs engineering has shown great promise in treating a number of hereditary diseases, yet the process remains cost and labor-intensive, as it requires specialized expertise. To overcome this issue, various groups have tried to engineer HSPCs in vivo by intraosseous or intravenous injection of viral vectors. However, this approach is limited by low targeting efficiency of these approaches, often insufficient for meaningful clinical outcomes.

[0637] A recent work by Milani et al. showed improved transduction efficiency when targeting circulating HSPCs via intravenous LV injection during hematopoiesis migration in newborns or mobilized juvenile mice (Milani et al, Nature pre-accepted). In this context, the fraction of transduced circulating HSPCs was higher when the therapeutic payload provided a selective advantage to corrected HSPCs, leading to their expansion and thus better disease correction, such in the case of Fanconi Anemia.

[0638] Building on this observation, the fraction of transduced HSPCs was enhanced by employing Comet LVs containing mRNAs that can promote transient cell proliferation and expansion. As proof of concept, two different proteins were tested: (1) HoxB4, a transcription factor involved in HSPCs self-renewal, and (2) KrasQ61R’ a gain-of-function variant of small GTPase KRAS involved in the signaling pathways regulating cell growth and differentiation.

[0639] Comet LV encoding a PGK. GFP expression cassette as integrating transgene and copackaging the murine form of either HoxB4 or KrasQ61RmRNA, hereon referred as HoxB4mGFP LV and KrasmGFP LV, respectively, were produced. Human CD34+ cells (n = 3 donors / group) were transduced with either Comet LV or with a control GFP LV at MOI 100 in presence of lentiboost and monitored GFP expression, cell growth and sample composition over time (Figure 8a). While all tested LV showed comparable infectivity, KrasmGFP LV provided treated cells with a proliferation advantage compared to other LV-treated groups, increasing the overall number of cells without depleting the most primitive HSPCs compartment of CD90+ and CD90+CD45RA- cells (Figure 8b-d). On the other hand, the lack of effect of HoxB4mGFP LV might stem from species mismatch (murine mRNA in human cells), as HoxB4 conservation between mouse and human is relatively low if compared to that of Kras.

[0640] These findings demonstrate the feasibility of employing Comet LV to deliver transient proliferation enhancers, expanding corrected HSPCs without compromising stem cell content or depleting long term repopulating HSCs fraction. This could be useful both to expand ex vivo engineered HSPCs before transplantation, or to enhance the efficacy of in vivo HSPCs gene therapy approaches by providing a selective advantage to transduced cells. Similarly, this approach could be extended to the in vivo correction of other poorly targeted cell populations, as it could improve target organ repopulation with corrected cells by selectively enhancing their proliferation.

[0641] Comet LVs can be employed to transiently express proteins to improve T cell fitness (trafficking, proliferation, activation)

[0642] To assess whether Comet LVs can be used in lymphocytes to achieve stable expression of one transgene and transient expression of another, the kinetics of fluorescent reporter expression in target cells transduced with either standard LVs or Comet LVs engineered to co-package GFP mRNA together with a mCherry or BFP transgene in the viral genome were compared. As surrogate B lymphocytes, Nalm6 cells were employed. Live-cell imaging revealed distinct expression profiles for the integrating mCherry transgene and the codelivered GFP mRNA (Figure 9a). mCherry fluorescence, reflecting integration and transcription of the transgene, gradually increased over time and plateaued approximately 96 hours post-transduction. In contrast, GFP fluorescence was detectable as early as 1 hour post-transduction, peaked between 24 and 48 hours at lower intensity than mCherry, and declined thereafter, consistent with transient expression from co-packaged mRNA. Notably, omission of the PLC. MCP protein during vector production impaired GFP mRNA copackaging, resulting in an absence of GFP signal in transduced cells. As expected, mCherry expression was not affected by the presence or absence of PLC. MCP in Comet LV preparations. A similar pattern was observed in activated primary human T cells transduced with an LV encoding BFP or a Comet LV encoding Blue Fluorescent Protein (BFP) and copackaged GFP mRNA (Figure 9b, c). Early GFP expression was detectable within 24 hours post-transduction, preceding the onset of stable BFP expression. GFP fluorescence declined after 48 hours and was nearly undetectable by day 7, whereas the frequency of BFP+cells increased progressively and plateaued around 72 hours. Consistently, co-packaged mRNA expression levels were lower and more transient than those of the integrating transgene across both cell models. Overall, these data confirm that co-packaging of mRNA enables rapid and transient protein expression that complements delayed transgene expression from vector integration also in primary human T cells.

[0643] As proof of concept of enhanced T cell migration capacity, the efficacy of a CXCR4 mRNA-encoding Comet LVs to restore CXCR4 expression in T cells was assessed (Figure 10a). Of note, CXCR4 regulates T cell trafficking towards lymphoid organs, sites of inflammation and tumors, and is involved in T cell activation by stabilizing the immunological synapse and providing direct co-stimulatory signals that synergize with TCR engagement. As such, CXCR4 is highly expressed in most subsets of T cells, but is quickly downregulated following T cell activation, resulting in reduced responsiveness to migration. CXCR4 downregulation following ex vivo T cell activation prior to LV transduction might thus impair their trafficking towards tumors and lymphoid organs, and its rescue with Comet LVs might provide better CAR T cell efficacy. Moreover, supraphysiological CXCR4 expression may confer T cells enhanced capacity to migrate to sites of inflammation, including tumors.

[0644] Contrary to primary human T cells, Jurkat T cells are not restrictive to LV transduction, but their ex vivo activation with aCD3-pseudotyped LVs similar to primary T cells results in CXCR4 downregulation directly correlated with transduction levels (Figure 10b, c). This is not observed with vesicular stomatitis virus G protein (VSV-G)-pseudotyped LVs, which are unable to transduce primary T cells without a pre-activation via CD3 / CD28 stimulation.

