AU2024264830A1

AU2024264830A1 - Gene therapy

Info

Publication number: AU2024264830A1
Application number: AU2024264830A
Authority: AU
Inventors: Luigi Naldini; Marco NOTARO; Mario Leonardo Squadrito
Original assignee: Univ Vita Salute San Raffaele; Fondazione Telethon; Ospedale San Raffaele SRL
Current assignee: Universita Vita Salute San Raffaele; Fondazione Telethon; Ospedale San Raffaele SRL
Priority date: 2023-05-03
Filing date: 2024-05-03
Publication date: 2025-11-13
Also published as: WO2024227946A1

Abstract

A product comprising: (a) a vector for liver and/or splenic phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence; and (b) an immune checkpoint inhibitor or a Tr1 cell inhibitor.

Description

GENE THERAPY FIELD OF THE INVENTION The present invention relates to vectors for phagocyte-specific expression, particularly liver and/or splenic phagocyte-specific expression, and combinations of the vectors with immune checkpoint inhibitors and/or Tr1 cell inhibitors. The invention also relates to cells, and pharmaceutical compositions comprising said vectors, and uses in therapy, including treatment or prevention of cancer, for example liver metastases. BACKGROUND TO THE INVENTION The liver is involved in several biological functions, including detoxification, clearance of protein and cells, and metabolic functions among others. In order to preserve the liver from immunological reactions that might damage it, the liver is characterized by an immunosuppressive environment that limits immune. Due to its immunosuppressive environment several tumour types are prone to spread towards the liver giving rise to liver metastases. The liver is one of the most common sites for cancer metastasis, accounting for nearly 25% of all cases. A variety of primary tumors may be the source for metastasis, however, colorectal adenocarcinomas are the most common considering the overall number of patients affected. Liver metastases are linked to poor prognosis and often constitute the cause of death of cancer patients. Surgical resection remains the gold standard for anatomically resectable liver metastases. Strategies to improve the chances of resection include neoadjuvant chemotherapy, portal vein embolization to increase the future liver remnant, or a two-stage resection versus a combined one-stage resection of the primary tumor, and hepatic lesions. However, the five-year survival after curative resection of hepatic lesions for patients with colorectal metastases has been reported as only 25% to 58% with a median survival length of 74 months. Thus, there is a significant need for improved treatments for cancers such as liver metastases. SUMMARY OF THE INVENTION The present inventors developed a lentiviral vector (LV) platform that, for example, enables engineering of liver resident macrophages (Kupffer cells) to deliver transgenes, such as interferon-alpha (IFNα), specifically to liver metastases. The inventors observed that gene- based IFNα delivery to distinct mouse models of colorectal and pancreatic ductal adenocarcinoma liver metastases significantly delayed tumor growth. While not wishing to be bound by theory, the inventors observed that response to IFNα was associated with tumor- associated macrophage (TAM) immune activation, enhanced MHCII-restricted antigen presentation by tumor-infiltrating dendritic cells, as well as reduced exhaustion of CD8 T cells. Conversely, increased IL10 signalling, enhanced expression of CTLA4 and expansion of Eomes CD4 T cells, a cell type displaying features of type I regulatory T (Tr1) cells, were associated with resistance to IFNα gene therapy. The inventors then observed that targeting of regulatory T cell function by immune checkpoint blockade and IFNα LV delivery resulted in a strong synergy attaining complete response in most mice. In one aspect, the invention provides a product comprising: (a) a vector for liver and/or splenic phagocyte-specific expression; and (b) an immune checkpoint inhibitor or a Tr1 cell inhibitor. Suitably, the phagocytes targeted in the present invention are selected from one or more of: a macrophage, such as a M2-like macrophage and/or MRC1+ macrophage; a dendritic cell; and an endothelial cell, such as a liver sinusoidal endothelial cell. Suitably, the phagocytes targeted in the present invention are selected from one or more of: a resident macrophage (e.g. a Kupffer cell); a liver sinusoidal endothelial cell; a splenic macrophage; a tumour- associated macrophage; and/or a monocyte-derived macrophage. In some embodiments, the phagocytes targeted in the present invention are Kupffer cells. The vector may comprise a transgene operably linked to one or more expression control sequence. In one aspect, the invention provides a product comprising: (a) a vector for liver and/or splenic phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence; and (b) an immune checkpoint inhibitor or a Tr1 cell inhibitor. In preferred embodiments, the product of the invention comprises the immune checkpoint inhibitor. In some embodiments, the product of the invention comprises the Tr1 cell inhibitor. In one aspect, the invention provides a product comprising: (a) a vector for liver and/or splenic phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence; and (b) an immune checkpoint inhibitor. In one aspect, the invention provides a product comprising: (a) a vector for liver and/or splenic phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence; and (b) a Tr1 cell inhibitor. In some embodiments, the product is in the form of a composition (e.g. a pharmaceutical composition) or a kit. In one aspect, the invention provides a vector for use in therapy, wherein the vector is for liver and/or splenic phagocyte-specific expression, and wherein the vector is used in combination with an immune checkpoint inhibitor or a Tr1 cell inhibitor. In preferred embodiments, the use of the invention comprises combination with the immune checkpoint inhibitor. In some embodiments, the use of the invention comprises combination with the Tr1 cell inhibitor. In one aspect, the invention provides a vector for use in therapy, wherein the vector is for liver and/or splenic phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence, and wherein the vector is used in combination with an immune checkpoint inhibitor or a Tr1 cell inhibitor. In one aspect, the invention provides a vector for use in therapy, wherein the vector is for liver and/or splenic phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence, and wherein the vector is used in combination with an immune checkpoint inhibitor. In one aspect, the invention provides a vector for use in therapy, wherein the vector is for liver and/or splenic phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence, and wherein the vector is used in combination with a Tr1 cell inhibitor. In one aspect, the invention provides an immune checkpoint inhibitor or a Tr1 cell inhibitor for use in therapy, wherein the immune checkpoint inhibitor or Tr1 cell inhibitor is used in combination with a vector for liver and/or splenic phagocyte-specific expression. In one aspect, the invention provides an immune checkpoint inhibitor or a Tr1 cell inhibitor for use in therapy, wherein the immune checkpoint inhibitor or Tr1 cell inhibitor is used in combination with a vector for liver and/or splenic phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence. In one aspect, the invention provides an immune checkpoint inhibitor for use in therapy, wherein the immune checkpoint inhibitor is used in combination with a vector for liver and/or splenic phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence. In one aspect, the invention provides a Tr1 cell inhibitor for use in therapy, wherein Tr1 cell inhibitor is used in combination with a vector for liver and/or splenic phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence. In some embodiments, the vector is for Kupffer cell-specific expression. In some embodiments, the one or more expression control sequence comprises: (a) a phagocyte-specific promoter and/or enhancer; and/or (b) one or more miRNA target sequence. In some embodiments, the one or more expression control sequence comprises a phagocyte- specific promoter and/or enhancer; and/or (b) one or more miRNA target sequence. In some embodiments, the one or more expression control sequence comprises: (a) a phagocyte-specific promoter and/or enhancer. In some embodiments, the one or more expression control sequence comprises one or more miRNA target sequence. In some embodiments, the one or more expression control sequence comprises: (a) a phagocyte- specific promoter and/or enhancer; and (b) one or more miRNA target sequence. In some embodiments, the phagocyte-specific promoter and/or enhancer is a liver and/or splenic phagocyte-specific promoter and/or enhancer. In some embodiments, the one or more miRNA target sequence suppresses expression in cells other than liver phagocytes. In some embodiments, the phagocyte is a liver and/or splenic phagocyte. In some embodiments, the phagocyte is a macrophage. In some embodiments, the phagocyte is an M2-like macrophage and/or MRC1+ macrophage; dendritic cell; or liver sinusoidal endothelial cell. In some embodiments, the phagocyte is a liver-resident phagocyte. In some embodiments, the phagocyte is a liver-resident macrophage. In some embodiments, the phagocyte is a Kupffer cell. In some embodiments, the phagocyte is a liver sinusoidal endothelial cell. In some embodiments, vector comprises from 5’ to 3’: the phagocyte-specific promoter and/or enhancer – the transgene – the one or more miRNA target sequence. In some embodiments, the phagocyte-specific promoter and/or enhancer, is selected from the group consisting of: a MRC1 promoter and/or enhancer; an ITGAM promoter and/or enhancer; a CD86 promoter and/or enhancer; a CD274 promoter and/or enhancer; a CD163 promoter and/or enhancer; a LYVE1 promoter and/or enhancer; a STAB1 promoter and/or enhancer; a ITGAX promoter and/or enhancer; a SIRPA promoter and/or enhancer; a TIE2 promoter and/or enhancer; a CHIL3 promoter and/or enhancer; a CD68 promoter and/or enhancer; a CSF1R promoter and/or enhancer; a VCAM1 promoter and/or enhancer; a PTGS1 promoter and/or enhancer; and a C1QA promoter and/or enhancer; a fragment thereof, or a combination thereof. In some embodiments, the phagocyte-specific promoter and/or enhancer is a MRC1 promoter and/or enhancer or a fragment thereof. In some embodiments, the MRC1 promoter and/or enhancer or fragment thereof comprises or consists of a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1, or a fragment thereof. In some embodiments, the MRC1 promoter and/or enhancer or fragment thereof comprises or consists of the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof. In some embodiments, the one or more miRNA target sequence suppresses expression in non-phagocyte (e.g. non-liver and/or splenic phagocyte) cells. In preferred embodiments, the one or more miRNA target sequence suppresses expression in non-liver phagocyte cells (i.e. in cells other than liver phagocyte cells). In some embodiments, the one or more miRNA target sequence suppresses transgene expression in hepatocytes and/or liver sinusoidal endothelial cells and/or splenic phagocytes. The one or more miRNA target sequence may suppress expression in some liver and/or spleen cell populations. In some embodiments, the one or more miRNA target sequence suppresses transgene expression in hepatocytes. In some embodiments, the one or more miRNA target sequence suppresses transgene expression in liver sinusoidal endothelial cells (LSECs). In some embodiments, the one or more miRNA target sequence suppresses transgene expression in splenic phagocytes. In some embodiments, the one or more miRNA target sequence suppresses transgene expression in splenic macrophages. In some embodiments, the one or more miRNA target sequence suppresses transgene expression in hepatocytes, liver sinusoidal endothelial cells (LSECs) and/or splenic phagocytes. In some embodiments, the one or more miRNA target sequence comprises: (a) one or more miR-126 target sequence; and/or (b) one or more miR-122 target sequence. In some embodiments, the one or more miRNA target sequence comprises one or more miR- 126 target sequence. In some embodiments, the one or more miRNA target sequence comprises one or more miR-122 target sequence. In some embodiments, the one or more miRNA target sequence comprises: (a) one or more miR-126 target sequence; and (b) one or more miR-122 target sequence. In some embodiments, the one or more miRNA target sequence comprises four miR-126 target sequences and/or four miR-122 target sequences. In some embodiments, the one or more miRNA target sequence comprises four miR-126 target sequences and four miR-122 target sequences. In some embodiments, the miR-126 target sequence comprises or consists of SEQ ID NO: 3. In some embodiments, the miR-122 target sequence comprises or consists of SEQ ID NO: 4. In preferred embodiments, the vector comprises a transgene operably linked to (a) a MRC1 promoter and/or enhancer, or a fragment thereof; and (b) one or more miR-126 target sequence and/or one or more miR-122 target sequence. In some embodiments, the transgene encodes a therapeutic polypeptide and/or an antigenic polypeptide. In some embodiments, the transgene encodes a therapeutic polypeptide. In some embodiments, the transgene encodes an antigenic polypeptide. In some embodiments, the transgene encodes a cytokine. In some embodiments, the cytokine is interferon-alpha, interferon-beta, interferon-gamma, IL2, IL12, TNF-alpha, CXCL9, IL1-beta, IL15, IL18, IL10, GMCSF, FLT3, IL7 or IL21. In some embodiments, the transgene encodes interferon-alpha. In some embodiments, the transgene encodes IL12. In some embodiments, the transgene encodes IL10. In some embodiments, the interferon-alpha comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 8. In some embodiments, the interferon-alpha comprises or consists of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the transgene encodes a tumour antigen. In some embodiments, the tumour antigen is carcinoembryonic antigen (CEA), TRP2, melanoma associated antigen (MAGE) family, cancer germline (CAGE) family, B melanoma antigen (BAGE-1), synovial sarcoma x breakpoint 20 (SSX-2), Sarcoma antigen (SAGE) family, LAGE1, NY-ESO-1, HER2, EGFR, MUC-1 or GAST. In some embodiments, the antigen is an MHC-I-restricted antigen. In some embodiments, the antigen is an MHC-II-restricted antigen. In some embodiments, the tumour antigen is TRP2. In some embodiments, the transgene encodes a cytokine, and the product or combination further comprises a second vector comprising a second transgene operably linked to one or more expression control sequence; and optionally a third vector comprising a third transgene operably linked to one or more expression control sequence. In some embodiments, the transgene encodes a cytokine, and the product or combination further comprises a second vector comprising a second transgene operably linked to one or more expression control sequence; and a third vector comprising a third transgene operably linked to one or more expression control sequence. The one or more expression control sequences may be as disclosed herein. In some embodiments, the transgene encodes a cytokine, and the vector further comprises a second transgene operably linked to one or more expression control sequence; and optionally a third transgene operably linked to one or more expression control sequence. In some embodiments, the transgene encodes a cytokine, and the vector further comprises a second transgene operably linked to one or more expression control sequence; and a third transgene operably linked to one or more expression control sequence. The one or more expression control sequences may be as disclosed herein. The third transgene may be comprised in a second vector. Preferably, the transgene and second transgene are different. Preferably, the transgene, second transgene and third transgene are different. The second and/or third vector and the second and/or third transgene may comprise further features or be operably linked to further features in the same manner as disclosed herein for the vector and/or transgene of the invention. In some embodiments, the cytokine is interferon-alpha, interferon-beta, interferon-gamma, IL2, IL12, TNF-alpha, CXCL9, IL1-beta, IL15, IL18, IL10, GMCSF, FLT3, IL7 or IL21. In some embodiments, the cytokine is interferon-alpha. In some embodiments, the second transgene encodes a second cytokine, wherein the cytokine is different to the second cytokine. In some embodiments, the second cytokine is IL12, interferon-alpha, interferon-beta, interferon-gamma, IL2, TNF-alpha, CXCL9, IL1-beta, IL15, IL18, IL10, GMCSF, FLT3, IL7 or IL21. In some embodiments, the second cytokine is IL12. In some embodiments, the cytokine is interferon-alpha and the second cytokine is IL12. In some embodiments, the second transgene encodes a tumour antigen. In some embodiments, the tumour antigen is carcinoembryonic antigen (CEA), TRP2, melanoma associated antigen (MAGE) family, cancer germline (CAGE) family, B melanoma antigen (BAGE-1), synovial sarcoma x breakpoint 20 (SSX-2), Sarcoma antigen (SAGE) family, LAGE1, NY-ESO-1, HER2, EGFR, MUC-1 or GAST. In some embodiments, the cytokine is interferon-alpha and the second cytokine is a tumour antigen. In some embodiments, the cytokine is IL12 and the second cytokine is a tumour antigen. In some embodiments, the third transgene encodes a tumour antigen. In some embodiments, the tumour antigen is carcinoembryonic antigen (CEA), TRP2, melanoma associated antigen (MAGE) family, cancer germline (CAGE) family, B melanoma antigen (BAGE-1), synovial sarcoma x breakpoint 20 (SSX-2), Sarcoma antigen (SAGE) family, LAGE1, NY-ESO-1, HER2, EGFR, MUC-1 or GAST. In some embodiments, the tumour antigen is a TRP2. In some embodiments, the second transgene encodes a second cytokine, wherein the cytokine is different to the second cytokine, and the third transgene encodes a tumour antigen. In some embodiments, the cytokine is interferon-alpha, the second cytokine is IL12 and the third transgene encodes a tumour antigen. In some embodiments, the cytokine is interferon-alpha, the second cytokine is IL12 and the tumour antigen is TRP2. In some embodiments, the transgene is further operably linked to one or more regulatory element. In some embodiments, the transgene is further operably linked to a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In some embodiments, the transgene is further operably linked to a destabilising domain. In some embodiments, the destabilising domain is a dihydrofolate reductase destabilising domain. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an integrating viral vector. In some embodiments, the vector is a non-integrating viral vector. In some embodiments, the vector is a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a herpes simplex viral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an integration-defective lentiviral vector (IDLV). In some embodiments, the viral vector is a viral vector particle. In some embodiments, the viral vector particle is VSV-G pseudotyped. In some embodiments, the viral vector is a VSV-G pseudotyped lentiviral vector particle. In some embodiments, the viral vector particle is produced in a viral particle producer or packaging cell which has been genetically engineered to decrease expression of CD47 and/or HLA on the surface of the cell. In some embodiments, the viral vector particle is substantially devoid of surface-exposed CD47 and/or HLA. The vector may specifically express the transgene in phagocytes. In some embodiments: (i) expression of the transgene in phagocytes transduced by the vector is greater than expression of the transgene in other cells transduced by the vector; and/or (ii) the transgene is substantially not expressed in cells other than the phagocytes, when transduced by the vector; and/or (iii) the transgene is substantially not expressed in lung cells, bone marrow cells and/or blood cells, when transduced by the vector; and/or (iv) the transgene is substantially only expressed in some liver cells and/or some splenic cells; and/or (v) expression of the transgene in Kupffer cells is at least ten times greater than expression in hepatocytes, when transduced by the vector; and/or (vi) the transgene is substantially not expressed in hepatocytes when transduced by the vector. In some embodiments, expression of the transgene in phagocytes transduced by the vector is greater than expression of the transgene in other cells transduced by the vector. In some embodiments, the transgene is substantially not expressed in cells other than the phagocytes, when transduced by the vector. In some embodiments, the transgene is substantially not expressed in lung cells, bone marrow cells and/or blood cells, when transduced by the vector. In some embodiments, expression of the transgene in Kupffer cells is at least ten times greater than expression in hepatocytes, when transduced by the vector. In some embodiments, the transgene is substantially not expressed in hepatocytes when transduced by the vector. In some embodiments, the transgene is substantially only expressed in liver cells and/or splenic cells, and optionally substantially not expressed in hepatocytes when transduced by the vector. In some embodiments, the transgene is substantially only expressed in liver cells and/or splenic cells. In some embodiments, the immune checkpoint inhibitor inhibits an inhibitory checkpoint molecule selected from the group consisting of CTLA-4 (Cytotoxic T-Lymphocyte-Associated protein 4; CD152), A2AR (Adenosine A2A receptor), B7-H3 (CD276), B7-H4 (VTCN1), BTLA (B and T Lymphocyte Attenuator; CD272), HVEM (Herpesvirus Entry Mediator), IDO (Indoleamine 2,3-dioxygenase), TDO (tryptophan 2,3-dioxygenase), KIR (Killer-cell Immunoglobulin-like Receptor), LAG3 (Lymphocyte Activation Gene-3), PD-1 (Programmed Death 1 receptor), PD-L1 (PD-1 ligand 1), PD-L2 (PD-1 ligand 2), TIM-3 (T- cell Immunoglobulin domain and Mucin domain 3), VISTA (V-domain Ig Suppressor of T cell Activation), B7-1 (CD80), B7-2 (CD86), a TGFB (Transforming growth factor beta) pathway- associated protein, Il13 (interleukin-13), IL4 (interleukin-4), FGL (Fibrinogen Like 1), TIGIT (T cell immunoreceptor with Ig and ITIM domains), CD96 (TACT protein), Ceacam-1 (Carcinoembryonic antigen related cell adhesion molecule 1), CD155 (PVR protein), CD112 (PVR-related protein 2 (PVRL2)), LGALS3 (Galectin 3) and CD47 (integrin associated protein). A combination of two or more immune checkpoint inhibitors may be used. In some embodiments, the immune checkpoint inhibitor inhibits PD-1. In some embodiments, the cytokine is interferon-alpha, the second cytokine is IL12 and the tumour antigen is TRP2, and the immune checkpoint inhibitor inhibits PD-1. In some embodiments, the TGFB pathway-associated protein is selected from the group consisting of TGFB1 (Transforming growth factor beta-1), TGFB2 (Transforming growth factor beta-2), TGFB3 (Transforming growth factor beta-3), LTBP1 (Latent Transforming Growth Factor Beta Binding Protein 1), TGFBR1 (Transforming growth factor beta receptor 1), TGFBR2 (Transforming growth factor beta receptor 2), Integrin αv, Integrin β5, Integrin β6, Integrin β8, and LRRC32 (Leucine Rich Repeat Containing 32). In some embodiments, the immune checkpoint inhibitor is an antibody. In some embodiments, the immune checkpoint inhibitor antibody is selected from the group consisting of an anti- CTLA4 antibody, an anti-PD1 antibody, an anti-PDL1 antibody, an anti-PDL2 antibody and an anti-LAG-3 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA4 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody. In some embodiments, the transgene encodes interferon-alpha and the immune checkpoint inhibitor is an anti-PD1 antibody. In some embodiments, the transgene encodes interferon- alpha and the immune checkpoint inhibitor is an anti-CTLA4 antibody. In some embodiments, the cytokine is interferon-alpha, the second cytokine is IL12 and the tumour antigen is TRP2, and the immune checkpoint inhibitor is an anti-PD1 antibody. In some embodiments, the Tr1 cell inhibitor inhibits a molecule selected from the group consisting of Cd4, Eomes, Gzmk, Lag3, Pdcd1, Ahr, Maf, Prdm1, Ctla4 and Il10ra. In one aspect, the invention provides a cell comprising the product of the invention. In one aspect, the invention provides a cancer vaccine comprising the product of the invention. In one aspect, the invention provides the product of the invention for use in therapy. In preferred embodiments, the use in therapy is treatment or prevention of cancer. In one aspect, the invention provides a method of treating or preventing cancer comprising administering to a subject in need thereof: (a) a vector for liver and/or splenic phagocyte- specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence; and (b) an immune checkpoint inhibitor or a Tr1 cell inhibitor. The components of the combination may be, for example, administered simultaneously, sequentially or separately. In some embodiments, the cancer is liver metastasis. The metastasis may, for example, derive from a colon rectal carcinoma or pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the cancer is a primary liver tumour. In some embodiments, the product, combination or components thereof is administered systemically. In some embodiments, the product, combination or components thereof is administered by intravenous injection, intraportal injection or intrahepatic artery injection. In one aspect, the invention provides a product comprising: (a) a vector for liver and/or splenic phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence; (b) a second vector for liver and/or splenic phagocyte-specific expression, wherein the second vector comprises a second transgene operably linked to one or more expression control sequence, wherein the transgene is different to the second transgene. In one aspect, the invention provides a vector for use in therapy, wherein the vector is for liver and/or splenic phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence, and wherein the vector is used in combination with a second vector for liver and/or splenic phagocyte-specific expression, wherein the second vector comprises a second transgene operably linked to one or more expression control sequence, wherein the transgene is different to the second transgene. In one aspect, the invention provides a method of treating or preventing cancer comprising administering to a subject in need thereof: (a) a vector for liver and/or splenic phagocyte- specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence; and (b) a second vector for liver and/or splenic phagocyte- specific expression, wherein the second vector comprises a second transgene operably linked to one or more expression control sequence, wherein the transgene is different to the second transgene. In one aspect, the invention provides a product comprising a vector for liver and/or splenic phagocyte-specific expression, wherein the vector comprises (a) a transgene operably linked to one or more expression control sequence; and (b) a second transgene operably linked to one or more expression control sequence, wherein the transgene is different to the second transgene. In one aspect, the invention provides a vector for use in therapy, wherein the vector is for liver and/or splenic phagocyte-specific expression, wherein the vector comprises (a) a transgene operably linked to one or more expression control sequence; and (b) a second transgene operably linked to one or more expression control sequence, wherein the transgene is different to the second transgene. In one aspect, the invention provides a method of treating or preventing cancer comprising administering to a subject in need thereof a vector for liver and/or splenic phagocyte-specific expression, wherein the vector comprises (a) a transgene operably linked to one or more expression control sequence; and (b) a second transgene operably linked to one or more expression control sequence, wherein the transgene is different to the second transgene. In some embodiments, the transgene encodes a cytokine. In some embodiments, the cytokine is IL12, interferon-alpha, interferon-beta, interferon-gamma, IL2, TNF-alpha, CXCL9, IL1-beta, IL15, IL18, IL10, GMCSF, FLT3, IL7 or IL21. In some embodiments, the cytokine is IL12. In some embodiments, the second transgene encodes a second cytokine, wherein the cytokine is different to the second cytokine. In some embodiments, the second cytokine is interferon-alpha, interferon-beta, interferon-gamma, IL2, IL12, TNF-alpha, CXCL9, IL1-beta, IL15, IL18, IL10, GMCSF, FLT3, IL7 or IL21. In some embodiments, the second cytokine is interferon-alpha. In some embodiments (a) the transgene encodes a cytokine (e.g. IL12, interferon-alpha, interferon-beta, interferon-gamma, IL2, TNF-alpha, CXCL9, IL1-beta, IL15, IL18, IL10, GMCSF, FLT3, IL7 or IL21); and (b) the second transgene encodes a second cytokine (e.g. interferon-alpha, interferon-beta, interferon-gamma, IL2, IL12, TNF-alpha, CXCL9, IL1-beta, IL15, IL18, IL10, GMCSF, FLT3, IL7 or IL21), wherein the cytokine is different to the second cytokine. In some embodiments, the transgene encodes IL12 and the second transgene encodes interferon-alpha. In some embodiments, the second transgene encodes a tumour antigen. In some embodiments, the tumour antigen is carcinoembryonic antigen (CEA), TRP2, melanoma associated antigen (MAGE) family, cancer germline (CAGE) family, B melanoma antigen (BAGE-1), synovial sarcoma x breakpoint 20 (SSX-2), Sarcoma antigen (SAGE) family, LAGE1, NY-ESO-1, HER2, EGFR, MUC-1 or GAST. In some embodiments (a) the transgene encodes a cytokine (e.g. IL12, interferon-alpha, interferon-beta, interferon-gamma, IL2, TNF-alpha, CXCL9, IL1-beta, IL15, IL18, IL10, GMCSF, FLT3, IL7 or IL21); and (b) the second transgene encodes a tumour antigen (e.g. carcinoembryonic antigen (CEA), TRP2, melanoma associated antigen (MAGE) family, cancer germline (CAGE) family, B melanoma antigen (BAGE-1), synovial sarcoma x breakpoint 20 (SSX-2), Sarcoma antigen (SAGE) family, LAGE1, NY-ESO-1, HER2, EGFR, MUC-1 or GAST). In some embodiments, the transgene encodes IL12 and the second transgene encodes a tumour antigen. In some embodiments, the product or combination further comprises a third vector for liver and/or splenic phagocyte-specific expression, wherein the third vector comprises a third transgene operably linked to one or more expression control sequence, wherein the third transgene is different to the transgene and the second transgene. In some embodiments, the vector further comprises a third transgene operably linked to one or more expression control sequence, wherein the third transgene is different to the transgene and the second transgene. In some embodiments, the product or combination further comprises a second vector for liver and/or splenic phagocyte-specific expression, wherein the second vector comprises a third transgene operably linked to one or more expression control sequence, wherein the third transgene is different to the transgene and the second transgene. The transgene, second transgene and third transgene may each be independently selected from a cytokine or a tumour antigen, for example a cytokine or a tumour antigen as disclosed herein. In one aspect, the invention provides a method of treating or preventing cancer comprising administering to a subject in need thereof: (a) a vector for liver and/or splenic phagocyte- specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence; (b) a second vector for liver and/or splenic phagocyte-specific expression, wherein the second vector comprises a second transgene operably linked to one or more expression control sequence, wherein the transgene is different to the second transgene; and (c) a third vector for liver and/or splenic phagocyte-specific expression, wherein the third vector comprises a third transgene operably linked to one or more expression control sequence, wherein the third transgene is different to the transgene and the second transgene. In one aspect, the invention provides a method of treating or preventing cancer comprising administering to a subject in need thereof a vector for liver and/or splenic phagocyte-specific expression, wherein the vector comprises (a) a transgene operably linked to one or more expression control sequence; (b) a second transgene operably linked to one or more expression control sequence; and (c) a third transgene operably linked to one or more expression control sequence. In some embodiments (a) the transgene encodes a cytokine (e.g. IL12, interferon-alpha, interferon-beta, interferon-gamma, IL2, TNF-alpha, CXCL9, IL1-beta, IL15, IL18, IL10, GMCSF, FLT3, IL7 or IL21); (b) the second transgene encodes a second cytokine (e.g. interferon-alpha, interferon-beta, interferon-gamma, IL2, IL12, TNF-alpha, CXCL9, IL1-beta, IL15, IL18, IL10, GMCSF, FLT3, IL7 or IL21), wherein the cytokine is different to the second cytokine; and (c) the third transgene encodes a tumour antigen (e.g. carcinoembryonic antigen (CEA), TRP2, melanoma associated antigen (MAGE) family, cancer germline (CAGE) family, B melanoma antigen (BAGE-1), synovial sarcoma x breakpoint 20 (SSX-2), Sarcoma antigen (SAGE) family, LAGE1, NY-ESO-1, HER2, EGFR, MUC-1 or GAST). The cytokines and tumour antigen may be as disclosed herein. In some embodiments, the transgene encodes IL12, the second transgene encodes interferon-alpha, and the third transgene encodes a tumour antigen. In one aspect, the invention provides a method of treating or preventing cancer comprising administering to a subject in need thereof (i) a vector for liver and/or splenic phagocyte-specific expression, wherein the vector comprises (a) a transgene operably linked to one or more expression control sequence; (b) a second transgene operably linked to one or more expression control sequence; and (c) a third transgene operably linked to one or more expression control sequence; and (ii) an immune checkpoint inhibitor. In some embodiments, the transgene encodes interferon-alpha, the second transgene encodes IL12, the third transgene encodes TRP2, and the immune checkpoint inhibitor inhibits PD-1. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody. DESCRIPTION OF THE DRAWINGS FIGURE 1 Generation of a LV platform enabling in vivo liver macrophage engineering. (A) Schematics of Mrc1.GFP and Mrc1.GFP.miRT LVs. (B) Schematics of the experiments shown in panels C-F. (C) LV copies per cell of the indicated organs by digital droplet PCR (ddPCR) analysis. (D and E) GFP expression in the indicated cell types in the indicated organs by flow cytometry (FC) analysis. (n = 5 mice/group, statistical analysis by Mann-Whitney test comparing only Mrc1.GFP LV vs Mrc1.GFP.miRT LV and p-values adjusted for multiple testing with Bonferroni’s correction). In E, Mrc1.GFP LV and Mrc1.GFP.miRT LV were used at 3*10¹⁰ TU/kg. (F) Representative immunofluorescence (IF) images obtained by confocal microscopy (CM) and relative GFP quantification of livers bearing metastases from MC38 cells in the left panel, mCherry (red), GFP (green), F4/80 (grey) and nuclei (blue, left panel) or AKTPF cells in right panel, GFP (green), F4/80 (grey) and nuclei (blue, right panel); metastasis (Met), peri metastatic area (dotted line) and intact liver are indicated. In the left panel, MC38 cells were injected 10 days after LV delivery, LV at 3*10¹⁰ TU/kg; in the right panel, Mrc1.GFP.miRT LV was used at 5*10⁹ TU/kg in NSG mice (n=5 mice/group; statistical analysis by bootstrap t test). FIGURE 2 In vivo LV-engineered KCs enable rapid, sustained and well-tolerated IFNα production. (A) Schematics of a KC engineered with the IFNα LV (top) or the Control LV (bottom). (B) Plasma IFNα levels by ELISA analysis at the indicated time points upon LV injection (n = 10, 10, 5 mice/group in Control LV, IFNα LV or untransduced, UT, respectively). (C) LV copies per cell by ddPCR analysis (n = 8, 8, 5 mice/group in Control LV, IFNα LV or UT, respectively; statistical analysis by Kruskal-Wallis with Dunn’s tests, adjusted p-value by Bonferroni’s correction). (D) Blood cell counts of B cells (left panel), eosinophils (middle panel) and neutrophils (right panel) at the indicated time points upon LV injection (n values as in B; statistical analysis by Mann-Whitney test). (E) Serum levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) at 126 days upon LV injection (n = 9, 10, 5 mice/group in Control LV, IFNα LV or UT, respectively, statistical analysis by Kruskal-Wallis test). (F) Histopathologic analysis of the indicated organs at day 366 upon LV injection (n values as in C). FIGURE 3 Gene-based enforced IFNα expression by KCs unleashes T cell activation and impairs liver metastasis growth. (A) Schematics of the experiments in panel B-N. (B and H) Plasma IFNα levels by ELISA at the indicated time points upon tumor challenge. Lower dose or higher dose, 1.5*10⁹ or 1.5*10¹⁰ TU/kg, respectively (in B, left panel, n = 10,10,5; in B, right panel, n = 10,10,5; in H, n = 11,10,3 mice/group in Control LV, IFNα LV or UT, respectively). (C, E, I and N) Tumor growth by magnetic resonance imaging (MRI) (C, left panel, n = 9,8,9,8; C, right panel, n = 10,10,10,10; in E, n =10,10,10,9; in I, n = 9,10,9,10, and in N, n = 7,8 mice/group from left to right; statistical analysis by Mann-Whitney). (D and J) Representative MRI of a Control LV- (left panel) and a IFNα LV-treated (right panel) mice bearing, in D, MC38 liver metastases, 20 days after tumor transplant, or, in J, AKTPF liver metastasis, 28 days after tumor transplant, complete responder (CR), healthy liver (white) and metastasis (Met, yellow) are indicated with a dotted line in D or arrows in J. (F, G, K and L) Percentage of the indicated cell types infiltrating MC38.OVA, in F and G, or AKTPF, in K and L, liver metastases by FC analysis (n = 10,7 mice/group in Control LV or IFNα LV, respectively; statistical analysis by Mann-Whitney test). (M) Representative IF images obtained by CM and relative CD8 T cell quantification of AKTPF liver metastases, CD4 (green), CD8 (red), E-Cadherin (grey) and nuclei (blue, n as in L; statistical analysis by Mann-Whitney test). FIGURE 4 Engineering of KCs by IFNα LV enables preferential IFNα signaling in peri metastatic areas. (A) Side-by-side comparison of representative liver sections containing metastatic lesions (Met) from the indicated treatment cohort analyzed by spatial transcriptomics showing the H&E-stained (left) or analyzed by using spatial transcriptomics (right). Spatial spots are indicated in a color associated to a spatial compartment. (B) Heatmap displaying gene set enrichment analysis (GSEA) normalized enrichment score (NES) for selected gene ontology (GO) terms across distinct spatial compartments by spatial transcriptomics (Visium). Gene sets are grouped into cytokine related effects (red), immune activation state (blue), tumor associated (black) and hepatic functions (olive; n = 3,3,2 mice/group in partial responder, resistant or control respectively). (C) Fold change over average gene expression level for the indicated genes belonging to the indicated gene categories in the spatial compartments and treatment cohorts (n values as in B). FIGURE 5 IFNα promotes APC immune activation and enhanced MHCII-restricted antigen presentation in responder mice. (A) GSEA analysis of scRNA sequencing data showing NES for selected GO terms calculated based on genes differentially expressed in antigen presenting cells (APCs) in the indicated comparisons (n as in A; statistical analysis by an adaptive multi-level split Monte-Carlo scheme; *: padj <0.05; **: padj <0.01; ***: padj <0.001; ****: padj <0.0001). (B) UMAP representation of scRNA sequencing data of APCs for the indicated groups (n = 3,3,2 mice/group in partial responder, resistant or control respectively). (C) Expression of selected genes showing the average expression (color scale) and percentage of cells expressing the indicated gene (size of the shape) belonging to the indicated gene categories in IFNα TAMs and TAMs. (D) GSEA analysis showing NES of selected GO terms on genes differentially expressed in IFNα TAMs vs TAMs (n = 8 mice/group; statistical analysis as in A). (E) Percentage of cells within the indicated populations belonging to the APC compartment (n = 3,3,2 mice/group in partial responder, resistant or control respectively). (F) Expression profile of genes belonging to indicated categories showing the average expression (color scale) and percentage of cells expressing the indicated gene (size of the shape). (G) Combined gene expression score of genes belonging to the indicated categories in the different cell populations from the indicated cohorts. FIGURE 6 Therapeutic response to IFNα is associated with T cell activation and is counteracted by Eomes CD4 T cell infiltration. (A) GSEA analysis showing NES for selected GO terms on genes differentially expressed in the indicated comparisons (n = 3,3,2 mice/group in partial responder, resistant or control respectively; statistical analysis as in Figure 5A). (B) UMAP representation of cells from AKTPF liver metastasis annotated as T and NK cells (n values as in A). (C) Expression of selected genes showing the average expression (color scale) and percentage of cells expressing the indicated gene (size of the shape) belonging to the indicated gene categories in Eomes CD4 T cells and all other T and NK cells pooled. (D) Percentage of the indicated cell populations for the indicated groups (n as in A; statistical analysis as in Figure 5D). (E) Gene expression profile showing average expression (color scale) and percentage of cells expressing the indicated gene (size of the shape) for all CD8 T cell subtypes pooled together with genes belonging to exhaustion-associated gene signature and effector/memory like- associated genes highlighted in yellow or green, respectively. FIGURE 7 IFNα from engineered KCs in combination with functional inhibition of regulatory T cells eradicates liver metastases. (A) Stratification of patients into IFNα-signaling low and high cohort based on their IFNα signature score by bulk RNA sequencing of the tumor (n = 21). (B) Tr1 cell signature score detected in bulk RNA sequencing data from human patient CRC derived liver metastasis stratified by their intrinsic IFNα signaling score (n = 21 patients per group, statistical analysis by Mann-Whitney test). (C) IF images of CRC liver metastases from 2 patients (pt.#31, IFNα high signaling; pt.#16, IFNα low signaling;) showing CD4 (green), LAG3 (red) and nuclei (blue). (D) Percentage of EOMES CD4+ T cells infiltrating AKTPF liver metastases, treated as indicated, by FC analysis (n = 7,8,5,10 mice/group from left to right; statistical analysis by Mann-Whitney test, p-values adjusted by Bonferroni’s correction). (E and H) Tumor growth by MRI analysis (in E, n = 9,9,9,10; in H, n = 7,8,9,9 mice/group from left to right; in E statistical analysis by ANCOVA, in H Mann-Whitney test, p-values adjusted by Bonferroni’s correction). (F) Schematics of the experiments shown in G and H. (G) Tumor growth assessed by tumor weight (n = 13,8,10,9 mice/group, Control consists of 10 Control LV mice and 3 UT mice; statistical analysis by Mann-Whitney test, p-values adjusted by Bonferroni’s correction). FIGURE 8 Generation of a LV platform enabling in vivo liver macrophage engineering. (A) Schematics (generated with the USCS Genome Browser) showing the murine putative Mrc1 promoter. (B) Representative FACS plots showing GFP expression in bone marrow- derived macrophages (BMDM) transduced as indicated. (C) Percentage of GFP+ BMDMs by flow cytometry (FC) analysis (n = 3 cell cultures/group). (D) Mean fluorescence intensity of PDL1 (left panel) and MRC1 (right panel) in BMDM polarized as indicated, untreated (NT), analyzed by using FC (n = 3 cell cultures/group). (E) LV copies per cell calculated by using ddPCR analysis (n = 3 cell cultures/group). (F) Schematics of bidirectional LV design without (top) and with (bottom) miRT sites which were delivered systemically (i.v.) to mice. (G) LV copies per cell calculated by using ddPCR analysis (n = 8,8,8,3 mice/group from left to right, statistical analysis by Kruskal-Wallis test). (H) Average number of GFP+ hepatocytes per frame detected in 5-6 CM images of the liver (n = 8 mice/group, statistical analysis by Mann- Whitney test). (I) Representative immunofluorescence (IF) images of the liver obtained by confocal microscopy (CM), GFP (green), F4/80 (red), nuclei (blue). Magnification of the indicated area showing GFP and nuclei (top), and F4/80 and nuclei (bottom). (J and K) Percentage (in J) and MFI (in K) of GFP- and dlNGFR-positive cells out of liver KCs and LSECs analyzed by using FC (n = as in A; statistical analysis by Kruskal-Wallis with Dunn’s test, adjusted p-value by Bonferroni’s correction). (L) GFP expression in distinct organs upon PBS (UT) or Mrc1.GFP.miRT LV delivery to mice challenged with liver metastases. Single channels of the images shown in F. Livers bearing metastases from MC38 cells (top) or AKTPF cells (bottom). (M) Representative CM images of the indicated organs from PBS (UT, top) or Mrc1.GFP.miRT LV (bottom) treated mice, GFP (green), F4/80 (grey) nuclei (blue). FIGURE 9 In vivo LV-engineered KCs enable rapid, sustained and well-tolerated IFNα production. (A) Blood cell counts of inflammatory monocytes, resident monocytes, CD8 T cells, CD4 T cells, eosinophils, platelets and red blood cells as well as amount of hemoglobin at the indicated time points upon LV injection (n = 10,10,5 mice/group in Control LV, IFNα LV or UT mice, respectively, statistical analysis by Mann-Whitney test). (B) Heatmap showing the fold change over the row mean of autoreactive antibodies against the indicated targets detected in the serum of mice at day 52 (n = 3,4 mice/group in IFNα LV or Control LV, respectively) and day 366 (n = 8,8,5 mice/group in IFNα LV, Control LV or UT, respectively) after LV injection. Positive control: plasma from a lupus mouse (18-week-old female NZB/NZW mouse, statistical analysis by Mann-Whitney test, adjusted p-value by Bonferroni’s correction). (C) Histopathologic analysis of the indicated organs at day 366 upon LV injection scored as not present (non), minimal, mild, moderate, marked and severe (n = 8,8,5 mice/group in Control LV, IFNα LV or UT mice, respectively). FIGURE 10 Gene-based enforced IFNα expression by KCs unleashes T cell activation and impairs liver metastasis growth. (A) LV copies per cell in the liver analyzed by using ddPCR (left panel, n = 9,10,3; right panel, 8,7,5 mice/group in Control LV, IFNα LV or UT, respectively). (B) B cell counts in the blood (left panel, n = 10,9,5; right panel, n = 9,9,5 mice/group in Control LV, IFNα LV or UT, respectively; statistical analysis by Mann-Whitney test). (C) Tumor volume measured by caliper at the indicated time points (left panel, n = 1,4, right panel n = 1,5 mice/group in the IFNα LV complete responder or UT cohort, respectively). (D) Plasma IFNα levels measured by using ELISA (Day 5, n = 9,6; Day 11, n = 9,7 mice/group, left panel) and LV copies per cell by using ddPCR analysis in the liver (n = 10,10 mice/group, right panel). (E) Representative image of an H&E staining of a human liver section containing CRC metastases (left) and a murine liver section containing AKTPF metastasis (right); to note the neoplastic glands (NG), neoplastic endothelium (NE) and dirty central necrosis (CDN) highlighted with black arrows. (F) Representative CM images of a murine liver containing AKTPF metastases stained for CD4 (green), CD8 (red), CD11c (green), F4/80 (red), CD31 (green), α-SMA (red), E-cadherin (grey) and nuclei (blue) as indicated in the figure. (G) Tumor growth by using MRI analysis (n = 10,9 mice/group in Control LV or IFNα LV mice, respectively; statistics by Mann-Whitney test), (H) Plasma IFNα levels by using ELISA (n =10,9,5 mice/group in Control LV, IFNα LV or UT, respectively, left) and LV copies per cell by ddPCR analysis in the liver (n =10,10,5 mice/group in Control LV, IFNα LV or UT, respectively, right). (I) LV copies per cell by using ddPCR analysis in the liver (n =11, 6 mice/group in Control LV or IFNα LV). (J) Plasma IFNα levels by using ELISA (Control LV, n = 7; IFNα LV, n = 8 mice/group). FIGURE 11 Engineering of KCs by IFNα LV enables preferential IFNα signaling in peri metastatic areas. (A) Gene expression analysis of tumors by using ddPCR analysis (n = 10, 7 mice/group in Control LV or IFNα LV respectively). (B) Schematics indicating the tumor volume at day 28 for 3 distinct cohorts: control (red), partial responder (blue) or resistant (green). (C) UMAP representation based on spatial transcriptomic spots of AKTPF liver metastasis (left) and a representative H&E image overlayed with the transcriptomic spots highlighted by the color according to UMAP clustering (right). (D) Gene expression of selected genes associated with CRC (cancer cell gene signature) or liver functions (liver gene signature). The average gene expression is shown by a color scale and the percentage of cells expressing the indicated gene is represented by the size of the shape (n = 8). (E) Lower right: schematics showing the spatial compartments A to H. The approximated distances to the tumor-liver boundary are indicated in mm as well as the color associated with the individual spatial compartment. Upper right and left: sections analyzed by using spatial transcriptomics excluding the sections shown in Figure 4A. FIGURE 12 IFNα promotes APC immune activation and enhanced MHCII-restricted antigen presentation in responder mice. (A) UMAP representation, as shown in Figure 4B, showing cells from AKTPF liver metastasis split into the different treatment cohorts (n as in A). (B) Fraction of cells belonging to