[0645] CXCR4 proficient Jurkat T cells transduced with aCD3-pseudotyped GFP LV showed a marked downregulation of CXCR4 expression, which was rescued by aCD3-pseudotyped CXCR4mGFP LV. It was also observed that CXCR4mGFP LV transduction can restore CXCR4 expression in CXCR4 KO Jurkat T, while reaching supraphysiological levels in CXCR4 WT Jurkat T cells (Figure 10d-f). These findings support the use of Comet LVs to co-deliver mRNAs for the transient expression of functional receptors that improve T cell migration in order to improve their fitness for in vivo use. Comet LVs incorporating transiently expressed cytokine payloads can be employed to fine tune-cytokine milieu for in vivo gene therapy

[0646] The possibility of delivering mRNA payloads for the transient and regulated expression of immune-modulatory cytokines whose sustained or unregulated expression might otherwise give rise to adverse effects (e.g. IL12 and DR18) was also tested. As proof of concept, a Comet LV encoding GFP as an integrating payload and co-delivering an mRNA for murine I L12 expression (IL12mGFP LV) was developed. As control to assess the carryover of free mlL12 present in the vector prep, a IL12mGFP LV produced in absence of PLC. MCP (PLC-LV) was employed. These vectors were employed to transduce HEK 293T cells, and after 24h media was refreshed to washout residual mlL12 that might be contained into the vector prep. Cell surnatant was subsequently collected 48h and 72h post-transduction to measure IL12 released from transduced cells (Figure 11a). At comparable transduction levels, surnatant collected from cells transduced with IL12mGFP LV showed higher levels of mlL12 compared to PLC- control (Figure 11b). These data thus support the use of Comet LV to promote the controlled release of immune-modulatory cytokines (such as IL12) from transduced cells, in particular for the engineering of immune cells such as CAR T cells and macrophages / dendritic cells for cancer immunotherapy. Similar to Il-12 other cytokines could be employed, in particular those whose expression is toxic or detrimental if employed long term (e.g. IL2, IL7, IL12A, IL12B, IL15, IL18, IL21, IL23A, IFNG, TNF, LTA, LTB, CSF2 (GM-CSF), CSF1, FLT3LG, TGFB1, VEGFA, CXCL9, CXCL10, CXCL11, CCL2, CCL3, CCL4, CCL5, CCL19, CCL21, IFNA1, IFNB1).

[0647] Comet LVs enable temporally resolved transgene expression

[0648] The kinetics of expression of transgenes encoded by the co-packaged mRNA or the genome of Comet LVs were evaluated. To this aim, a Comet LV driving the expression of a nonsignalling, low-affinity NGFR (LNGFR) from its integrating genome and co-packaging GFP. MS2. Wpre mRNA was produced. As controls, Comet LVs lacking either PLC. MCP (No MCP) or MS2 loops within the GFP mRNA (No MS2), a standard LV encoding LNGFR, and a viral-like particles (VLP, i.e. lacking an integrating genome) co-packaging GFP. MS2. Wpre mRNA were included (Figure 12a,b). As expected, GFP expression by Comet LVs was quickly induced, peaking 1 to 2 days after vector transduction and diluting completely after 5 to 7 days, while NGFR expression increased more gradually, reaching its peak after 3 to 5 days (Figure 12c-e). The experiment was repeated including more vector dilutions and extending the time frame of analysis to monitor transgene integration within the transduced cell DNA. Importantly, integrated vector copies increasing proportionally with vector dose 14 days after transduction were detected, while integrated GFP DNA sequence was virtually undetected (Figure 12f). Taken together, these results further confirm that Comet LVs are able to simultaneously deliver both mRNA, which is a transiently expressed, and the viral genome, which stably drives the expression of a transgene. Furthermore, they showcase how the peak of expression of co-delivered mRNA happens priorly and independently from retrotranscription and integration of the LV genome, thus indicating how mRNA-encoded transgene could be exploited to overcome antiviral responses within the target cells that may impede these events, thus restricting transduction (e.g. VPX for human macrophages transduction, kinases or CD28 for T cell activation and transduction).

[0649] Materials and methods

[0650] 1. Vector production and vector prep quality assessment

[0651] Plasmid design, cloning and amplification

[0652] Transfer plasmids PGK. GFP LV, PGK.mCherry LV, Mrc1.lfna.miRT122-126, and Mrc1.liOva.miRT122-126 were used.

[0653] GFP. MS2 mRNA plasmid was synthetized ex novo by Twinhelix and used to as template for the generation subsequent mRNA transfer constructs by restriction enzyme-based cloning (Table 1). The SRC. PC. MCP, the SRC. MCP and the PLC. MCP was synthetized ex novo by Twinhelix (Table 1). The MCP. Gagpol plasmid was ordered from Addgene (#166031).

[0654] The plasmid for the expression of the aCD3 scFv described in Nicolai etal. (Blood 144, 977-987 (2024)) was synthetized ex novo by GeneArt, ThermoFisher (Table 1).

[0655] The B3_E. O7 plasmid for gRNA expression was used as template for the generation of all the Atp6C gRNA expression plasmid employed for CAS9 VLP generation. MMLV Gag-Cas9 plasmid employed for CAS9 VLP generation was provided by Lombardo lab in SR-Tiget.

[0656] Table 1. Sequences of plasmids employed for Comet LVs

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663]

[0664]

[0665]

[0666]

[0667]

[0668]

[0669]

[0670] LV production

[0671] In this study, third generation VSV-G pseudotyped LVs were used. LV stocks were produced in laboratory scale as described previously (Soldi, et al., 2020, Mol Ther Methods Clin Dev 19, 411-425). The titer of the LV stocks was measured in HEK 293T cell-transducing units (TU / mL), as described in following sections and in Soldi et al, 2020.

[0672] To produce aCD3 pseudotyped LVs, 12 μg / plate of a plasmid for the expression of the αCD3 scFv described in Nicolai et al. were added standard mix for LV production described above. To produce Comet LVs, 3.125ug / plate of PLC. MCP plasmid and 25ug / plate of MS2 mRNA expression plasmid were added to standard mix for LV production described above.

[0673] Alternatively, 3.125ug / plate of PLC. PCP plasmid and 30ug / plate of PP7 mRNA expression plasmid were added to standard mix for LV production described above.

[0674] Retroviral VLPs production

[0675] MMLV stocks were produced in laboratory scale with plasmids listed in Table 2, employing calcium phosphate transfection as described for LV production. Plasmid quantities per 150mm dish of HEK293T are reported in Table 2.

[0676] Table 2. Plasmid quantities in MMLV production

[0677]

[0678] Vector copy number determination

[0679] Genomic DNA was extracted from cell culture samples using Maxwell RSC 48 Instrument (Promega / AS8500) with Maxwell RSC Cultured Cells DNA Kit (Promega / AS1620). Genomic DNA from whole tissue samples was extracted by using the DNeasy Blood and Tissue Kit (Qiagen / 69504) according to manufacturer’s instruction. LV copy number was determined using a QX200 Droplet Digital PCR System (Biorad / 1864003) apparatus, and the digital droplet PCR (ddPCR) was performed according to manufacturer’s instructions. Briefly, for each sample a reaction was prepared containing ddPCR Supermix for Probes (No dUTP) (Biorad / 1863024), 10-50ng of genomic DNA, primers at a final concentration of 900nM and the detection probes at 250nM. Primers and probes for the detection of HIV genome and mouse normalizer Sema3a gene are reported in Table 3, while for human normalizer GAPDH a commercially available expression assay was used (Invitrogen / Hs00894322_cn). After droplet generation, the plate was sealed and amplified in a thermal cycler with the following protocol:

[0680]

[0681] Amplified droplets were acquired using the BioRad QX200 Droplet Reader and analyzed by using the QuantaSoft software (Biorad). LV copies per genome were calculated by the formula:

[0682]

[0683] Table 3. Primer or probe sequences for ddPCR

[0684]

[0685] Viral RNA extraction, retrotranscription and quantification by ddPCR

[0686] Viral RNA was extracted from vector stocks using MagMAX viral RNA isolation kit (Applied Biosystems / AM1939), following manufacturer’s instructions. DNase treatment and retrotranscription of 100-200ng of extracted viral RNA were performed using SuperScript IV VILO kit (I nvitrogen / 11766050) following manufacturer’s instructions. Quantification of the retrotranscribed DNA was performed with ddPCR, using a QX200 Droplet Digital PCR System (Biorad / 1864003) apparatus. Briefly, for each sample a reaction was prepared containing 200-300fg of retrotranscribed DNA, primers at a final concentration of 900nM and, when needed, the detection probes at 250nM. For GFP detection, ddPCR Supermix for Probes (No dUTP) (Biorad / 1863024) was used, togetherwith primers and probes for the detection of HIV genome and GFP gene (Table 3). After droplet generation, the plate was sealed and amplified in a thermal cycler with the following protocol:

[0687]

[0688] For Ad5 quantification, ddPCR EvaGreen Supermix (Biorad / 1864034) was used, together with primers for the detection of HIV genome and Ad5 gene (Table 3). After droplet generation, the plate was sealed and amplified in a thermal cycler with the following protocol:

[0689]

[0690] Amplified droplets were acquired using the BioRad QX200 Droplet Reader and analyzed by using the QuantaSoft software (Biorad).

[0691] Dynamic light scattering (DLS)

[0692] DSL measurements were employed to assess particle concentration (particles / mL) and size (nm) of different vector preps, using a Z sizer ultra apparatus (Malvern Panalytical). Samples were prepared by filtering PBS with 0,20pm filters and diluting 10pL of vector prep in 990pL of filtered PBS using 10x10x45mm polystyrene cuvettes (Sarstedt / 67.754). Samples were acquired with ZS Xplorer software (Malvern Panalytical), setting PBS as dispersant (R.l. 1,33; viscosity 0,8882 mPa.s; temperature 25°C and protein (refractive index 1,45; adsorption 0,001) as material. Acquisitions were performed in triplicate and results were obtained calculating the average of each measurement.

[0693] 2. Cell culture

[0694] HEK293T cells

[0695] Human embryonic kidney 293T (HEK293T) cells were employed to produce and titer LVs. They were purchased from ATCC and their authenticity is supported by their capability to produce high titer LV stocks. Cells were cultured in adherence at 37°C in IM DM medium (Corning / 10-016-CV) supplemented with 10% fetal bovine serum (FBS; HyClone / SH30066.03), 100 IU / mL penicillin, and 100pg / mL streptomycin. Cells were split three times a week 1:5-1:7 by removing culture medium, washing cells with Phosphate Buffered Saline (PBS; Corning / 21-031-CVR) and detaching them with a solution of 0.05% trypsin and EDTA (4mM) in PBS (ATV). Cells were then resuspended in fresh medium and transferred into a new plate.

[0696] For experiments, cells were cultured as described above but seeded 50.000 cells / well in a MW24 with a final volume 0,5mL / well and transduced by the addition of the volume of vector corresponding to desired MOI.

[0697] Immortalized KCs (iKC cells)

[0698] The in vitro model of murine KCs was obtained by systemic injection of 5 weeks old female C57bl / 6 mice with a 1,75E8 TU / mouse of LV enforcing the expression of SV40 Large T antigen under PGK promoter. Mice were sacrificed 7 days after LV injection, and liver was collected, smashed, and filtered through a 40pm pore filter. Single cell suspension was pelleted at 30g for 5 minutes at room temperature to remove hepatocytes. Supernatant was collected and pelleted again at 300g for 5min at room temperature. Pellet was finally resuspended in RPMI medium with L-Glutamine (Corning / 10-040-CV) supplemented with 10% FBS (HyClone / SH30066.03), and 100ng / mL of mCSF (Peprotech / 3015-02), and kept in culture refreshing the media 3 times per week until non-immortalized cell were completely depleted. The cells thus obtained were termed iKCs and were used for subsequent in vitro experiments as a model of murine KCs.

[0699] For maintenance, cells were cultured in adherence at 37°C in RPMI medium with L- Glutamine (Corning / 10-040-CV) supplemented with 10% FBS (HyClone / SH30066.03), 100 IU / mL penicillin, 100pg / mL streptomycin, and 100ng / mL of mCSF (Peprotech / 3015-02). Cells were split twice a week 1:3-1:4 by removing culture medium, washing cells twice with PBS (Corning / 21-031- CVR), and detaching them with a solution of Trypsin-EDTA (0.25%) and phenol red in PBS (Gibco / 25200056). Trypsin solution was inactivated by adding fresh RPMI 10%FBS 1% P / S, cells were pelleted 5 minutes 300g 4°C and resuspended in fresh medium before being transferred into a new plate. For experiments, cells were cultured as described above but seeded 50.000 cells / well in a MW24 with a final volume 0,5mL / well and transduced by the addition of the volume of vector corresponding to desired MOI.