the identified cell populations for each sample (n =3,3,2 mice/group in control, partial responder or resistant, respectively). (C) UMAP representation showing all cells from all groups (n as in A), displaying the expression level of the indicated gene on a scale from gray (low expression) to blue (high expression). (D) Heatmap showing the log fold change in expression of the top 20 upregulated genes in each cluster (n = 8). (E) Gene expression of selected genes associated with indicated features in the different clusters of the APC compartment showing the average expression (color scale) and percentage of cells expressing the indicated gene (size of the shape; n = 8). FIGURE 13 Therapeutic response to IFNα is associated with T cell activation and is counteracted by Eomes CD4 T cell infiltration. (A) Heatmap showing the log fold change in expression of the top 20 upregulated genes in each cluster identified in the T and NK cell compartment (n = 8). (B) Gene expression of selected genes associated with indicated features in the different clusters (representation as in Figure 12E; n = 8). (C) GSEA analysis, showing NES for selected GO terms, on genes differentially expressed in CD8 T cells in the indicated comparisons (n as in Figure 4A; statistical analysis as in Figure 5A). FIGURE 14 IFNα from engineered KCs in combination with functional inhibition of regulatory T cells eradicates liver metastases. (A) Correlation of the IFNα signature score and the Tr1 signature score (n = 42; statistical analysis by Spearman’s correlation coefficient). (B) IF image of a section of human liver containing metastases, stained for CD4 (green), LAG3 (red) and nuclei (blue). Liver, metastasis (Met), neoplastic glands (NG), neoplastic endothelium (NE) and central dirty necrosis (CDN) are indicated. CD4+ LAG3+ cells are indicated with arrows. (C) Plasma IFNα levels by using ELISA (left panel, n = 10,9,9,9 mice/group from left to right) and LV copies per cell by using ddPCR in the liver (right panel, n = 9,8,9,10 mice/group from left to right). (D) MFI of PD1 expression on CD8 (left panel) or CD4 (right panel) T cells in blood circulation analyzed by using FC (n = 10,9,10,10 mice/group from left to right; statistical analysis by Mann-Whitney test, adjusted p-values by Bonferroni’s correction). (E and F) Plasma IFNα levels by using ELISA (in E, left panel, n = 10,8,10,9,3; in F, left panel, n = 10,9,9,9 mice/group from left to right) and LV copies per cell in the liver analyzed by using ddPCR (in E, right panel, n = 10,8,10,9,3; in F, right panel, n = 6,7,9,9 mice/group from left to right). FIGURE 15 Combination of IL12 and IFNα promotes CD8 T cell activation and liver metastasis clearance. (a) Schematics of the experiment. (b) Tumor weight at day 22 from tumor cell inoculation in the indicated groups. (c) Digital droplet PCR analysis showing OVA expression in whole tumor lysates in the indicated groups. (d) Blood analysis by flow cytometry at day 15 showing the percentage of tetramer (anti-OVA) CD8 T cells out of total number of circulating CD8 T cells. (e and f) Percentage of CD8 T cells out of total CD45 cells, in e; and percentage of progenitor exhausted (PEX) tetramer CD8 T cells out of all tetramer CD8 T cells, in f, infiltrating MC38.OVA liver metastases. PEX were identified as indicated in the methods section. (g to i) Flow cytometry analysis of the liver showing terminally exhausted (TEX) CD8 T cells out of total tetramer CD8 T cells, in g; PEX CD8 T cells out of total tetramer CD8 T cells, in h; and median fluorescence intensity of PD1 marking in tetramer CD8 T cells, in i. TEX were identified as indicated in the methods section. FIGURE 16 Combination of IL12, IFNα and anti-PD1 monoclonal antibody promotes CD8 T cell activation and liver metastasis clearance. (a) Schematics of the experiment. (b) Tumor weight at day 22 from tumor cell inoculation in the indicated groups. (c) Digital droplet PCR analysis showing OVA expression in whole tumor lysates in the indicated groups. (d) Table showing treatments groups displaying no tumor (complete responders) or OVA expression (clearing all OVA expressing cells from tumors). (e) Blood analyses by flow cytometry at day 14 showing the percentage of tetramer (anti-OVA) CD8 T cells out of total number of circulating CD8 T cells. (f and g) Percentage of CD8 T cells out of total CD45 cells, in f; and percentage of PEX tetramer CD8 T cells out of all tetramer CD8 T cells, in g, infiltrating MC38.OVA liver metastases. (h to j) Flow cytometry analysis of the liver showing terminally exhausted (TEX) CD8 T cells out of total tetramer CD8 T cells, in h; PEX CD8 T cells out of total tetramer CD8 T cells, in i; and median fluorescence intensity (MFI) of PD1 marking in tetramer CD8 T cells, in j. FIGURE 17 Expression from liver macrophages of IL12 and IFNα in combination with a melanoma- associated antigen promotes melanoma liver metastasis clearance. (a) Schematics of the experiment. (b and c) Magnetic resonance imaging (MRI) analysis at days 13 and 19 after melanoma inoculation in the indicated groups. (d) Representative photographs of livers of mice from the indicated treatment groups. (e and f) Percentage of CD4, in e, and CD8, in f, T cells out of total CD45 cells infiltrating B16 liver metastases. (f) Flow cytometry analysis of the liver showing percentage of CD4 T cells expressing PD1 out of total CD4 T cells. FIGURE 18 a,b, UMAP projection of single cell RNA sequencing (scRNA-seq) of the whole dataset for the indicated tissues. c,d, UMAP projection of scRNA-seq of APCs subcluster for the indicated tissues. e,f, Expression of selected genes belonging to the indicated categories and tissues in APCs (n = 2, 3, 3, 3 mice/group for liOVA, OVA.Ifna, OVA.Il12 and OVA.Combo; Statistical analysis by Wilcoxon test with Bonferroni correction, compared with the liOVA; *: padj <0.05; **: padj <0.005; ***: padj <0.0005). g, Combined gene expression score for genes belonging to the indicated categories in the different cell populations from the indicated tissue and cohort. FIGURE 19 a,b, UMAP projection of scRNA-seq of the whole dataset for the indicated group and tissue. c,d, GSEA of scRNA-seq data showing NES for selected GO terms calculated based on genes differentially expressed in KCs, macrophages and monocytes in the indicated comparisons (n = 2, 3, 3, 3 mice/group for liOVA, OVA.Ifna OVA.Il12 and OVA.Combo; statistical analysis by an adaptive multi-level split Monte-Carlo scheme; *: padj <0.05; **: padj <0.005; ***: padj <0.0005). FIGURE 20 a, UMAP projection of scRNA-seq of liver T and NK cells subclustered. b, Gene set enrichment analysis (GSEA) of scRNA-seq data showing normalized enriched score (NES) for selected gene ontology (GO) terms calculated based on genes differentially expressed in T and NK cells in the indicated comparisons (n = 2, 3, 3, 3 mice/group for liOVA, OVA.Ifna, OVA.Il12 and OVA.Combo; statistical analysis by an adaptive multi-level split Monte-Carlo scheme; *: padj <0.05; **: padj <0.005; ***: padj <0.0005). c, Combined gene expression score for genes belonging to the indicated categories in the different cell populations from the indicated tissue and cohort. d, Expression of selected genes belonging to the indicated categories in liver CD8⁺ T cells (number of mice as in b, statistical analysis by Wilcoxon test with Bonferroni correction, compared with the liOVA; *: padj <0.05; **: padj <0.005; ***: padj <0.0005). e, UMAP projection of liver scRNA-seq indicating cells bearing OVA specific TCRs. f, GSEA of scRNA-seq data showing NES for selected GO terms calculated based on genes differentially expressed in OVA specific CD8⁺ T cells in the indicated comparisons (mice number and statistic as in b). g, Combined gene expression score of genes belonging to the indicated categories in the different cell populations from the indicated tissue and cohort. h, Clonotype sharing between liver and tumor CD4⁺ T cells, grouped by TCR clonotype. i, UMAP projection of liver scRNA sequencing indicating CD4⁺ T cells clonotypes shared between liver and tumor tissue. j, Percentage of cells within the CD4⁺ T cells populations in the liver. k, Number of CD4+ T cells divided by TCR clonotype frequency. FIGURE 21 a, Clonotype sharing between liver and tumor of OVA specific and bystander CD8⁺ T cells, grouped by TCR clonotype. b, Expression of selected genes belonging to the indicated categories in liver OVA specific CD8⁺ T cells (n = 2, 3, 3, 3 mice/group for liOVA, OVA.Ifna, OVA.Il12 and OVA.Combo; statistical analysis by Wilcoxon test with Bonferroni correction, compared with the liOVA. *: padj <0.05; **: padj <0.005; ***: padj <0.0005). c, Expression of selected genes belonging to the indicated categories in liver shared vs non shared CD4⁺ T cells in OVA.Combo or OVA.Il12 treated animals (number of mice and statistical analysis). FIGURE 22 a, Schematics of the antigen prediction pipeline exploited for the identification of neoantigens in the AKTPF LM model. b, Schematic of the TA33 LV. c, Schematic of the experiment shown in panels d-h. d-h, Treatment of mice bearing established AKTPF LM with TA33 of TA33.Combo after 7 days post tumor challenge (TA331*10⁷ TU/mouse, TA33.Combo total dose 1.2*10⁸ TU/mouse). In d, quantification of LM volume by MRI at day 27 post tumor injection (n= 6, 6, 9 mice/group, for Control untreated, TA33 or TA33.Combo treated mice; horizontal line represents median, statistical analysis by Kruskal-Wallis with Dunn’s tests). In e,f, FC analysis of the liver (n= 6, 6, 8 mice/group, for Control untreated, TA33 or TA33.Combo treated mice; statistics as in I). In g and h, IFNg ELISPOT assay performed on CD8⁺ T cells isolated from the spleen of indicated mice (n= 3,3 mice/group). FIGURE 23 a, Plasma levels of IFN ^ and IL-12 measured by ELISA at 7 days post treatment (n= 6, 6, 9 mice/group, for Control untreated, TA33 or TA33.Combo treated mice; horizontal line represents median). b, FC analysis of the blood, performed at day 14 post tumor injection (number of mice as in a, horizontal line represents median, statistical analysis by Kruskal- Wallis with Dunn’s tests). c, Correlation between circulating Ly6c+ CD44+ CD8 T cells and tumor volume measured by MRI at day 27 (n of mice as in a, statistical analysis by Spearman correlation) FIGURE 24 a-g, Treatment of mice bearing established B16-F10 LM at day 5 post tumor challenge with Trp2.Combo (Control mice were left untreated, OVA.Combo total dose of 1.2*10⁸ TU/mouse). After 3- and 10-days post LV injection, mice were injected with 0.2mg of a-PD1 or left untreated. In b, quantification of LM volume MRI at the indicated time points (n= 5, 8, 6, 7 mice/group, for Control untreated, Control + a-PD1, Trp2.Combo or Trp2.Combo + a-PD1 treated mice; horizontal line represents median, statistical analysis by Kruskal-Wallis with Dunn’s tests). In c, representative MRI images of liver from Control untreated or Trp2.Combo+ a-PD1 treated mice. In d-g, FC analysis of the indicated tissues (n= 3, 5, 6, 7 mice/group, for Control untreated, Control + a-PD1, Trp2.Combo or Trp2.Combo + a-PD1 treated mice; horizontal line represents median, statistical analysis by Kruskal-Wallis with Dunn’s tests) FIGURE 25 a, FC analysis of the blood (n= 5, 8, 6, 7 mice/group, for Control untreated, Control + a-PD1, Trp2.Combo or Trp2.Combo + a-PD1 treated mice; horizontal line represents median, statistical analysis by Kruskal-Wallis with Dunn’s tests). b, Correlation between circulating Ly6c+ CD44+ CD8⁺ T cells and tumor volume measured by MRI at day 13 (n of mice as in c, statistical analysis by Spearman correlation) DETAILED DESCRIPTION OF THE INVENTION The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including” or “includes”; or “containing” or “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or steps. The terms “comprising”, “comprises” and “comprised of” also include the term “consisting of”. PHAGOCYTES The present invention relates to phagocyte-specific transgene expression, particularly liver and/or splenic phagocyte-specific transgene expression. As used herein, a “phagocyte” is a specialised cell which is capable of phagocytosis. Phagocytosis may consist in recognition and ingestion of particles larger than 0.5 μm into a plasma membrane derived vesicle, known as phagosome. Phagocytes can ingest microbial pathogens and apoptotic cells. Thus, phagocytosis is essential not only for microbial elimination, but also for tissue homeostasis (Rosales, C. and Uribe-Querol, E., 2017. BioMed research international, 2017). Suitably, the phagocytes targeted in the present invention are liver and/or splenic phagocytes. As used herein, “liver phagocytes” may be phagocytes which are predominantly present in liver tissue and “splenic phagocytes” may be phagocytes which are predominantly present in spleen tissue. Suitably, the phagocytes may be monocytes, macrophages, neutrophils, dendritic cells, eosinophils, fibroblasts, epithelial cells and/or endothelial cells. Suitably, the phagocytes may be macrophages, dendritic cells and/or liver sinusoidal endothelial cells. For example, the phagocytes may be liver and/or splenic macrophages, liver and/or splenic dendritic cells, and/or liver sinusoidal endothelial cells. Suitably, the phagocytes may be professional phagocytes (e.g. liver and/or splenic professional phagocytes), such as monocytes, macrophages, neutrophils, dendritic cells and eosinophils. In some embodiments, the phagocytes are macrophages and/or dendritic cells. Suitably, the phagocytes may be non-professional phagocytes, such as fibroblasts, epithelial cells and/or endothelial cells. In some embodiments, the phagocytes are endothelial cells. “Professional phagocytes” include monocytes, macrophages, neutrophils, dendritic cells, osteoclasts and eosinophils. These cells are in charge of eliminating microorganisms and of presenting them to cells of the adaptive immune system. In addition, fibroblasts, epithelial cells and endothelial cells can also perform phagocytosis. These “non-professional” phagocytes cannot ingest microorganisms but are important in eliminating apoptotic bodies (Rosales, C. and Uribe-Querol, E., 2017. BioMed research international, 2017). Macrophages In some embodiments, the phagocytes are macrophages (e.g. liver and/or splenic macrophages). Macrophages are innate immune cells that clear tissue from pathogens or other biological material. In adult mammals, macrophages are found in all tissues where they display great anatomical and functional diversity. In tissues, they are organized in defined patterns with each cell occupying its own territory. Macrophages have roles in almost every aspect of an organism’s biology ranging from development, homeostasis, to repair through to immune responses to pathogens. In particular, tumours are abundantly populated by macrophages and they play an important role in tumour initiation, progression, and metastasis. (Ta, W., Chawla, A. and Pollard, J.W., 2013. Nature, 496, pp.445-455). Liver macrophages may include liver-resident macrophages, infiltrating macrophages (e.g. bone marrow (BM)-derived macrophages), avascular peritoneal macrophages, and splenic- derived monocytes. Splenic macrophages may include marginal zone macrophages (MZMΦs), marginal metallophilic macrophages (MMMΦs), and red pulp macrophages (RpMΦs). In some embodiments, the phagocytes are M2-like macrophages and/or MRC1+ macrophages (e.g. liver and/or splenic M2-like and/or MRC1+ macrophages). According to the activation state and functions of macrophages, they can be divided into M1- like (classically activated macrophage) and M2-like (alternatively activated macrophage). The M1 activation is induced by intracellular pathogens, bacterial cell wall components, lipoproteins, and cytokines such as interferon gamma and tumour necrosis factor alpha. M1- like macrophages are characterized with inflammatory cytokine secretion and production of nitric oxide (NO), resulting in an effective pathogen killing mechanism. M2 activation is induced by fungal cells, parasites, immune complexes, complements, apoptotic cells, macrophage colony stimulating factor, IL-4, IL-13, IL-10, tumour growth factor beta. M2-like macrophages have high phagocytosis capacity, producing extracellular matrix (ECM) components, angiogenic and chemotactic factors, and IL-10. In addition to the pathogen defence, M2-like macrophages clear apoptotic cells, can mitigate inflammatory response, and promote wound healing. M2-like macrophages are commonly known as anti- inflammatory, pro-resolving, wound healing, tissue repair, and trophic or regulatory macrophages (Rőszer, T., 2015. Mediators of inflammation, 2015). M2-like