[0700] bEnd.3

[0701] Cells were cultured in adherence at 37°C in IMDM medium (Corning / 10-016-CV) supplemented with 10% fetal bovine serum (FBS; HyClone / SH30066.03), 100 IU / mL penicillin, and 100pg / mL streptomycin. Cells were split three times a week 1:4-1:5 by removing culture medium, washing cells with Phosphate Buffered Saline (PBS; Corning / 21-031-CVR) and detaching them with a solution of 0.05% trypsin and EDTA (4mM) in PBS (ATV). Cells were then resuspended in fresh medium and transferred into a new plate.

[0702] For experiments, cells were cultured as described above but seeded 50.000 cells / well in a MW24 with a final volume 0,5mL / well and transduced by the addition of the volume of vector corresponding to desired MOI.

[0703] Hepa 1-6

[0704] Cells were cultured in adherence at 37°C in IMDM medium (Corning / 10-016-CV) supplemented with 10% fetal bovine serum (FBS; HyClone / SH30066.03), 100 IU / mL penicillin, and 100pg / mL streptomycin. Cells were split three times a week 1:8-1:10 by removing culture medium, washing cells with Phosphate Buffered Saline (PBS; Corning / 21-031 -CVR) and detaching them with a solution of 0.05% trypsin and EDTA (4mM) in PBS (ATV). Cells were then resuspended in fresh medium and transferred into a new plate.

[0705] For experiments, cells were cultured as described above but seeded 50.000 cells / well in a MW24 with a final volume 0,5mL / well and transduced by the addition of the volume of vector corresponding to desired MOI.

[0706] Jurkat cells

[0707] Jurkat and Jurkat CXCR4 KO cells were provided by Naldini group in SR-Tiget. Cells were cultured in suspension at 37°C in RPMI medium with L- Glutamine (Corning / 10-040-CV) supplemented with 20% FBS (HyClone / SH30066.03), 100 IU / mL penicillin, 100pg / mL streptomycin. Cells were split three times a week 1:3-1:5 by dilution in fresh medium. For experiments, cells were cultured as described above but seeded 100.000 cells / well in a MW24 with a final volume 0,5mL / well and transduced by the addition of the volume of vector corresponding to desired MOI.

[0708] Nalm6 cells

[0709] Nalm6 cells were maintained in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were transduced with the indicated vectors at an MOI of 3.

[0710] Primary human T cells

[0711] Primary human T cells were isolated from peripheral blood of healthy donors, cultured in X-VIVO™ 15 medium (Lonza) supplemented with 5% human AB serum and 100 U / mL recombinant human IL-2, and activated using anti-CD3 / CD28 Dynabeads in the presence of IL-2 (100 U / mL) for 48 hours prior to transduction. Cells were transduced with the indicated vectors at an MOI of 3.

[0712] MC38 OVA

[0713] MC38 OVA were obtained by mixing of MC38 cell line untransduced or transduced with an OVA LV, at a ratio of 1:9, respectively. Cells were cultured in adherence at 37°C in IMDM medium (Corning / 10-016-CV) supplemented with 10% fetal bovine serum (FBS; HyClone / SH30066.03), 100 IU / mL penicillin, and 100pg / mL streptomycin. Cells were split three times a week 1:8-1:12 by removing culture medium, washing cells with Phosphate Buffered Saline (PBS; Corning / 21-031-CVR) and detaching them with a solution of 0.05% trypsin and EDTA (4mM) in PBS (ATV). Cells were then resuspended in fresh medium and transferred into a new plate.

[0714] Human CD34+ HSPCs

[0715] G-CSF mPB CD34+ HSPCs were purchased from Mobilized Leukopak (AllCells) according to TIGET-HPCT protocol approved by the San Raffaele Institute Bioethical Committee and purified with the CliniMACS CD34 Reagent System (Miltenyi Biotec) according to the manufacturer’s instructions. HSPCs were seeded at the concentration of 1x106cells / mL in serum-free StemSpan medium (StemCell Technologies) supplemented with 100 IU / mL penicillin, 100pg / mL streptomycin, 2% glutamine, 300ng / mL hSCF, 300ng / mL hFlt3-L, 100ng / mL hTPO, 1pM SR1, 35nM UM 171 and 10pM PGE2. All cells were cultured in a 5% CO2 humidified atmosphere at 37°C. In vitro, the human HSC population was defined as CD34+ CD90+. For experiments, cells were cultured as described above but seeded 100.000-300.000 cells / well with a final concentration of 1 mln cells / ml and transduced by the addition of the volume of vector corresponding to desired MOI. For retronectin-based transduction, MW96 was coated with 60ul / well of retronection at concentration 100ug / ml and incubated 2h at RT. Subsequently either transduction was carried on as usual (Retro_SN) or vector was previously attached to plate surface by 2h centrifugation at 2000g at 37°C before cell plating (Retro_RBV). For PGE2, cells were incubated with final concentration of PGE2 for 2h before transduction. For all other transduction enhancers, they were added together with vector during transduction at concentration annotated in Table 4.

[0716] Table 4. List of transduction enhancers

[0717]

[0718] Electroporation of hCD34+ cells was performed as previously described by Omer-Javed et al. (Omer-Javed et al. (2022). Cell 185, 2248-2264 e2221).

[0719] 3. Mouse procedures

[0720] Female C57BI / 6N mice were purchased from Charles River Laboratory. All experiments and procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) at San Raffaele Hospital animal facilities (IACUC number: 1383) and authorized by the Italian Ministry of Health and local authorities according to the Italian law. Mice were used between 5 and 10 weeks of age and were maintained in Specific Pathogen-free (SPF) animal research facilities with a 12h / 12h dark / light cycle and standardized temperature (22°C + / - 2°C) and humidity (55% + / - 5%).