macrophages may be identified based on the gene transcription or protein expression of a set of M2 markers as described in Rőszer, T., 2015. Mediators of inflammation, 2015. These markers include transmembrane glycoproteins, scavenger receptors, enzymes, growth factors, hormones, cytokines, and cytokine receptors. Suitably, M2-like macrophages express one or more M2 macrophage markers such as MRC1 (CD206), CD163, CD209, Arginase-1, Chi3l3, FIZZ1, MGL-1, and Dectin-1. In some embodiments, the phagocytes are MRC1+ macrophages. Mannose receptor C-type 1 (MRC1) is also known as CD206, CLEC13D, and CLEC13DL. MRC1 is a C-type lectin primarily present on the surface of macrophages, immature dendritic cells and liver sinusoidal endothelial cells and mediates the endocytosis of glycoproteins. An example human MRC1 sequence is described under accession number UniProtKB P22897. An example mouse MRC1 sequence is described under accession number UniProtKB Q61830. In mouse and humans, M2-like polarized macrophages, including tumour-associated macrophages (TAMs), or some resident macrophage populations such as Kupffer cells (KCs), some splenic macrophages, and adipose tissue macrophages express high levels of MRC1. MRC1 is also expressed by some dendritic cell (DC) populations and liver sinusoidal endothelial cells (LSECs) (Pandey, E., A.S. Nour, and E.N. Harris, Front Physiol, 2020.11: p. 873). In some embodiments, the phagocytes are resident macrophages (e.g. liver-resident macrophages or splenic-resident macrophages). The majority of tissues in the body contain tissue-resident macrophage populations. Tissue- resident macrophages are known for their role as immune sentinels in the frontline of tissue defence where they are discretely positioned and transcriptionally programmed for the encounter with pathogens or environmental challenges (Davies, L.C., et al., 2013. Nature immunology, 14(10), p.986). Liver-resident macrophages (also called “liver macrophages”) include Kupffer cells and motile liver macrophages. Kupffer cells are maintained in the adult independently of the bone marrow and function to clear microorganisms and cell debris from the blood, and clear aged erythrocytes. Kupffer cell phenotypic markers may include F4/80^hi, CD11b^lo, CD169⁺, CD68⁺, Galectin-3⁺, and CD80^lo/−. Motile liver macrophages have an immune surveillance function and phenotypic markers may include F4/80⁺, CD11b⁺, and CD80^hi ((Davies, L.C., et al., 2013. Nature immunology, 14(10), p.986). Splenic-resident macrophages include marginal zone macrophages (MZMΦs), marginal metallophilic macrophages (MMMΦs), and red pulp macrophages (RpMΦs). Microanatomically, the spleen is divided into the white pulp and the red pulp (Rp), separated by the marginal zone (MZ). RpMΦs form a vast network inside the Rp and are characterized in mice by expression of F4/80^highCD68⁺CD11b^low/− and intense autofluorescence. Inside the MZ, two populations of macrophages can be discerned. The MZMΦs typically express in their surface the C-type lectin SIGN-related 1 (SIGNR1) and a type I scavenger receptor called Macrophage Receptor with Collagenous structure (MARCO). MMMΦs are defined, among other molecules, by the expression of Sialic acid-binding Ig-like Lectin-1 (Siglec-1, Sialoadhesin, CD169) and MOMA-1. In some embodiments, the phagocytes are infiltrating macrophages (e.g. liver-infiltrating macrophages or splenic-infiltrating macrophages), e.g. bone marrow (BM)-derived macrophages. In some embodiments, the phagocytes are avascular peritoneal macrophages (PMs). PMs reside in the peritoneal cavity with self-renewal abilities and exist as two distinct PM subsets i.e., large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs). LPMs originate from embryonic precursors and represent the most abundant subset under steady conditions that display F4/80^high CD11b^high MHCII^low phenotype. While SPMs are the minor subset with F4/80^low CD11b^low MHCII^high phenotype and originate from BM-derived myeloid precursors and predominantly appear during infection. In some embodiments, phagocytes are monocyte-derived macrophages (e.g. liver and/or splenic monocyte-derived macrophages). Monocytes circulate in the blood and are recruited to mucosal tissues or inflammation sites, where they can differentiate into monocyte-derived macrophages or monocyte-derived dendritic cells. MerTK, CD68, CD163, and the transcription factor MAFB are considered robust markers of macrophages, while dendritic cells express CD1a, CD1b, FcεRI, and CD226. Macrophages are large cells containing many phagocytic vesicles. By contrast, dendritic cells are smaller and display dendrites on their surface (Segura, E. and Coillard, A., 2019. Frontiers in immunology, 10, p.1907). In some embodiments, the phagocytes are tumour-associated macrophages (e.g. liver and/or splenic tumour-associated macrophages). Tumour-associated macrophages (TAMs) are a class of macrophage present in high numbers in the microenvironment of solid tumours. Tumour-associated macrophages (TAMs) contribute to tumour progression at different levels: by promoting genetic instability, nurturing cancer stem cells, supporting metastasis, and taming protective adaptive immunity. TAMs can have a dual supportive and inhibitory influence on cancer, depending on the disease stage, the tissue involved, and the host microbiota (Mantovani, A., et al., 2017. Nature reviews Clinical oncology, 14(7), p.399). In some embodiments, the phagocytes are MRC1+ liver macrophages (e.g. Kupffer cells) and/or MRC1+ splenic macrophages. In some embodiments, the phagocytes are Kupffer cells. Dendritic cells In some embodiments, the phagocytes are dendritic cells (e.g. liver and/or splenic dendritic cells). Dendritic cells (DCs) are antigen-presenting cells of the mammalian immune system. Their main function is to process antigen material and present it on the cell surface to the T cells of the immune system. In normal liver, DCs typically reside only around portal triads and, like DC in other peripheral sites, are able to efficiently capture, process, and transport antigens to regional lymphoid tissues. Compared to LSECs and KCs, freshly isolated hepatic DC are predominantly immature cells, expressing surface MHC but few costimulatory molecules necessary for T cell activation (Lau, A.H. and Thomson, A.W., 2003. Gut, 52(2), pp.307-314). Both conventional/myeloid DCs (cDC) and plasmacytoid DCs (pDC) at different maturation stages and different subsets are present in human spleen (Velásquez-Lopera, M.M., et al., 2008. Clinical & Experimental Immunology, 154(1), pp.107-114). Endothelial cells In some embodiments, the phagocytes are endothelial cells (e.g. liver and/or splenic endothelial cells). For example, the phagocytes may be liver sinusoidal endothelial cells (LSECs). LSECs have one of the highest endocytic capacities in the human body and can clear soluble macromolecules and small particles through endocytic receptors. Features used to identify LSECs include: (a) their high and rapid endocytic capacity, (b) fenestrae without diaphragm and organized in sieve plate, and (c) surface markers such as VEGFR3⁺ CD34⁻ VEGFR2⁺ VE-Cadherin⁺ FactorVIII⁺ CD45⁻ or CD31⁺, LYVE-1⁺, L-SIGN⁺, Stabilin-1⁺, CD34⁻, PROX-1⁻ (Poisson, J., et al., 2017. Journal of hepatology, 66(1), pp.212-227). In some embodiments, the phagocytes are LSECs. In another aspect, the invention provides a vector for LSEC-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence VECTOR In one aspect, the present invention provides a product comprising a vector for phagocyte- specific expression, particularly liver and/or splenic phagocyte-specific expression. Phagocyte-specific expression The vector may be a phagocyte-specific expression vector, particularly a liver and/or splenic phagocyte-specific expression vector. The terms “phagocyte-specific expression”, “liver phagocyte-specific expression” and “splenic phagocyte-specific expression”, as used herein, may refer to the preferential or predominant expression of a transgene (e.g. as polypeptide or RNA) in the phagocytes as compared to other cells (e.g. blood, lung and bone marrow cells). In some embodiments, at least 50% of transgene expression occurs in the phagocytes. In some embodiments, at least 60%, 70%, 80%, 90% or 95% of transgene expression occurs in the phagocytes. In some embodiments, the transgene is substantially exclusively expressed in the phagocytes. For example: (i) expression of the transgene in phagocytes transduced by the vector may be greater than expression of the transgene in other cells transduced by the vector; and/or (ii) the transgene may be substantially not expressed in cells other than the phagocytes, when transduced by the vector; and/or (iii) the transgene may be substantially not expressed in lung cells, bone marrow cells and/or blood cells, when transduced by the vector; and/or (iv) the transgene may be substantially only expressed in some liver cells and/or some splenic cells; and/or (v) expression of the transgene in Kupffer cells may be at least ten times greater than expression in hepatocytes, when transduced by the vector; and/or (vi) the transgene may be substantially not expressed in heptocytes when transduced by the vector. Expression of the transgene may be determined by any suitable method known to the skilled person. For example, if the transgene is a reporter gene (e.g. GFP) flow cytometry analysis may be used to determine expression levels in different cell types. Alternatively, if the transgene is a reporter gene (e.g. GFP) immunofluorescent analysis (e.g. by confocal imaging analysis) may be used to determine expression levels in different cell types. Suitably, expression of the transgene in phagocytes transduced by the vector may be greater than expression of the transgene in other cells transduced by the vector. For example, expression of the transgene in phagocytes transduced by the vector may be at least 10 times, at least 20 times, or at least 50 times, or at least 100 times greater than in other cells transduced by the vector. Suitably, the transgene is substantially not expressed in cells other than the phagocytes, when transduced by the vector. For example, the percentage of the cells other than the phagocytes which express the transgene may be 5% or less, 2% or less, 1% or less, or 0%. For example, expression of the transgene in cells other than the phagocytes may be undetectable. Suitably, the transgene is substantially not expressed in lung cells, bone marrow cells and/or blood cells, when transduced by the vector. For example, the percentage of lung cells, bone marrow cells and/or blood cells which express the transgene may be 5% or less, 2% or less, 1% or less, or 0%. For example, expression of the transgene in lung cells, bone marrow cells and/or blood cells may be undetectable. Suitably, the transgene is substantially only expressed in liver cells and/or splenic cells. For example, the percentage of the cell types other than liver cells and/or splenic cells which express the transgene may be 5% or less, 2% or less, 1% or less, or 0%. For example, expression of the transgene in cell types other than liver cells and/or splenic cells may be undetectable. Suitably, expression of the transgene in Kupffer cells may be at least ten times greater than expression in hepatocytes, when transduced by the vector. For example, expression of the transgene in Kupffer cells may be at least ten times greater, at least twenty times greater, or at least fifty times greater than expression in hepatocytes. Suitably, the transgene may be substantially not expressed in hepatocytes when transduced by the vector. For example, the percentage of hepatocytes which express the transgene may be 5% or less, 2% or less, 1% or less, or 0%. For example, expression of the transgene in hepatocytes may be undetectable. Suitably, expression of the transgene in Kupffer cells may be at least ten times greater than expression in LSECs, when transduced by the vector. For example, expression of the transgene in Kupffer cells may be at least ten times greater, at least twenty times greater, or at least fifty times greater than expression in LSECs. Suitably, the transgene may be substantially not expressed in LSECs when transduced by the vector. For example, the percentage of LSECs which express the transgene may be 5% or less, 2% or less, 1% or less, or 0%. For example, expression of the transgene in LSECs may be undetectable. If the vector is an integrating vector (e.g. integrase proficient) then copies of the vector may be, for example, specifically integrated into phagocytes, particularly liver and/or splenic phagocytes. For example: (i) integration of the vector in liver and spleen may be greater than integration of the vector in other organs (e.g. lymph node, brain, small intestine, blood, bone marrow); and/or (ii) integration of the vector may substantially occur in liver, spleen, optionally blood and optionally bone marrow; and/or (iii) integration of the vector may substantially not occur in lymph node, brain, small intestine. Integration of the vector may be determined by any suitable method known to the skilled person. For example, viral copy number analysis, e.g. by quantitative digital droplet PCR of different organs. Suitably, integration of the vector in liver and spleen is greater than integration of the vector in other organs, such as lymph node, brain, small intestine, blood, bone marrow. For example, the viral copy number of liver and spleen may be at least 10 times, at least 20 times, or at least 50 times, or at least 100 times greater than in other organs. Suitably, integration of the vector substantially occurs in liver, spleen, optionally blood and optionally bone marrow. For example, integration of the vector in the liver and spleen, optionally blood and optionally bone marrow, may be at least detectable. Suitably, integration of the vector substantially does not occur in lymph node, brain, small intestine. For example, integration of the vector in in lymph node, brain, small intestine may be undetectable. All these biological compartments host resident macrophage populations that could potentially express the transgene upon systemic delivery of the vector. Viral vector Suitably, the vector of the present 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, K., 2018. Diseases, 6(2), p.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. The vector of the present invention may be in the form of a viral vector particle. Suitably, the viral vector of the present invention is in the form of a lentiviral vector particle. 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. Methods of preparing and modifying viral vectors and viral vector particles, such as lentiviral vectors, are well known in the art. Suitable methods are described in Merten, O.W., et al., 2016. Molecular Therapy-Methods & Clinical Development, 3, p.16017; Nadeau, I. and Kamen, A., 2003. Biotechnology advances, 20(7-8), pp.475-489; Ayuso, E., et al., 2010. Current gene therapy, 10(6), pp.423-436; and Goins, W.F., et al., 2008. Methods Mol Biol. 433, pp.97-113. Retroviral and lentiviral vectors The vector of the present invention may be a retroviral vector or a lentiviral vector. The vector of the present invention may be a retroviral vector particle or a lentiviral vector particle. 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). 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. 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. 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. 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. In a defective retroviral vector genome gag, pol and env may be absent or not functional. In a typical retroviral vector, at least part of one or more protein coding regions essential for replication may be removed from the virus. This makes the viral vector replication-defective. Portions of the viral genome may also be replaced by a library encoding candidate modulating moieties operably linked to a regulatory control region and a reporter moiety in the vector genome in order to generate a vector comprising candidate modulating moieties which is capable of transducing a target host cell and/or integrating its genome into a host genome. 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). 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. 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. 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. As examples of lentivirus-based vectors, HIV-1- and HIV-2-based vectors are described below. 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. 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. Optionally, the viral vector used in the present invention has a minimal viral genome. By “minimal viral genome” it is to be understood that the viral vector has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in WO 1998/017815. 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. 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). 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. The vectors may be integrase-defective (i.e. integrase-deficient). 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. In some embodiments, the vector is an integrase-defective lentiviral vector. In some embodiments, the vector is an integrase-proficient lentiviral vector. Vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral vectors (LV) upon systemic delivery may efficiently and specifically target the liver and are preferentially internalized by liver and splenic phagocyte populations, although other cell types including endothelial cells and, hepatocytes, are also transduced (Milani, M., et al., Sci Transl Med, 2019.11(493)). Thus, VSV-G-pseudotyped LVs constitute excellent tools to deliver genes of interest to the liver cell populations. Suitably, the vector is VSV-G-pseudotyped. In some embodiments, the vector is a VSV-G- pseudotyped lentiviral vector particle. Gene transfer into professional phagocytes and antigen presenting cells (APCs) is constrained by the presence of the CD47 molecules on LV particles. CD47-free LV show preserved infectivity and substantially increased susceptibility to phagocytosis. CD47-free LV more efficiently transduce professional phagocytes both ex vivo and in vivo, and induce a substantially higher rise in cytokine response upon systemic administration to mice, compared to CD47-bearing LV. CD47-free LV allow increased gene transfer efficiency into human primary monocytes, and have increased susceptibility to phagocytosis both ex vivo by primary human macrophages and in vivo when administered systemically to mice, compared to previously available LV. For example, VSV-G-pseudotyped LVs lacking CD47 molecules on their surface are even more efficiently uptaken by professional phagocytes of liver and spleen than CD47-bearing VSV-G-pseudotyped LVs. An allogeneic human leukocyte antigen (HLA) e.g. MHC-I may also be recognised by the immune system. For example, antibodies may bind HLA epitopes directly. As a result, cells and enveloped viruses that comprise HLA proteins originating from an allogeneic source may be targeted and neutralised by the immune system. A decreased number or lack of surface- exposed HLA molecules is advantageous in viruses for use as vaccines, as the viruses will be less likely to be neutralised by antibodies binding to HLA. Suitable methods of producing CD47-free and/or HLA-free vectors are described in WO 2019/219836. In some embodiments, the vector is substantially devoid of surface-exposed CD47 and/or HLA molecules. In some embodiments the vector is a VSV-G-pseudotyped lentiviral vector particle substantially devoid of surface-exposed CD47 and/or HLA molecules. The term “substantially devoid” as used herein means that there is a substantial decrease in the number of molecules that are expressed on the surface, in comparison to the number of molecules that are expressed on the surface of a vector produced in cells which have not been genetically engineered to reduce expression of the molecule (but under otherwise substantially identical conditions), such that the vectors 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. In some embodiments, the vector does not comprise any surface-exposed CD47 molecules and/or HLA molecules. In some embodiments, the vector is a VSV-G-pseudotyped lentiviral vector particle which does not comprise any surface-exposed CD47 molecules and/or HLA molecules. Adenoviral vector The vector of the present invention may be an adenoviral vector. The vector of the present invention may be an adenoviral vector particle. The adenovirus is a double-stranded, linear DNA virus that does not go through an RNA intermediate. There are over 50 different human serotypes of adenovirus divided into 6 subgroups based on the genetic sequence homology. The natural targets of adenovirus are the respiratory and gastrointestinal epithelia, generally giving rise to only mild symptoms. Serotypes 2 and 5 (with 95% sequence homology) are most commonly used in adenoviral vector systems and are normally associated with upper respiratory tract infections in the young. Adenoviruses have been used as vectors for gene therapy and for expression of heterologous genes. The large (36 kb) genome can accommodate up to 8 kb of foreign insert DNA and is able to replicate efficiently in complementing cell lines to produce very high titres of up to 10¹². Adenovirus is thus one of the best systems to study the expression of genes in primary non- replicative cells. The expression of viral or foreign genes from the adenovirus genome does not require a replicating cell. Adenoviral vectors enter cells by receptor mediated endocytosis. Once inside the cell, adenovirus vectors rarely integrate into the host chromosome. Instead, they function episomally (independently from the host genome) as a linear genome in the host nucleus. Hence the use of recombinant adenovirus alleviates the problems associated with random integration into the host genome. Adeno-associated viral vector The vector of the present invention may be an adeno-associated viral (AAV) vector. The vector of the present invention may be in the form of an AAV vector particle. The AAV vector or AAV vector particle may comprise an AAV genome or a fragment or derivative thereof. An AAV genome is a polynucleotide sequence, which may encode functions needed for production of an AAV particle. These functions include those operating in the replication and packaging cycle of AAV in a host cell, including encapsidation of the AAV genome into an AAV particle. Naturally occurring AAVs are replication-deficient and rely on the provision of helper functions in trans for completion of a replication and packaging cycle. Accordingly, the AAV genome is typically replication-deficient. The AAV genome may be in single-stranded form, either positive or negative-sense, or alternatively in double-stranded form. The use of a double-stranded form allows bypass of the DNA replication step in the target cell and so can accelerate transgene expression. AAVs occurring in nature may be classified according to various biological systems. The AAV genome may be from any naturally derived serotype, isolate or clade of AAV. AAV may be referred to in terms of their serotype. A serotype corresponds to a variant subspecies of AAV which, owing to its profile of expression of capsid surface antigens, has a distinctive reactivity which can be used to distinguish it from other variant subspecies. Typically, an AAV vector particle having a particular AAV serotype does not efficiently cross- react with neutralising antibodies specific for any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11. AAV may also be referred to in terms of clades or clones. This refers to the phylogenetic relationship of naturally derived AAVs, and typically to a phylogenetic group of AAVs which can be traced back to a common ancestor, and includes all descendants thereof. Additionally, AAVs may be referred to in terms of a specific isolate, i.e. a genetic isolate of a specific AAV found in nature. The term genetic isolate describes a population of AAVs which has undergone limited genetic mixing with other naturally occurring AAVs, thereby defining a recognisably distinct population at a genetic level. Typically, the AAV genome of a naturally derived serotype, isolate or clade of AAV comprises at least one inverted terminal repeat sequence (ITR). An ITR sequence acts in cis to provide a functional origin of replication and allows for integration and excision of the vector from the genome of a cell. ITRs may be the only sequences required in cis next to the therapeutic gene. Suitably, one or more ITR sequences flank the transgene. The AAV genome may also comprise packaging genes, such as rep and/or cap genes which encode packaging functions for an AAV particle. A promoter may be operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19 and p40 promoters. For example, the p5 and p19 promoters are generally used to express the rep gene, while the p40 promoter is generally used to express the cap gene. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof. The cap gene encodes one or more capsid proteins such as VP1, VP2 and VP3 or variants thereof. The AAV genome may be the full genome of a naturally occurring AAV. For example, a vector comprising a full AAV genome may be used to prepare an AAV vector or vector particle. Suitably, the AAV genome is derivatised for the purpose of administration to patients. Such derivatisation is standard in the art and the invention encompasses the use of any known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art. The AAV genome may be a derivative of any naturally occurring AAV. Suitably, the AAV genome is a derivative of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of a transgene from an AAV vector of the invention in vivo. Typically, it is possible to truncate the AAV genome significantly to include minimal viral sequence yet retain the above function. This may reduce the risk of recombination of the vector with wild-type virus, and avoid triggering a cellular immune response by the presence of viral gene proteins in the target cell. Typically, a derivative will include at least one inverted terminal repeat sequence (ITR), optionally more than one ITR, such as two ITRs or more. One or more of the ITRs may be derived from AAV genomes having different serotypes, or may be a chimeric or mutant ITR. A suitable mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows for continued replication of the genome to generate a single-stranded genome which contains both coding and complementary sequences, i.e. a self-complementary AAV genome. This allows for bypass of DNA replication in the target cell, and so enables accelerated transgene expression. The AAV genome may comprise one or more ITR sequences from any naturally derived serotype, isolate or clade of AAV or a variant thereof. The AAV genome may comprise at least one, such as two, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 ITRs, or variants thereof. The one or more ITRs may flank the transgene at either end. The inclusion of one or more ITRs is can aid concatamer formation of the AAV vector in the nucleus of a host cell, for example following the conversion of single-stranded vector DNA into double-stranded DNA by the action of host cell DNA polymerases. The formation of such episomal concatamers protects the AAV vector during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo. Suitably, ITR elements will be the only sequences retained from the native AAV genome in the derivative. Suitably, a derivative may not include the rep and/or cap genes of the native genome and any other sequences of the native genome. This may reduce the possibility of integration of the vector into the host cell genome. Additionally, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) within the vector in addition to the transgene. The following portions could therefore be removed in a derivative of the invention: one inverted terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes. However, derivatives may additionally include one or more rep and/or cap genes or other viral sequences of an AAV genome. Naturally occurring AAV integrates with a high frequency at a specific site on human chromosome 19, and shows a negligible frequency of random integration, such that retention of an integrative capacity in the AAV vector may be tolerated in a therapeutic setting. The invention additionally encompasses the provision of sequences of an AAV genome in a different order and configuration to that of a native AAV genome. The invention also encompasses the replacement of one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from more than one virus. Such chimeric genes may be composed of sequences from two or more related viral proteins of different viral species. The AAV vector particle may be encapsidated by capsid proteins. Suitably, the AAV vector particles may be transcapsidated forms wherein an AAV genome or derivative having an ITR of one serotype is packaged in the capsid of a different serotype. The AAV vector particle also includes mosaic forms wherein a mixture of unmodified capsid proteins from two or more different serotypes makes up the viral capsid. The AAV vector particle also includes chemically modified forms bearing ligands adsorbed to the capsid surface. For example, such ligands may include antibodies for targeting a particular cell surface receptor. Where a derivative comprises capsid proteins i.e. VP1, VP2 and/or VP3, the derivative may be a chimeric, shuffled or capsid-modified derivative of one or more naturally occurring AAVs. In particular, the invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones, or isolates of AAV within the same vector (i.e. a pseudotyped vector). The AAV vector may be in the form of a pseudotyped AAV vector particle. Chimeric, shuffled or capsid-modified derivatives will be typically selected to provide one or more desired functionalities for the AAV vector. Thus, these derivatives may display increased efficiency of gene delivery and/or decreased immunogenicity (humoral or cellular) compared to an AAV vector comprising a naturally occurring AAV genome. Increased efficiency of gene delivery, for example, may be effected by improved receptor or co-receptor binding at the cell surface, improved internalisation, improved trafficking within the cell and into the nucleus, improved uncoating of the viral particle and improved conversion of a single-stranded genome to double-stranded form. Chimeric capsid proteins include those generated by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This may be performed for example by a marker rescue approach in which non-infectious capsid sequences of one serotype are co-transfected with capsid sequences of a different serotype, and directed selection is used to select for capsid sequences having desired properties. The capsid sequences of the different serotypes can be altered by homologous recombination within the cell to produce novel chimeric capsid proteins. Chimeric capsid proteins also include those generated by engineering of capsid protein sequences to transfer specific capsid protein domains, surface loops or specific amino acid residues between two or more capsid proteins, for example between two or more capsid proteins of different serotypes. Shuffled or chimeric capsid proteins may also be generated by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be created by randomly fragmenting the sequences of related AAV genes e.g. those encoding capsid proteins of multiple different serotypes and then subsequently reassembling the fragments in a self-priming polymerase reaction, which may also cause crossovers in regions of sequence homology. A library of hybrid AAV genes created in this way by shuffling the capsid genes of several serotypes can be screened to identify viral clones having a desired functionality. Similarly, error prone PCR may be used to randomly mutate AAV capsid genes to create a diverse library of variants which may then be selected for a desired property. The sequences of the capsid genes may also be genetically modified to introduce specific deletions, substitutions or insertions with respect to the native wild-type sequence. In particular, capsid genes may be modified by the insertion of a sequence of an unrelated protein or peptide within an open reading frame of a capsid coding sequence, or at the N- and/or C-terminus of a capsid coding sequence. The unrelated protein or peptide may advantageously be one which acts as a ligand for a particular cell type, thereby conferring improved binding to a target cell or improving the specificity of targeting of the vector to a particular cell population. The unrelated protein may also be one which assists purification of the viral particle as part of the production process, i.e. an epitope or affinity tag. The site of insertion will typically be selected so as not to interfere with other functions of the viral particle e.g. internalisation, trafficking of the viral particle. The capsid protein may be an artificial or mutant capsid protein. The term “artificial capsid” as used herein means that the capsid particle comprises an amino acid sequence which does not occur in nature or which comprises an amino acid sequence which has been engineered (e.g. modified) from a naturally occurring capsid amino acid sequence. In other words the artificial capsid protein comprises a mutation or a variation in the amino acid sequence compared to the sequence of the parent capsid from which it is derived where the artificial capsid amino acid sequence and the parent capsid amino acid sequences are aligned. Herpes simplex viral vector The vector of the present invention may be a herpes simplex viral vector. The vector of the present invention may be a herpes simplex viral vector particle. Herpes simplex virus (HSV) is a neurotropic DNA virus with favourable properties as a gene delivery vector. HSV is highly infectious, so HSV vectors are efficient vehicles for the delivery of exogenous genetic material to cells. Viral replication is readily disrupted by null mutations in immediate early genes that in vitro can be complemented in trans, enabling straightforward production of high-titre pure preparations of non-pathogenic vector. The genome is large (152 Kb) and many of the viral genes are dispensable for replication in vitro, allowing their replacement with large or multiple transgenes. Latent infection with wild-type virus results in episomal viral persistence in sensory neuronal nuclei for the duration of the host lifetime. The vectors are non-pathogenic, unable to reactivate and persist long-term. The latency active promoter complex can be exploited in vector design to achieve long-term stable transgene expression in the nervous system. HSV vectors transduce a broad range of tissues because of the wide expression pattern of the cellular receptors recognized by the virus. Increasing understanding of the processes involved in cellular entry has allowed targeting the tropism of HSV vectors. Other viral vectors Other suitable viral vectors include those described in Lundstrom, K., 2018. Diseases, 6(2), p.42. The vector of the present invention may be an alphaviral vector. The vector of the present invention may be an alphaviral vector particle. The vector of the present invention may be a flaviviral vector. The vector of the present invention may be a flaviviral vector particle. Self-amplifying ssRNA viruses comprise of alphaviruses (e.g. Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis virus, and M1) and flaviviruses (e.g. Kunjin virus, West Nile virus, and Dengue virus) possessing a genome of positive polarity. Alphaviruses have been mainly applied in preclinical gene therapy studies for cancer treatment. Alphavirus vectors can be delivered in the form of naked RNA, layered plasmid DNA vectors and recombinant replication-deficient or -proficient particles. The vector of the present invention may be a rhabdoviral vector. The vector of the present invention may be a rhabdoviral vector particle. The vector of the present invention may be a measles viral vector. The vector of the present invention may be a measles viral vector particle. Rhabdoviruses (e.g. rabies and vesicular stomatitis virus) and measles viruses carry negative strand genomes. Among rhabdoviruses, recombinant vesicular stomatitis virus (VSV) has been applied for preclinical gene therapy studies. Measles viruses (e.g. MV-Edm) have found a number of gene therapy applications. The vector of the present invention may be a Newcastle disease viral vector. The vector of the present invention may be a Newcastle disease viral vector particle. The ssRNA paramyxovirus Newcastle disease virus (NDV) replicates specifically in tumour cells and has therefore been frequently applied for cancer gene therapy. The vector of the present invention may be a poxviral vector. The vector of the present invention may be a poxviral vector particle. The characteristic feature of poxviruses is their dsDNA genome, which can generously accommodate more than 30 kb of foreign DNA. Poxviruses have found several applications as gene therapy vectors. For instance, vaccinia virus vectors have demonstrated potential for treatment of cancer. Vaccinia virus is large enveloped poxvirus that has an approximately 190 kb linear, double-stranded DNA genome. Vaccinia virus can accommodate up to approximately 25 kb of foreign DNA, which also makes it useful for the delivery of large genes. A number of attenuated vaccinia virus strains are known in the art that are suitable for gene therapy applications, for example the MVA and NYVAC strains. The vector of the present invention may be a picornaviral vector. The vector of the present invention may be a picornaviral vector particle. Picornoviruses are non-enveloped ssRNA viruses. Coxsackieviruses belonging to Picornaviridae, have been applied as oncolytic vectors. EXPRESSION CONTROL SEQUENCES The vector of the present invention may comprise one or more expression control sequence. Suitably, the transgene is operably linked to one or more expression control sequence. As used herein an “expression control sequence” is any nucleotide sequence which controls expression of a transgene, e.g. to facilitate and/or increase expression in some cell types and/or decrease expression in other cell types. The expression control sequence and the transgene may be in any suitable arrangement in the vector, providing that the expression control sequence is operably linked to the transgene. The term “operably linked”, as used herein, means that the parts (e.g. transgene and one or more expression control sequence) are linked together in a manner which enables both to carry out their function substantially unhindered. The expression control sequence may be a phagocyte-specific expression control sequence, particularly a liver and/or splenic phagocyte-specific expression control sequence (e.g. such that the vector specifically expresses a transgene in phagocytes, particularly liver and/or splenic phagocytes). Expression control sequences include promoters, enhancers, and 5’ and 3’ untranslated regions (e.g. miRNA target sequences). The one or more expression control sequence may comprise: (a) a phagocyte-specific promoter and/or enhancer; and/or (b) one or more miRNA target sequence. In some embodiments, the one or more expression control sequence comprises a phagocyte- specific promoter and/or enhancer, and, optionally, one or more miRNA target sequence. The vector may, for example, comprise from 5’ to 3’: a phagocyte-specific promoter and/or enhancer – a transgene – one or more miRNA target sequence. MRC1-derived expression control sequences Suitably, the vector of the present invention may comprise one or more MRC1-derived expression control sequence. As used herein, a “MRC1-derived expression control sequence” is an expression control sequence which includes any of the regulatory features present in the MRC1 gene. An example human MRC1 gene is NCBI gene ID: 4360 and GeneCard GCID: GC10P017809. Aliases include CLEC13D. In assembly GRCh38.p13, the human MRC1 gene is located at Chr 10: 17809348..17911164. The MRC1 gene is conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, zebrafish, and frog. Regulatory features which are present in the MRC1 gene may be identified by any suitable method known to the skilled person. For example, regulatory elements can be identified in GeneHancer which is a database of genome-wide enhancer-to-gene and promoter-to-gene associations. Regulatory features which are present in the MRC1 gene include a MRC1 promoter, a MRC1 enhancer, and MRC1 5’ and 3’ UTRs. Mannose receptor regulatory sequences are located, at least in part, immediately upstream to the site of transcriptional start (Eichbaum, Q., et al., Blood, 1997.90(10): p.4135-43). Phagocyte-specific promoters The vector of the present invention may comprise a phagocyte-specific promoter, particularly a liver and/or splenic phagocyte-specific promoter. Suitably, the transgene is operably linked to a phagocyte-specific promoter, particularly a liver and/or splenic phagocyte-specific promoter. A “promoter” is a region of DNA that leads to initiation of transcription of a gene. Promoters are located near the transcription start sites of genes, upstream on the DNA (towards the 5' region of the sense strand). As used herein, a “phagocyte-specific promoter” may be a promoter that enables phagocyte- specific expression of a transgene which is operably coupled to the promoter Exemplary phagocyte-specific promoters include a MRC1 promoter; an ITGAM promoter; a CD86 promoter; a CD274 promoter; a CD163 promoter; a LYVE1 promoter; a STAB1 promoter; a ITGAX promoter; a SIRPA promoter; a TIE2 promoter; a CHIL3 promoter; a CD68 promoter; a CSF1R promoter; a VCAM1 promoter; a PTGS1 promoter; and a C1QA promoter. An engineered promoter variant derived from any of these promoters may be used, provided that the variant retains the capacity to drive phagocyte-specific expression of a transgene which is operably linked to the promoter. A skilled person will be arrive at such variants using methods known in the art. The variant may have at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to any of the promoters. A fragment of any of these promoters (or variants thereof) may be used, provided that the fragment retains the capacity to drive phagocyte-specific expression of a transgene which is operably linked to the promoter. A skilled person will be able to arrive at such fragments using methods known in the art. The fragment may be, for example, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, or at least 1000 nucleotides in length. In some embodiments, the phagocyte-specific promoter is selected from the group consisting of: a MRC1 promoter; an ITGAM promoter; a CD86 promoter; a CD274 promoter; a CD163 promoter; a LYVE1 promoter; a STAB1 promoter; a ITGAX promoter; a SIRPA promoter; a TIE2 promoter; a CHIL3 promoter; a CD68 promoter; a CSF1R promoter; a VCAM1 promoter; a PTGS1 promoter; and a C1QA promoter; or a variant and/or fragment thereof. In preferred embodiments, the phagocyte-specific promoter is a MRC1 promoter or a variant and/or fragment thereof. MRC1 promoter In one aspect, the present invention provides a vector comprising an MRC1 promoter. Suitably, the transgene is operably linked to an MRC1 promoter. Any suitable method may be used to identify an MRC1 promoter, for example by using promoter prediction tools or by using a sequence immediately upstream to the MRC1 open reading frame. Suitably, an MRC1 promoter may be about a 0.2-5 kb, 0.5-5 kb, 1-2 kb, or about 1.8 kb sequence immediately upstream to the MRC1 open reading frame. In some embodiments, the MRC1 promoter comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 1 or a fragment thereof. Suitably, the MRC1 promoter 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: 1 or a fragment thereof. In some embodiments of the invention, the MRC1 promoter comprises or consists of the nucleotide sequence SEQ ID NO: 1 or a fragment thereof. Exemplary human MRC1 promoter: CCCTGAATGTGATTATATACATAATTCAATTAAATGTATTTGCTTCTGAAATATATATAAATGTAAAT TAGGCAGTCACTTTTGTATATGATTTATTTATATTTGAAAGCCACAAATGACCCATTTAAACTATTAT TTTCATAAGCCAGTGAAACAATGTCTGAGAAACATTTTTGTTTTGTCTGTTCTGTTCTATAACCATCA TTTTTTTTTTCAGTCATGTACAGCCTTAGTGACAAAGAAACTTTGGTCCTCTGTCCTACATTTTCACT ATCTTTTTCCCTCCGGTCAGGATAATCTCAAATTTACATGTTAAAAACAATCAGTAAGAGAACTACAT CACATTTCTAATAGGATGGAAACTTTTCAACTTTATCACAAAGACAACGAATGTGGAGGCTTTCCGTT TGAAGATAAAACTATTCATTTAAAAAATTTTAAAAATTACAATGTTTCCAGTAGCTTCTTTTTGAATT ACTAACATATTCCACACTCTAGTAACGGTTTGGCCAGCTAATCGTTAGTTTCTGCTTTAAAATGTTCT AAATTCCTGTTCTACTTTTGAAAAATGACAACATAAATGTTTGGAGGGTTATTTTCTGCTTAATGAAA GATCTAGAAACATATTTTATTCTAAGAAAGAATTCCACTTGCCTTTAAATAAAGATATACCTTTTGAC CAAACAATCAGATTTTCTTTTTCTTTTTTTTCTTTTCTTTTTTTTTTTTGAGATGGAGTTTCGCGTCT GTCGCCCAGGCTGGAGTGTAGTGGTGCGATCCTGACTCACTGTAACTTCCACTTCCCAGGTTCAAACG ATTCTGTTGCCTCAGCCTCCTGAGTAGCTGGGCTTACAGGTGTGCATGATCACACCCGGCTAACTTTT GTATTTTTAGTAGAGACGGGTTTTTGCCATGTTGACCAGGCTGGTTTCAAACTCCTGACCTCGGGTGA TCTGACTGCCTCGGCCTCCCAAACTGCTGGGATTGCAGGCGTGAGCCATTGTGCCTGGCCAGATTTTC TTTTTCTAGCAAGGGGACCCACTTAAACTTGAAGAGGACCGGGATGGTTGAGGCTGGGCAGCAAGGCT TTACTGCAAATCCTTTACCACTGTTTTTTCTGGCTTTCTAGAGAACGTTCTAGCAAAAGGTTTCTAGA ACTTTCTCCTTCCTGGCCTGACTGACATTCCCTCTTAGGTGTAGCCTCCTTTTCACTTTTCTTCTGCC TGGAGGAAATGAAGCTCCACGGAACTTTCTGTTGAAACTTTCCAAGAAAAAAAAGAAAGGCTCTAAGC ACTGAATGTGGAAACTGAAGGGGATGAGCTTCAACTCTGAAGTGTTTCCAGCGTAAAACTGTCCTTTC CAGGGCCCGTGTGGCTGTCACTTCAGAGTGGAGGTTGTCTGCTGAGGGACCCCTGACTCAGCTGCTTC CCAGGGGAAGCTCCGTCTTCCGGCACAGGTAATGGCCTGCAGCTTGATCTCCACCCAGCCCCATCTGA GCAGGCCGGGAGCTCCCAGGCTGTTTCACTTCTCTCCTTCCTGACTCCTCACCATCACCATCGCCCTC TCTCCTCCCCACCCCGCCACTCCTCTCCCACACGTGTCCCTTTCTCCCCTTCCTCTGCGTCTGCTCTT CTCAGAAGTTAGCTTACGAAGCAAAGTTGTTACTTTGAATTCCTGTTTTTCCAGCCACCCTCATGTGA CAGGATGTCTCCTCAGTAGAGGCTTTCCCTAAATTCAGGAGCCCTTTAAAAGGGAGGGCTTCCTCTGT AGTTCTTTTCAGCTGGGCAGCTCTGGGAACTTGGATTAGGTGGAGAGGCAGTTGGGGGGCCTCGTTGT TTTGCGTCTTAGTTCCGCCCTCCTGTCCATCAGGAGAAGGAAAGGATAAACCCTGGGCC (SEQ ID NO: 1) In some embodiments of the invention, the MRC1 promoter comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 2 or a fragment thereof. Suitably, the MRC1 promoter 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: 2 or a fragment thereof.. In some embodiments of the invention, the MRC1 promoter comprises or consists of the nucleotide sequence SEQ ID NO: 2 or a fragment thereof. Exemplary mouse MRC1 promoter: CTCGAGCCGAGCTCTGAAATGGATGCTTCAAGGATTTGAAGAGACACCAGAAGTGAAAAACGTGCTAT TTTCCCACAGTTCCTGGCAATACAAAGATTGTTTTAAGGCCTATGGAAATTCCTCTTCCTCCGTTACC TGAAATTACAGATTTGTGTTGACTTGCTCACCCCTCCTAACCTGATAAAATCTTCCAATAAGATAAAA ATGATGGAGACAAATCCTTTGTGGGATGTTGGACTTCACTTTATATCACATCCAGCGTCTCGTTACTG ATTCTGATTTTATTCCTGTGCATGTAAGACACGTTGACATAATAAAACCATGGATATACAGATGCCTG CAATTCAGTTAACTCTTTTTTTTCCTCTTCAAATAAGTCAAAGCAAACCCCAATTAGGCAAAACAATT TGAATGGCTTGCATTTAAAAGACCAATTAAAACATTTTTTGGTCAGCAAGCATGATGGGACACACTTA TAATCCCAGCTCTCAGAAAGTCAAAACAGAGGAACCAAGAATTCAAGGCCAGCCTGCGCTACAAACGC AAGACTGTTTCGGTGTTCCTGTGATAAGTCAGTTACGCAGTGATTGAAAAGGAAACGTTTGCAGCCTC TCACCAGTTGTGGGAGAATTTTCTTTGTCAGTTAAGCCTTGATAGAATGAAAAAGAACGGTGGGTCCC TTCTCAGAATCTTCCTAATTTAGGCTTTTTAAAAAGAAAATTCTTGAGAGAAACCACAGCTTATTGGG AAATGAGTGTGTACCTGCCTCAGCGTGGATGGGTCTGAACAGCTTTTCACTTGAAGGTAAACCATCTG TTTACAACTTCTAAGTCGCCAGTGTTTCCAGAGCTTCTTTTTGAAACGATGACATTTCCCACGCTCCA GTTTCAGGTCTTCCCTGACTAACCACAAATATCCATTTCTAAATATTCTTAATTCTTGTTGAACGTCT GGAAAAAAAAAATCAGTGTTTAGGTGGGTTGTGTGGTGCTTTGTGAACGACCCTGCAAAATCATGAAG ACGAAACCCCACTGTCATCGAATCAACAAGCAACTTTTGGACTCAAGCCAGGCTTTCTTTTGCAAGAG AGAGAGAGAGGTCTTCCCTTTTTCAAACTCTGAGGACTGTAATGGTTGAGGCCTGGCAGCGAACCGAC AACAAAGCTATTGCCACTATTTCCTCTGGCTTTCTAAGGAAAGCTGCTAGAACTTTCTATCCCTGGGC TTCATTGAGGTTGTCTTAAAATTAACTTCTGTCATTTTCCTTCTAGAGACAGGGGCAAAACTCTACGT GAACCATACCTTTGATCCTTTCCAAGGAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT GTGTGTGTGTTGGTGCTCGGGCTCTAAGCCTGAGCAGGAAGAGCTTCTGATGCTTTCCAGCGAGTGTC CTCCCTTTCTGACTGTAGAATTGTGGGTGAGAGCCTCCACAGCTGCCTCCTGGAGACTTTTTCCCACC CAGATAATGGCCTCCGTTTGGTTACTGCCCAGCACCTGTGGAGAGCTCAGCAGGGCTGCCTCTCCCTG CTGCTCATGGCCTGGGTCCTCACTTCTCCCCACTTCCTGCGTTTTCTCCTCTCCTACACATGTTCCTC TCTCCCCTTCCTCCTGTGCCTTAGCTTACGAAGCAAAGTTGTAACTTTGAATTCCTGTTTTTCTAACC GCCCCCATGTGACAGGATATCTCTCAATTGGAGGGTTTTCCTAAATTCAGGAGTCCTTTAAAAGGGAC AGCTTCCTCTGTCCTCCTTTTCAGTCAGGCAGCTCCCAGACCTTGGACTGAGCAAAGGGGCAACCTGG GGACCTGGTTGTATTCTTTGCCTTTCCCAGTCTCCCTCTTCTCCCTCATTGGAACCGGT (SEQ ID NO: 2) In some embodiments of the invention, the MRC1 promoter comprises or consists of a nucleotide sequence which is at least 40% identical to SEQ ID NO: 1 and SEQ ID NO: 2 or a fragment thereof. Suitably, the MRC1 promoter comprises or consists of a nucleotide sequence which is at least 50%, at least 60%, or at least 70% identical to SEQ ID NO: 1 and SEQ ID NO: 2 or a fragment thereof. In some embodiments, the MRC1 promoter comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 31 or a fragment thereof. Suitably, the MRC1 promoter 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: 31 or a fragment thereof. In some embodiments of the invention, the MRC1 promoter comprises or consists of the nucleotide sequence SEQ ID NO: 31 or a fragment thereof. Exemplary XhoI-Human.MRC1.promoter CTCGAGCCCTGAATGTGATTATATACATAATTCAATTAAATGTATTTGCTTCTGAAATATATATAAAT GTAAATTAGGCAGTCACTTTTGTATATGATTTATTTATATTTGAAAGCCACAAATGACCCATTTAAAC TATTATTTTCATAAGCCAGTGAAACAATGTCTGAGAAACATTTTTGTTTTGTCTGTTCTGTTCTATAA CCATCATTTTTTTTTTCAGTCATGTACAGCCTTAGTGACAAAGAAACTTTGGTCCTCTGTCCTACATT TTCACTATCTTTTTCCCTCCGGTCAGGATAATCTCAAATTTACATGTTAAAAACAATCAGTAAGAGAA CTACATCACATTTCTAATAGGATGGAAACTTTTCAACTTTATCACAAAGACAACGAATGTGGAGGCTT TCCGTTTGAAGATAAAACTATTCATTTAAAAAATTTTAAAAATTACAATGTTTCCAGTAGCTTCTTTT TGAATTACTAACATATTCCACACTCTAGTAACGGTTTGGCCAGCTAATCGTTAGTTTCTGCTTTAAAA TGTTCTAAATTCCTGTTCTACTTTTGAAAAATGACAACATAAATGTTTGGAGGGTTATTTTCTGCTTA ATGAAAGATCTAGAAACATATTTTATTCTAAGAAAGAATTCCACTTGCCTTTAAATAAAGATATACCT