[0721] Intravenous injection

[0722] For systemic LV injection, LVs were diluted in PBS to obtain the desired TU to be injected per mouse in a volume ranging from 200 to 300pL. Mice were warmed under an infrared / red-light lamp and the LVs were intravenously injected via the tail vein. All vectors were used between 1-1.5*1O10TU / kg. In all experiments untransduced (UT) mice were injected with a volume of PBS ranging from 200 to 300pL. Blood collection and analysis

[0723] Blood was withdrawn either from the retroorbital vein plexus and collected in Microvette with EDTA (Sarstedt / NC9990563). Hemocytometer analysis was performed on whole blood by using the ProCyte DXTM (IDEXX). For the collection of plasma, blood was centrifuged at 850g for 10 minutes at room temperature and precipitated red and white blood cells were discarded.

[0724] hCD34+ cells transplantation

[0725] hCD34+ cells transplantation, including mobilization-based conditioning regiment, was performed as previously described by Omer-Javed et al. (Omer-Javed et al. (2022). Cell 185, 2248-2264 e2221).

[0726] MC38 OVA tumor implant

[0727] Briefly, for subcutaneous MC38 OVA tumor implant, mice were anesthetized with Isoflurane (Iso-Vet) at a concentration of 3 % in flow of oxygen at 1.5 L / min. MC38 OVA were delivered subcutaneously by injecting 106MC38. OVA cells into the flank of mice in a volume of 50 pL of Matrigel (BD Biosciences) diluted 1:2 in PBS. Tumor growth was monitored by measuring the dimensions (larger diameter, x, and lower diameter, y) of the subcutaneous lesions using a caliper. Tumor volume was calculated with the formula:

[0728] Volume = diameter (x)2* diameter (y) / 2

[0729] For intrahepatic MC38 tumor implant, mice were shaved and, immediately prior to surgery, injected with 50 pL carprofen (2.5 mg / mL) for pain management. Mice were anesthetized as previously described and 100,000 or 500,000 cells / mouse were injected in 5 pL / mouse of PBS with an Hamilton syringe in the left liver lobe, for therapeutic or prophylactic setting respectively. Following surgery, mice were subjected to antibiotic treatment for one week by adding Baytril (Bayer) at a concentration of 0.5 mg / mL to the drinking water. Liver metastasis growth was measured by tumor weight (i.e. by dissecting the liver metastasis upon experiment termination and measuring its weight in a 10 mg precision digital bench scale).

[0730] Sacrifice and necropsy

[0731] For endpoint analysis, mice were euthanized by cervical dislocation. The liver was perfused by injecting 10mL of PBS containing 5mM of UltraPure EDTA pH8 (I nvitrogen / 15575020) through the inferior vena cava and cutting the portal vein to allow exiting of the solution containing most circulating blood cells from the liver. All organs were collected and immediately stored on ice (for flow cytometry) or dry ice (for DNA extraction). 4. Flow cytometry

[0732] Sample preparation

[0733] For FC analysis of in vivo samples, organs were smashed into small pieces and then incubated 15 minutes at 37°C in agitation with a tissue digestion solution composed of 1mL IMDM (Corning) supplemented with 0.35mg / mL collagenase type IV (Sigma-Aldrich / SCR103), 1mg / mL dispase II (Gibco / 17105041) and 0.2mg / mL DNAse (Roche / 11284932001). The tissue was then further dissociated by pipetting and filtered using 40pm cell strainers (Corning / 352340). Single cell solution was washed with 30mL of autoMACS Running Buffer (Miltenyi Biotec / 130-091 -221) and pelleted for 5 minutes at 300g 4°C to remove excess enzymes. Samples were transferred to FACS tubes and stained as described in section “sample staining”. For blood samples, after collection 70 pL of blood were moved to a FACS tube and 100pL of FBS were added prior to red blood cell lysis with TQ-Prep Workstation (Beckman Coulter).

[0734] For FC analysis of in vitro samples, cells were collected by ATV incubation (as described in relative section of chapter), moved to FACS tubes or 96-well plates U-bottom and washed with 1-5mL of autoMACS Running Buffer (Miltenyi Biotec / 130-091 -221) pelleting for 5 minutes 300g 4°C to remove excess culture medium.

[0735] Live-cell imaging and fluorescence quantification

[0736] For kinetic analysis in Nalm6 cells, images were acquired every hour for up to 192 hours posttransduction with standard lentiviral vectors encoding mCherry or CometLVs (with or without PLC MCP, as indicated) encoding mCherry and co-packaging GFP mRNA, using an Incucyte live-cell imaging system (Sartorius). mCherry and GFP fluorescence intensities were quantified automatically as orange and green intensity, respectively, using the Incucyte software (n=4 technical replicates per well), normalized to cell confluency, and reported as median ± SEM of 3 biological replicates per condition.

[0737] Sample staining

[0738] Upon single cell suspension preparation (see “sample preparation”), Fc Block (BD Pharmagen / 553142) was added to the cells to prevent unspecific staining through binding of the FC receptor. For membrane bound antigens, samples were stained for 15 minutes on ice. For staining of intracellular proteins, cells were fixed, permeabilized and stained using the True-Nuclear™ Transcription Factor Buffer Set (BioLegend) according to manufacturer’s recommendation. For the staining of TCRs specific for the SIINFEKL peptide loaded on MHC class I (H2-Kb), samples were stained with an SIINFEKL-loaded MHC class I tetramer (NIH tetramer core facility) according to manufacturer’s instruction. All antibodies were employed at concentration described in Table 5. After staining, cells were washed with 5m L of autoMACS Running Buffer (Miltenyi Biotec / 130-091 -221) and resuspended in 100-500pL of live / dead staining. Viability of cells was assessed by using either LIVE / DEAD™ Fixable Blue Dead Cell Stain Kit (Invitrogen), 7AAD (BioLegend / 420404) or Dapi (BioLegend / 422801) nuclear staining. Samples were acquired by using either a BD CytoflexS or a FACSymphony A5 Cell Analyzer (BD Biosciences).

[0739] Table 5. List of monoclonal antibodies

[0740]

[0741] 5. ELISA

[0742] Quantification of IFNa content in the blood was performed on plasma using the Mouse IFN Alpha All Subtypes ELISA KIT High Sensitivity (pbl Assay Science) according to manufacturer’s instruction. Quantification of IL12 content was performed on cell medium using the Legend MAX™ Mouse IL-12 (p70) ELISA Kit (Biolegend) according to manufacturer’s instruction.

[0743] 6. Integration site analysis

[0744] Sample preparation

[0745] Integration site analysis was performed on DNA extracted from human cells-enriched murine bone marrow. Briefly, bone marrow was harvested by flushing femurs and tibias of mice with 10mLautoMACS Running Buffer (Miltenyi Biotec / 130-091 -221) on a 50pm cell strainer. Single cell suspension was then enriched in human cells with Mouse cell depletion kit (Miltenyi Biotec / 130-104-694) following manufacturer's instruction. The mouse-depleted the cell suspension was pelleted for 10min at 300g, and genomic DNA was extracted with QIAmp DNA Microkit (QIAgen / 56304) following manufacturer’s instructions.

[0746] Retrieval of vector IS

[0747] Genomic DNA was subjected to custom amplification to recover vector integrations sites (ISs) using a Sonication Linker-Mediated PCR (SLiM-PCR) approach78. SLiM-PCR was performed with the NEBNext® UltraTM II DNA Library Prep Kit (New England Biolabs, ref: E7645) according to the manufacturer’s instructions. Briefly, the protocol comprised the following steps: 1) fragmentation of approximately 30 ng of genomic DNA per sample by sonication; 2) subdivision of each sample into three technical replicates; 3) end-repair, A-tailing, and ligation of fragmented DNA to a linker cassette (LC); 4) two sequential PCR amplifications (25 and 10 cycles, respectively) to selectively enrich vector-genome junctions using primers annealing to the vector long terminal repeats (LTR) and the LC.

[0748] A clean-up and concentration step was performed between the two amplification rounds to prepare the products for the second PCR. Each primer included a unique DNA barcode enabling unambiguous samples indexing, as well as adapters compatible with multiplexed paired-end sequencing on Illumina or MGI platforms. The final sequencing library, comprising 145 amplicons, was sequenced on an MGI G400 platform, generating over 1.96x108 raw reads.

[0749] Identification of vector IS

[0750] IS retrieval and annotation were performed using VISPA2 (Spinozzi, etal., BMC Bioinformatics 18, 1-12 (2017)) on PCR-amplified libraries sequenced with MGI paired-end technology. Paired-end reads underwent quality control filtering, followed by barcode recognition for sample demultiplexing. Vector sequences were trimmed, and the remaining genomic fragments were aligned to the human reference genome (GRCh38 / hg38, February 2022 realese).

[0751] Quantification of clonal abundance was carried out using SonicLength (Berry, et al., Bioinformatics 28, 755-762 (2012)) approach, which estimates the number of contributing genomes based on the count of distinct DNA fragment lengths obtained with the sonication, associated with each IS. This method ensures that IS abundance reflects the relative contribution of individual clones within the initial cell population. The final dataset comprised unique ISs that were precisely mapped and annotated with their nearest RefSeq gene.

[0752] Downstream analyses were conducted using the R package ISAnalytics (Pais, et al. Brief Bioinform 24, (2023)), which integrates VISPA2 outputs and provides comprehensive IS data processing, including quality control, filtering, and assessment of shared ISs across samples. A detailed report and source code for ISAnalytics are available at https: / / github.com / calabrialab / ISAnalytics. To address instances of collisions, identical ISs erroneously detected across independent samples, we applied a previously described disambiguation strategy6. Briefly, when an IS was found in two independent groups, it was assigned to the group in which its relative abundance was at least tenfold higher. Collision detection and resolution were implemented via ISAnalytics.

[0753] For the identification of Common Insertion Sites (CIS), we employed the Grubbs outliers test (Biffi, et al., Science 341, (2013)), as implemented in ISAnalytics. For each vector, we computed the targeting frequency of individual genes by counting ISs located within the gene body or ± 100 kbp from it, normalized by gene length. The Iog2-transformed gene frequency distribution was then analyzed using the Grubbs test to detect genes exhibiting significantly higher targeting frequencies compared to the overall background.

[0754] Clonal population diversity

[0755] An ecological system is maintained stable if the populating species are in equilibrium, suggesting a healthy environment. Populations dynamics can be characterized in terms of species diversity, using for example a quantitative measure such as the Shannon diversity index (H-index). H-index accounts for the number of distinct species (richness) and their relative abundance with the following formula:

[0756]

[0757] Where i is a clone (an IS), p_i is the clonal abundance, R is the set of clones.

[0758] Several clonal studies have investigated the heterogeneity and complexity of vector-marked cells across time, tissues, and differentiated lineages, using IS a proxy for different species and the IS abundance as an indicator of species prevalence. Richness and evenness were measured over time to assess long-term efficacy (reflected by the maintenance of a high H-index) or to detect malignant events (indicated by a sharp decline in the H-index over time). This diversity index was calculated using the R package Vegan and incorporated into ISAanalytics.

[0759] The invention is further described by the following numbered paragraphs:

[0760] 1. A fusion protein comprising a membrane-targeting domain and an RNA-binding domain, wherein the membrane-targeting domain and RNA-binding domain are linked by a peptide cleavable by a lentiviral protease, and wherein the fusion protein is not operably linked to a lentiviral structural protein.

[0761] 2. A fusion protein comprising, in order from N-terminus to C-terminus, a membranetargeting domain, a peptide cleavable by a lentiviral protease, and an RNA-binding domain.

[0762] 3. The fusion protein according to para 1 or para 2, wherein the membrane-targeting domain comprises a phospholipase C-δ1 pleckstrin homology domain or a membrane-targeting domain of a proto-oncogene tyrosine-protein kinase Src.

[0763] 4. The fusion protein according to any preceding para, wherein the membrane-targeting domain comprises a phospholipase C-δ1 pleckstrin homology domain.

[0764] 5. The fusion protein according to para 3 or para 4, wherein the phospholipase C-δ1 pleckstrin homology domain comprises or consists of an amino acid sequence according to SEQ ID NO: 3, or a sequence having at least 80% sequence identity thereto.

[0765] 6. The fusion protein according to any preceding para, wherein the RNA-binding domain comprises an aptamer-binding protein.

[0766] 7. The fusion protein according to any preceding para, wherein the RNA-binding domain comprises an MS2 phage capsid protein.

[0767] 8. The fusion protein according to para 7, wherein the MS2 phage capsid protein comprises or consists of an amino acid sequence according to SEQ ID NO: 5, or a sequence having at least 80% sequence identity thereto. 9. The fusion protein according to any preceding para, wherein the peptide cleavable by a lentiviral protease is an HIV protease cleavage sequence.

[0768] 10. The fusion protein according to para 9, wherein the HIV protease cleavage sequence comprises or consists of the amino acid sequence VSQNYPIVQ (SEQ ID NO: 6).

[0769] 11. The fusion protein according to any preceding para, wherein the fusion protein comprises or consists of an amino acid sequence according to SEQ ID NO: 1, or a sequence having at least 80% sequence identity thereto.

[0770] 12. A polynucleotide encoding a fusion protein according to any preceding para.

[0771] 13. The polynucleotide according to para 12, wherein the polynucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 11, or a sequence having at least 80% sequence identity thereto.

[0772] 14. A cell comprising the polynucleotide according to para 12 or para 13, optionally wherein the cell is a lentiviral packaging cell or a lentiviral producer cell.

[0773] 15. A lentiviral vector comprising:

[0774] a) a fusion protein according to any one of paras 1 to 11;

[0775] b) an mRNA comprising a first transgene and a target site for the RNA-binding domain;

[0776] and

[0777] c) a lentiviral genome, optionally wherein the lentiviral genome comprises a second transgene.

[0778] 16. The lentiviral vector according to para 15, wherein the target site for the RNA-binding domain comprises an aptamer.

[0779] 17. The lentiviral vector according to para 15 or para 16, wherein the target site for the RNA-binding domain comprises an MS2 stem-loop, preferably six MS2 stem-loops.

[0780] 18. The lentiviral vector according to any one of paras 15 to 17, wherein the mRNA comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).

[0781] 19. The lentiviral vector according to any one of paras 15 to 18, wherein the mRNA comprises an miRNA target sequence. 20. The lentiviral vector according to para 19, wherein the mRNA comprises an miR-122 target sequence, an miR-126 target sequence, and / or an miR-142 target sequence.

[0782] 21. The lentiviral vector according to para 20, wherein the miR-122 target sequence comprises or consists of SEQ ID NO: 53, the miR-126 target sequence comprises or consists of SEQ ID NO: 51, and / or the miR-142 target sequence comprises or consists of SEQ ID NO: 59.

[0783] 22. The lentiviral vector according to any one of paras 15 to 21, wherein the first transgene encodes a therapeutic polypeptide, an antigenic polypeptide, a cytokine, a chemokine receptor (such as CXCR4), a viral accessory protein (such as Vpx), a transduction enhancer, or an enzyme involved in dNTP pool enhancement.

[0784] 23. The lentiviral vector according to any one of paras 15 to 22, wherein the second transgene encodes a therapeutic polypeptide, an antigenic polypeptide, an immune activating molecule, or a cytokine.

[0785] 24. The lentiviral vector according to any one of paras 15 to 23, wherein the lentiviral vector is an integration defective lentiviral vector (IDLV).

[0786] 25. The lentiviral vector according to para 24, wherein the first transgene encodes a gene editing enhancer, a p53 inhibitor, a transduction enhancer, a cell proliferation enhancer, an enzyme involved in dNTP pool enhancement, and / or a cell cycle booster, and wherein the second transgene encodes a guide RNA and / or a corrective DNA template.

[0787] 26. The lentiviral vector according to para 24, wherein the first transgene encodes a p53 inhibitor, a transduction enhancer, an enzyme involved in dNTP pool enhancement, and / or a cell cycle booster, and wherein the second transgene encodes a guide RNA and / or a corrective DNA template.

[0788] 27. A cell comprising a lentiviral vector according to any one of paras 15 to 26.

[0789] 28. A pharmaceutical composition comprising a lentiviral vector according to any one of paras 15 to 26, or a cell according to para 27, and a pharmaceutically acceptable carrier, diluent or excipient.

[0790] 29. A method for producing a lentiviral vector, comprising the steps of:

[0791] a) introducing into a cell: a. a vector encoding a fusion protein according to any one of paras 1 to 11, b. a vector encoding an mRNA comprising a first transgene and a target site for the RNA-binding domain; and

[0792] c. a transfer vector and optionally one or more helper vector,

[0793] b) culturing the cell under conditions suitable for the production of the lentiviral vector.

[0794] 30. A lentiviral vector according to any one of paras 15 to 23, a cell according to para 27, or a pharmaceutical composition according to para 28 for use in a method of treating and / or preventing a disease in a subject.

[0795] 31. The lentiviral vector, cell or composition for use according to para 30, wherein the first transgene is transiently expressed by a cell, and optionally the second transgene is stably integrated into the genome of the cell.

[0796] 32. A lentiviral vector according to any one of paras 24 to 26, a cell according to para 27, or a pharmaceutical composition according to para 28 for use in a method of treating and / or preventing a disease in a subject.

[0797] 33. The lentiviral vector, cell or composition for use according to para 32, wherein the first transgene encodes a gene editing enhancer, a p53 inhibitor, a cell proliferation enhancer, and / or a cell cycle booster, and the second transgene encodes a corrective DNA template; and wherein the method further comprises administering a Cas ribonucleoprotein, or a guide RNA and a polynucleotide encoding a Cas protein.

[0798] 34. The lentiviral vector, cell or composition for use according to para 32, wherein the first transgene encodes a p53 inhibitor and / or a cell cycle booster, and the second transgene encodes a corrective DNA template; and wherein the method further comprises administering a Cas ribonucleoprotein, or a guide RNA and a polynucleotide encoding a Cas protein.

[0799] 35. The lentiviral vector, cell or composition for use according to para 33 or para 34, wherein the method comprises administering a virus like particle (VLP) comprising a Cas ribonucleoprotein.

[0800] 36. A kit of vectors, comprising:

[0801] a) a vector encoding a fusion protein according to any one of paras 1 to 11,

[0802] b) a vector encoding an mRNA comprising a first transgene and a target site for the RNA- binding domain; and

[0803] c) a transfer vector and optionally one or more helper vector.

Claims

CLAIMS1. A fusion protein comprising a membrane-targeting domain and an RNA-binding domain, wherein the membrane-targeting domain and RNA-binding domain are linked by a peptide cleavable by a lentiviral protease, and wherein the fusion protein is not operably linked to a lentiviral structural protein.

2. A fusion protein comprising, in order from N-terminus to C-terminus, a membranetargeting domain, a peptide cleavable by a lentiviral protease, and an RNA-binding domain.

3. The fusion protein according to claim 1 or claim 2, wherein:(a) the membrane-targeting domain comprises a phospholipase C-51 pleckstrin homology domain or a membrane-targeting domain of a proto-oncogene tyrosineprotein kinase Src, preferably a phospholipase C-51 pleckstrin homology domain, optionally wherein the phospholipase C-δ1 pleckstrin homology domain comprises or consists of an amino acid sequence according to SEQ ID NO: 3, or a sequence having at least 80% sequence identity thereto;(b) the RNA-binding domain comprises an aptamer-binding protein;(c) the RNA-binding domain comprises an MS2 phage capsid protein, optionally wherein the MS2 phage capsid protein comprises or consists of an amino acid sequence according to SEQ ID NO: 5, or a sequence having at least 80% sequence identity thereto; and / or(d) the peptide cleavable by a lentiviral protease is an HIV protease cleavage sequence, optionally wherein the HIV protease cleavage sequence comprises or consists of the amino acid sequence VSQNYPIVQ (SEQ ID NO: 6).

4. The fusion protein according to any preceding claim, wherein the fusion protein comprises or consists of an amino acid sequence according to SEQ ID NO: 1, or a sequence having at least 80% sequence identity thereto.

5. A polynucleotide encoding a fusion protein according to any preceding claim, optionally wherein the polynucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 11, or a sequence having at least 80% sequence identity thereto.

6. A cell comprising the polynucleotide according to claim 5, optionally wherein the cell is a lentiviral packaging cell or a lentiviral producer cell.

7. A lentiviral vector comprising:a) a fusion protein according to any one of claims 1 to 4;b) an mRNA comprising a first transgene and a target site for the RNA-binding domain;andc) a lentiviral genome, optionally wherein the lentiviral genome comprises a second transgene.

8. The lentiviral vector according to claim 7, wherein the target site for the RNA-binding domain comprises an aptamer, and / or wherein the target site for the RNA-binding domain comprises an MS2 stem-loop, preferably six MS2 stem-loops.

9. The lentiviral vector according to any one of claims 7 to 8, wherein the mRNA comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) and / or an miRNA target sequence, optionally wherein the mRNA comprises an miR-122 target sequence, an miR-126 target sequence, and / or an miR-142 target sequence, optionally wherein the miR-122 target sequence comprises or consists of SEQ ID NO: 53, the miR-126 target sequence comprises or consists of SEQ ID NO: 51, and / or the miR-142 target sequence comprises or consists of SEQ ID NO: 59.

10. The lentiviral vector according to any one of claims 7 to 9, wherein:(a) the first transgene encodes a therapeutic polypeptide, an antigenic polypeptide, a cytokine, a chemokine receptor (such as CXCR4), a viral accessory protein (such as Vpx), a transduction enhancer, or an enzyme involved in dNTP pool enhancement;(b) the second transgene encodes a therapeutic polypeptide, a chimeric antigen receptor (CAR), an antigenic polypeptide, an immune activating molecule, or a cytokine; and / or(c) the lentiviral vector is an integration defective lentiviral vector (IDLV), optionally wherein the first transgene encodes a gene editing enhancer, p53 inhibitor, a transduction enhancer, a cell proliferation enhancer, an enzyme involved in dNTP pool enhancement, and / or a cell cycle booster, and wherein the second transgene encodes a guide RNA and / or a corrective DNA template.

11. A cell comprising a lentiviral vector according to any one of claims 7 to 10.

12. A pharmaceutical composition comprising a lentiviral vector according to any one of claims 7 to 10, or a cell according to claim 11, and a pharmaceutically acceptable carrier, diluent or excipient.

13. A method for producing a lentiviral vector, comprising the steps of:a) introducing into a cell:a. a vector encoding a fusion protein according to any one of claims 1 to 4, b. a vector encoding an mRNA comprising a first transgene and a target site for the RNA-binding domain; andc. a transfer vector and optionally one or more helper vector,b) culturing the cell under conditions suitable for the production of the lentiviral vector.

14. A lentiviral vector according to any one of claims 7 to 10, a cell according to claim 11, or a pharmaceutical composition according to claim 12 for use in a method of treating and / or preventing a disease in a subject, optionally wherein:(a) the first transgene is transiently expressed by a cell, and optionally the second transgene is stably integrated into the genome of the cell; or(b) the first transgene encodes a gene editing enhancer, a p53 inhibitor, a cell proliferation enhancer, and / or a cell cycle booster, and the second transgene encodes a corrective DNA template; and wherein the method further comprises administering a Cas ribonucleoprotein, or a guide RNA and a polynucleotide encoding a Cas protein, optionally wherein the method comprises administering a virus like particle (VLP) comprising a Cas ribonucleoprotein.

15. A lentiviral vector according to any one of claims 7 to 10 for use in a method of therapy in vivo, optionally wherein the therapy is CAR T cell therapy.

16. A method of transducing a cell, comprising contacting the cell with the lentiviral vector of any one of claims 7 to 10, optionally wherein the cell is a T cell.

17. A cell obtainable by the method of claim 16, optionally wherein the cell is a T cell.

18. A kit of vectors, comprising:a) a vector encoding a fusion protein according to any one of claims 1 to 4,b) a vector encoding an mRNA comprising a first transgene and a target site for the RNA- binding domain; andc) a transfer vector and optionally one or more helper vector.

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