TTTGACCAAACAATCAGATTTTCTTTTTCTTTTTTTTCTTTTCTTTTTTTTTTTTGAGATGGAGTTTC GCGTCTGTCGCCCAGGCTGGAGTGTAGTGGTGCGATCCTGACTCACTGTAACTTCCACTTCCCAGGTT CAAACGATTCTGTTGCCTCAGCCTCCTGAGTAGCTGGGCTTACAGGTGTGCATGATCACACCCGGCTA ACTTTTGTATTTTTAGTAGAGACGGGTTTTTGCCATGTTGACCAGGCTGGTTTCAAACTCCTGACCTC GGGTGATCTGACTGCCTCGGCCTCCCAAACTGCTGGGATTGCAGGCGTGAGCCATTGTGCCTGGCCAG ATTTTCTTTTTCTAGCAAGGGGACCCACTTAAACTTGAAGAGGACCGGGATGGTTGAGGCTGGGCAGC AAGGCTTTACTGCAAATCCTTTACCACTGTTTTTTCTGGCTTTCTAGAGAACGTTCTAGCAAAAGGTT TCTAGAACTTTCTCCTTCCTGGCCTGACTGACATTCCCTCTTAGGTGTAGCCTCCTTTTCACTTTTCT TCTGCCTGGAGGAAATGAAGCTCCACGGAACTTTCTGTTGAAACTTTCCAAGAAAAAAAAGAAAGGCT CTAAGCACTGAATGTGGAAACTGAAGGGGATGAGCTTCAACTCTGAAGTGTTTCCAGCGTAAAACTGT CCTTTCCAGGGCCCGTGTGGCTGTCACTTCAGAGTGGAGGTTGTCTGCTGAGGGACCCCTGACTCAGC TGCTTCCCAGGGGAAGCTCCGTCTTCCGGCACAGGTAATGGCCTGCAGCTTGATCTCCACCCAGCCCC ATCTGAGCAGGCCGGGAGCTCCCAGGCTGTTTCACTTCTCTCCTTCCTGACTCCTCACCATCACCATC GCCCTCTCTCCTCCCCACCCCGCCACTCCTCTCCCACACGTGTCCCTTTCTCCCCTTCCTCTGCGTCT GCTCTTCTCAGAAGTTAGCTTACGAAGCAAAGTTGTTACTTTGAATTCCTGTTTTTCCAGCCACCCTC ATGTGACAGGATGTCTCCTCAGTAGAGGCTTTCCCTAAATTCAGGAGCCCTTTAAAAGGGAGGGCTTC CTCTGTAGTTCTTTTCAGCTGGGCAGCTCTGGGAACTTGGATTAGGTGGAGAGGCAGTTGGGGGGCCT CGTTGTTTTGCGTCTTAGTTCCGCCCTCCTGTCCATCAGGAGAAGGAAAGGATAAACCCT (SEQ ID NO: 31) Inducible promoter Suitably, the phagocyte-specific promoter may be an inducible promoter As used herein, an “inducible promoter” is a promoter which is only active under specific conditions. For example, expression of the transgene may be induced by a small molecule or drug (e.g. which binds to a promoter, regulatory sequence or to a transcriptional repressor or activator molecule) or by using an environmental trigger. Types of inducible promoter include chemically-inducible promoters (e.g. a Tet-on system); temperature-inducible promoters (e.g. Hsp70 or Hsp90-derived promoters); and light-inducible promoters. Suitably, the promoter is chemically-inducible. Any suitable method for engineering an inducible phagocyte-specific promoter may be used. Alternatively, the phagocyte-specific promoter may be a constitutive promoter. As used herein, a “constitutive promoter” is a promoter which is always active. Phagocyte-specific enhancers The vector of the present invention may comprise a phagocyte-specific enhancer. Suitably, the transgene is operably linked to a phagocyte-specific enhancer. An “enhancer” is a region of DNA that can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur. Enhancers are cis-acting. They can be located up to 1 Mbp (1,000,000 bp) away from the gene, upstream or downstream from the start site. As used herein, a “phagocyte-specific enhancer” may be an enhancer that enables phagocyte- specific expression of a transgene which is operably linked to the enhancer. Exemplary phagocyte-specific enhancers include a MRC1 enhancer; an ITGAM enhancer; a CD86 enhancer; a CD274 enhancer; a CD163 enhancer; a LYVE1 enhancer; a STAB1 enhancer; a ITGAX enhancer; a SIRPA enhancer; a TIE2 enhancer; a CHIL3 enhancer; a CD68 enhancer; a CSF1R enhancer; a VCAM1 enhancer; a PTGS1 enhancer; and a C1QA enhancer. An engineered enhancer variant derived from any of these enhancers may be used, provided that the variant retains the capacity to drive phagocyte-specific expression of a transgene which is operably linked to the enhancer. A skilled person will be arrive at such variants using methods known in the art. The variant may have at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to any of the enhancers. A fragment of any of these enhancers (or variants thereof) may be used, provided that the fragment retains the capacity to drive phagocyte-specific expression of a transgene which is operably linked to the enhancer. A skilled person will be able to arrive at such fragments using methods known in the art. The fragment may be at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, or at least 1000 nucleotides in length. In some embodiments, the phagocyte-specific enhancer is selected from the group consisting of: a MRC1 enhancer; an ITGAM enhancer; a CD86 enhancer; a CD274 enhancer; a CD163 enhancer; a LYVE1 enhancer; a STAB1 enhancer; a ITGAX enhancer; a SIRPA enhancer; a TIE2 enhancer; a CHIL3 enhancer; a CD68 enhancer; a CSF1R enhancer; a VCAM1 enhancer; a PTGS1 enhancer; and a C1QA enhancer; or a variant and/or fragment thereof. In preferred embodiments, the phagocyte-specific enhancer is a MRC1 enhancer or a variant and/or fragment thereof. The vector of the present invention may comprise a phagocyte-specific promoter and/or a phagocyte-specific enhancer, i.e. a phagocyte specific promoter and/or enhancer. Suitably, the transgene is operably linked to a phagocyte-specific promoter and/or enhancer. In some embodiments, the phagocyte-specific promoter and/or enhancer is selected from the group consisting of: a MRC1 promoter and/or enhancer; an ITGAM promoter and/or enhancer; a CD86 promoter and/or enhancer; a CD274 promoter and/or enhancer; a CD163 promoter and/or enhancer; a LYVE1 promoter and/or enhancer; a STAB1 promoter and/or enhancer; a ITGAX promoter and/or enhancer; a SIRPA promoter and/or enhancer; a TIE2 promoter and/or enhancer; a CHIL3 promoter and/or enhancer; a CD68 promoter and/or enhancer; a CSF1R promoter and/or enhancer; a VCAM1 promoter and/or enhancer; a PTGS1 promoter and/or enhancer; and a C1QA promoter and/or enhancer; or a variant and/or fragment thereof. The phagocyte-specific promoter and the phagocyte-specific enhancer may be a combination of any of the above, for example a MRC1 promoter and an ITGAM enhancer. In preferred embodiments, the phagocyte-specific promoter and/or enhancer is a MRC1 promoter and/or enhancer or a variant and/or fragment thereof. Exemplary MRC1 enhancers may include: Mouse Mrc1 enhancer 1 ACAGAACCAGCAGTATAGGGAAGGCCGTGGTGTTGTGGGACTCACATGATATTATTTATGATATCTTG GAAATTAGAGCAAAGACAGGTTAGGCATTGTGGTCAGAGGAGCTGGGTTATGACACCGAGGAAACAAG CTGACCCTTGAATTAAAACATATTGACGCCATAGCAATAAGAGGATGGAACCACATTGCCCTCTGCTG TTGGGGAATCATGGCCGCTGCCCCCATTCTGCAGTTAAGAGACCCGGTACTGCCCTCTGCTGGCTGGA TGCACATGTTTCCACATTCTGGATTAGTATCCTTTTGAATTTAAATTTAAAAACAGTCTCCTGCTGCC TGCCAGTGACTCACTGTGGCCTCTTTATGTTGTTAGTAGCTTTGTTTTACTCTGGCAGATAGAAAATA TGTTACAGGTCGCCATCTTGGTTCCGGGACTCAGCA (SEQ ID NO: 17) Human MRC1 enhancer 1 AGCCCCACCATGTTATTGATGGCCAAACAATACGCATGCTGACAGCCATTATCTGTGGCCTCTGATGC TATTAGCCAAACCATGTTATTGATGGTCAAACAATACGCATGCTGACAGCCATTATCTGGGACTCAGA AAGTTCTGCATATTCAAGTCAGGCCAGAGGATCCGAGTTCTAATGTTAAGAGAAACCAACACACCAAC AAGCAAATAAACAAACCTACCCTTGAACCAAAATATACATCAATACCTCCGTTGCAAATGGATAAATG GAACTGCATTGCCCTCTGCTGTTGGGGAATCTTGGCAACCATTTCAACTCTATGGCTGGAGATGACTT ACTGCTCTGTTTATTTTCCATCCTCCTGCTTAGATTATTGCTTTCAAAGTTTCCAGAATAGAAGAAGT CAGTGGTGGCCAGTTGTCCTTTAATGGTCTCTTATCTACCAATGGCTAGTATCCTTTTTGCATTATCG TAGCTCTACTCTTGTAGATGTTAAATT (SEQ ID NO: 18) Mouse Mrc1 enhancer 2 ACATGGGAGGCAAGGCGGAAGGAGCATGAGGCTGACCTAGCAGGCAGGAAGCACAGAAATCACATTTT GAGCTACATAGAAGAAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG AGAGAGAGAGAGAGAGAGAAATCAGGAAGTGAGACTAGTCTATAAAACTGCAAAGCCTACTCCCACTG ACATACTTCCTTTAGCAATGCACAGCTGCCACAACCCTCCCAAATCCTGCCACCAACTGGAGACCAAG GGTTACAATAAGTAGACCTAAGGGAGGGGTACTTTTCTTTTCAACCACTGCAGTGGAGCACACCTCTA TGTCCAACATGAAGGAAATAGAGGCTGGAAGACCAGAAATTCAAGGTCACCCACCAGCTCATCGCCAG TTGCAGATCAGTTTGAGCTACAGGCTATCTGCCTCAAATATAAAACTAAACAGAAAGTCAATAAAAAG GCCACACTTGGGGAAGTGGATAATAGGGTCAAATATTAGTAAACACCTCTTCTTCCCCATTGTTAAAG CCTGCTCCCTCCAGTTCCTCTGACTTTACTGTTACATAACAGATCTTGGACCTGTGACTGCTGTGTTT ACAACATACTCAGTGACCCCTAACTTCTAATCATGAAACACATTTACCCGGTTCCAGGATGCCATCTC TCCACCTACAGCTCACCATGGAAGCATTTTGCCTCTTAGCAAAGGTCTTTGGTTTCTCGTGGGTGGCA (SEQ ID NO: 19) Human MRC1 enhancer 2 TTTATTAGATTGTGAAGACTAAAAGGGCTAAAGTTTGCTTGAGAAGAGAGTATCCGTTTTAGCTTCTA AAATATACACTTGGGGAAACAGAGAGATTAGGATCAGAAATTAGTGTGGGAGCAAAAGCATCCCCGAT TTCTTGCCCCCAAAATCTCCTCCCTCCAGTTTTTCTGGACACGGCTATATTAATATTAGTCTGTCTCT GCTCCTATGACTGCCATGTTCAAAAAACCCCTTGTGGCTTCTCATTTCCCATCAAATAATTAACAAAA TACATTTTGCCTGACATTCAAGATTGCCACCTGCTGAACATCTGTACTATTCCCGGTCTTGCATTTTG CGGTCACAACAAAAGAAAAGTCAGTCTTGGATTCCTTGTTTTTCCTCATCCATTCTCACCCTCACATC TTTTCCCATGTCATTTCTTTTGCCTGGAATTCTCACGATTGTGTACAGTTGAAATGCAATTCTATTTC CATCTGCATTTCTCTTATTTGCTTATTACCACTTTTATTAGTATAATAGCTACTTACACATATGTCCT GCTTCCTTGACAACACTATAAATACATTAATTTGTTATTATTTATTTACTTATTCATTCATTCCCAAG ATATCTTGTACCCATGTGCCAGGCTTTGTGCTAGGTCCTGGGGACATCAAAGCTGACCCTGTCTTGTC CACATGCAGCTTAAAAGTCCAATGGGAAGAAACAGATGTAAACAAGAAAACAAACAAATGAATAGGTA CCAATTATAATAAGTCCTGCATGGAAAGCAAACTGAAGGCCATGTGGAGATCGCGTCAGAGGGGGAAA TAGCTGAAAACGGGGAATGTCTTGTGAATATTTATGACCCCTCTTCCCCCAAGGAGGTTCACACAGAG CCTTCCAAGGAAGGGTCTCACCAAACATTTGCTGACTGACATGTTAACATGCAACAAAATAAAAATAC TTAGAAATTCAGTCTCCTGTTTGGAAAACTAGACAGTCATGGCAAGAAGACAGTAAATGCAGTGGTTC TTCTGTGTGAAGTGGCTTATTCATCACCTTGGAATTCTGTTAAAGTGATGATGCCTGGGCTGAGATCC CGAAGACTCTAGATCAGTAGGTGTGACACAGGGCACAAGAATCTGCCTTTTGTCAAACTCCTGGGTGA TTCTGGGGCAGCTGGTTGAAAGACCATATCTGGCAAAACACTGTGTTAGTTCCTGCTGTATCCAGGTG CTAACACCAGGCAGCAGAGCAGAGAGGCTGAGAGCAAAGATTCTGCAGCCAGACTGCCTAGGTCCCAG CTCTGTGAA (SEQ ID NO: 20) Mouse Mrc1 enhancer 3 GGTCAACTATATAGTAATGAACACCTATCAATTATTTTCCTTAATATATTAGATTTTATTCCTCTTAA TTCAGCATCACTTGCATTCTAATGAAGATCTCTATGTCCTTCCAGCCATGTACTCCTTACTGGGCAAT GCAAATGGAGCCGTCTGTGCATTTCCATTCAAGTTTGAAAACAAGTGGTATGCAGACTGCACCTCTGC CGGGCGCTCGGACGGATGGCTCTGGTGTGGAACCACCACTGACTACGACAAAGACAAGCTGTTTGGAT TTTGTCCATTGCACTGTAAGTAACTGAAAACAGCACACCTGGGACATTCAGTATGGTCACATGATGGT AGGGTGGACTTTATGTACCCTCTATCTACCTTTCTTTGTTTCTTGTTTCACTTTCACTTCTCTCTCTC TCTCTCTTTCCTTCCCCATCTTTCTGTTTGCTAAGGATCAAACCCAGCCTTGCACATGCTAAACAAAC ACTCTACTACTGAGCTACATATCCTGACCTTATTAGTTATTTGCTAAGACTTTAGGGCAAATTATACT GAATATAGCATTATATATAGTCAGTGCTGGAGGTAGGTACATCGTTCTCCCAAACCCCAAGTGTTTTA GTTTTAAAAAGCCATAGGTTAAAGCAGGAATTTAAATAACACCACAAACGAGTTTGGTGGGAGTCTGT GAAGGCTCTTGCATTTTGCATCACCATGTGCTGGAGCTCCTTCTAGTACAGATGATACTGATGGAGGT TTGTGAATCTCAACGTAAGAAGGGTGGAATTCAGCTGAGTTGGCAATCAAGGGAAGTGAGTAATCTAT GCTTCAGTCCTTTGAAAGCAGAAGTTTGGTGTTACTAGAGCATGCAAGACCACATAAAGTACCAGAAC TTGAATTCTTGAGGTTTTATCCATTCGTAAGAATCTGTAAGAAAATATGTGGCAGCTTAGGTGGGGCT AGGGAGGGCAGCTGGGAGTCAGAGCTAGGGCTGAGGGAGGAGAAGGTTGAGGTCTTGGCTTAACTTCT GTATCTCTGAACATACTTTCTGAA (SEQ ID NO: 21) Human MRC1 enhancer 3 CAGCCTCCCGAGTAGCTGGGACTACAGGCGCCCGCCACCACGCCTGGCTAATTTTTTGTATTTTTAGT AGAGACGGGGTTTCACTGTGTTAGCCAGGATGGTCTCGATCTGCTAACCTCGTGATCCGCCCACCTCG CCTCCCAAAGTGTTGGGTTTACAGGCATGAGCCGGCGCGCCCGACTATGACCTCTTTATTTTTACCAA CTATCAGTTACTCAGCGAAAATTATCCTGTACACAATATAGTATATACTTAACACGGGAGGTAGGTAG GCATGCTATTATTCCCTCATCTCTGAAATGCCTTACCTTTATGAAACCATAGATCAAAAACAGAATTC AAAGGAAGCCACAAGCATGTGGTTTGATGGAGAAAGACGTGAAGGTCCTGAATTTTGTGTCAATGTCA TCAAAAGTGTTCTTCACAGTGAAAATGATAGTCAGGATACTCTTCTTATGTTTAAATTAAGAAGGGTG CCATTTAGTTTCACACAGCTAGAAATCAATGGAACTCGGTAATTTATATGCTTCAAGGTTTCTTAAAA ATGAAAATTCAGGTTTTATTGCAAGGATGCAAAATAAGCAGTATCGCATTATCTTAGTGGGAAAGCAC CAGAACAGATGAATTCTTGGTGTTCTTCCACGCTTAAAAATCTGTAGGCAAATAGTGAAGCACATTTT AGGTGGAGTGGAGGAGGGCGTCATGTTGAAGGCAGAGCTGGGGCTGAGGAAAAAGGAAAAGGAAGTAT TCCTCTTAGCTTCACGTTCCCCATCACCAGACACCCTCCTCCTGATGCTGGCTCCACCCTTCCCAAAC TTCTTACCCCCGACCTCTCACCTGCTACTTTAGACCAGATCAGAGTAGCTCTTGTTTCCTGGTTATCA CCCAGAACTCTTTCTCCTGCTGCCCTGCAAAGGGACTGGGCAGAGCAAAGAGCATTCGATATGGTCTG GGATGATTGTGACACCACCTGAGTAACAATAGAATCACGACTATCACAACTCAACTTTCCAGACCACA AATCCACAAGTAATCACACTATTTCAAGCATTATTGTAAAACAGAACAACTTAAAAAATACCTGAATT TGACGAACAAAAGCCAGAATTCTAAGAATTGTACTTATTTATCTCTCTGGATTTATAATCCCTAATTA TCACACTAAAAGTAAATTTAATTTCTGAGCCCCATATACTATTGTAATTGTCTTCAGAGTGCAGTCTC TCCAATCCGAATGAATACTCACAAAAGCCCATAGGCTTTCTGTTCATAGCGACACTGCTGCCTCGGTC TTAACTGAGGTAGTTCTATTTGTCTCTCTTATGTCAATCTTTAGAAAGACATTTGATTTCCATTCAAG GTTTTTAGATGTCGAATTTTGTATTCGAAGTATTTTTGTCTGAAACACATTGAGCAATTTTTTTCTAA GATAAAGCAATACTTGGTTTTCAAGTGATTGAAAGTGTCTTTCTCCTTTACTTAATAGGAATGATATT TTCTTAATCTGTTTCATGGACTTTCTTAAGGGTATATATTTCATGGGTCCAACTATAGCATCCTCCAC ATCCTTTGAAATTGACAAAGGAGTTAGATGAATGTGTGATTTCCTGAATGAAATGTGGAGGACAAGTG GTAAGTTACTAATCACAAAGAAAACTCACAATCTTGGAAATCCTTGGATGTGTGTTGGAGACGTATCT TGAGTTTGTTCAGTGGAATAATTTTTTAGTCTTATTACTTGTATTTATGCCTTCACTGTCAAATTATA TATTTTTTCCTGTTAAATGTAAAATAATCGTAGAAAATAAATTGATTTGGTTTCAATATGCATTAAAA TTTTAAATCACGTTTTGTACATTTAATATCTTTCTTAAAGGGCTTTATAGTCTTCCAGTCTGTTTCAT TTTGTGTTCTTTTCAAAAGAGTTTTTATTGTATTTATTTATTTATTTATTTTTGAGACAGGGTCTCAC TCTGTCATCCAGACTGGAGTGCATTAGCATGATCTTGGCTCACCACAACCTCTGCCTCCCAGGCTCAA GTGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGAT (SEQ ID NO: 22) Mouse Mrc1 enhancer 4 AATAAACGTCTAGGAACATTTACCCTAAAGTACTGCCCTCTCTATGTGAACAAACTTAAGCCTGTGTT CTTTCCTTTTTGTGAACAGACGCGAGGCAATTTTTAATCTATAATGAAGATCACAAGCGCTGCGTGGA CGCTCTAAGTGCCATCTCAGTTCAGACGGCAACTTGCAACCCGGAAGCTGAATCCCAGAAATTCCGCT GGGTGTCAGATTCTCAGATCATGAGTGTTGCTTTCAAATTATGTTTGGGAGTGCCATCAAAAACTGAC TGGGCTTCCGTCACCCTGTATGCCTGTGATTCGAAAAGTGAATATCAGA (SEQ ID NO: 23) Human MRC1 enhancer 4 TGGAAGAGTTGGAAACTTTTGACCTAAAAGATCGTCCTTGTTACATGAATCCACTTAGCCATGCTTGC TTTCTTCTTCTTTTCCTGCTTCTTTCTTTTTAAACAGACACCAGGCAATTTTTAATCTATAATGAAGA TCACAAGCGCTGCGTGGATGCAGTGAGTCCCAGTGCCGTCCAAACCGCAGCTTGCAACCAGGATGCCG AATCACAGAAATTCCGATGGGTGTCCGAATCTCAGATTATGAGTGTTGCATTTAAATTATGCCTGGGA GTGCCATCAAAAACGGACTGGGTTGCTATCACTCTCTAT (SEQ ID NO: 24) Mouse Mrc1 enhancer 5 TGTCAGGTTCTCTGGAGCACCCTCTCACCTGTTCAGACTAATTTCCTAAGTTCGGCGGGTCCCGGACC AAGATGGCGACCCGCTACATTTCATTCTTACATGCAGGGGATGAGCGCACTGTTTCACCACTTTGATT GCCTTTTTTGAGCATGGTAGATATTCAGTAAGCAACCCATGGATTGAATTCTACTTTATGTTTAATGC AGGACGAAAGGCGGGATGTGTTGCCATGAAAACCGGAGTGGCAGGTGGCTTATGGGATGTTTTGAGTT GTGAAGAAAAGGCAAAATTTGTGTGCAAACATTGGGCAGAAGGAGTGACTCGCCCACCAGAGCCCACA ACAACTCCTGAACCCAAATGTCCAGAAAACTGGGGTACCACCAGTAAAACCAGCATGTGTTTCAAAGT AAGGATCACTCGCCAAAT (SEQ ID NO: 25) Human MRC1 enhancer 5 CATCCTCATTTTATTTTATGTACTTCTTTGTTCGTTAAAGCTGGCATTCCTTACAGTTCTATGAGGCA GGTCTTGGTATTTGCATTTGGAGAGGAGAAAGCAAGTTCAGAGCGTTTGAGTAACTTACCTAAAATCT CTAGTTGAGACGTGTCTCATTTTGAAATCTGTGAAAAACTTTGGTCCTGGAAAACCTACGTAGACCTT GGGAAGAAGGAAGGAAAAAGGGAAGGAAGGAGGGAGGGAGAGAGAAGCAGTAAACTATTTTTGCCATT ATGGTGAATTTGATAATATAAAATATTTTATCATTAAATGCCTGTGTAGGGGGCACTTTGCCAAATGT TAGAAATATAAAGTGTTACAAACCCCCCTGCATCTGAGATCATAATTGGGCATCAGAACCCTGATGCT CGGTTCTGAGTGCCTTCTGTGAGCACGGCAGGCCTTCAGCAGGCACCTGTCAAGTGAATTCTACTTCA TATATTTAATGCAGGGCGAAAGCCAGGGTGTGTTGCCATGAGAACCGGGATTGCAGGGGGCTTATGGG ATGTTTTGAAATGTGATGAAAAGGCAAAATTTGTGTGCAAGCACTG (SEQ ID NO: 26) Mouse Mrc1 enhancer 6 GAGTGATTGTGCATGAACTTGTGGAGACCTCAATTGTTCTTGCAACTTGTCTCTTCTATTACTATTGC AAAAGGAATGGCTAAGTCTTTCTTGAAAGAATTCATATAGTTCTCTTTCAGAGACCTGCAGCAGTTAC CACTTTGGGGAACTAGAGAAAAGTTATTTTTAAGTTTCTCTGGAATGAAAGGCACAATTCTATAATTT GGCCTTATTGCTTAATCCACCAGTTTTAAGTTCCTTGTTTGTAAAATATGAATGTTAGTAACTCTTCT TCTTTAAAATCTCGTTATATCATCAAGCTTG (SEQ ID NO: 27) Human MRC1 enhancer 6 TTTGGCCAAGATCCTAAATAGATATAGATGCGGGACCTGGATGTTGGGTTTGATTATCCTTTACAGGC TCTCCATAGTGACGGTGGGTATCTTTAGAGAAAGCTCACCATTTTTGCATTTTACCTCTACTATTCCT CCTCTAGGAGAAATAGTGTATTTTTTCCTTTTTTGGAAGCCTTCATTACAATTCTCTTTCTAAGACTT GGAATTTCCATGTTGCCAAAGAGGAGAATAGTTACTTTATAGTTTCTCTGGTACAGCACTCAGAATTC TGTAACTTGGCCTTACTGCTTAACCTGCCGGTGCTGGGATCCTCATGTGTAAAATGGAAATGTTCATG ACTCTTCTTCCTGAGATAAAATTTTGTTCATTTCATCAAACACTCAGTACATTCTTATTCCTCCATGA TGCCTTCTTCACTCGCTAACCACTTGATGTCAGTTTCTGAACATCTCTATACTCCCTGGATTAATGAT TCTGTTTTATCTATAAACTCAAATAAACCAGAGCTTGGAAAAGCGTATCAGAGTTCAAATTATGCAGC ATACGGGATTAGCAACAGCCTTAGGCAAGAATTCAACCTCAAACCCTGTGAATTATTGTAACACTAAC CCGTTTGTCCATAATCCTCAGGTCTCTAGGGCTGTACTCTCTGGCTTAGCAGCCACTTAACCGCATGT GGCCACTGAGCACATGAGCTGTGGCTAGAGGAACAAATCATCTTCTGTGGCTGCCCCAGGGAACTCCC CTTCATTTCACTCAAGTTGGTTGTTTTCATGCTTTCAAATATGTTTAAAGTTCATCATTTCAGTTTTT GAAGGACAGCATTGGCTGATACTATTTTCAATTTCCTAGGTAGCAAAATTAAAATAACCCACCAGAGG GCTCCAAAGCTGTACTAAGCTTGCTTTCTTTTTCTTTTTCTTCTTTTTTTTTTTGTAACCACTGGAAG TGCACAGAATCTAAATTGTATTGAGGGAATAGAATTTTTTTAAATATGC (SEQ ID NO: 28) Mouse Mrc1 enhancer 7 ATGAGACGTCTGTCCTGGTTTGAACTTTGCCAACTGAGCCTTATTGCCAGCCTGACTGTTACTAGGAA TGGGTCATGAAATAAATGCTTCTGTCAGAATAGTTTATTCGGATTGAATGTGCTCTGCAACCTCTGCT GACAGCCATCTTCCCGAGTGTGTGCAGCAACCAGCCCGAATGTGTCAGCAATGGCTTTCAGGCACCTG TGACACACGTATCACAAGTAGGATGTTTTGATGTTTGCAGGGTTATAGCTTCTTCAGGCAAAACTTGC AGGGCATGAAGAAAGCAGATTCAGCAAGGACCTTAGCCTGAGCAGCTGACTCGAATCGACTGCCAAGT AGCAAGGAATCTGGCACGCGTTCTGAGCTCCTTGGCCAGCCTGAACCGGCTGAAGCTCAAGCCTCAAG CTCGCCTCTGCACCCCCGCACCTCCCCCCGCCCCCCACCAGTGCAGACAGTTTTCCTGCTTTTTGTTG TTGTTCCTTTCTTGTTTTATTTAAAAGCCAGATTCCTTTCATGAAGGGCAGCAAACATGTGAGCTCTG CACATGCGCAGCAGTGAAGAAATTAGCTGAGGAAGTTGAGGCTGTGTCAGGGCACCTTTCCTGAAGTG GATCCTTGGACATCCAAGCCACTGTGTTCTTTTGGCCTGTCTTCAACGGAGTACGTTGTATGGTGCCA AGCCTCAGGATACCAAAGAACTGCTTACAAAACACTTGCTCCTTCACAAGAAGCACAGCAGTTTAGCC AAGATAACATCGCTGCCAAGAACTCTCACATAGGCTAAGATAAAAACTGAAAGCCCCAGCACATGAGA GTGAGTTCTTGGTAGGAAAAAGCACAGTAAGTTCTTCTCAGCCCTCACCGTCAAGATGGCTGGCACGT GCCACCTACTCAGCAGAGATCTGGATGTCTCAACAGTATAGTTCACCTTTCTGTGTGGATGGGCATCT CCCTGTCCACTGAGGGCCTAGGAAGAACAAAAGCAGCAGCAGGAGGAGTCCACTCTTTTGCTTCTAGT CTTCCTGTTTAAGCCGGGAGCTTCCCACCTCACTGTCCTTGGAATAGGTTTCACGCCACTGGCTGGCT TCCCTGCTTTGTGTGTCTTAGAACTTAGATTGAATTCTACCACCTGCTCCTCTGGGTCTTCAACTTGC AGGGAGGTCTTGGAATTATCAGCCTTCACAATTTGGCCAGCCTGAACCGCTCAAGCTCAAGCCTCAAG CTCGCCTCTGCAACCCCGCACCTCCCCCCCCCCGCCCCAGTGCAGACAGTTTTCCTGTTTTTGTTGTT GTTCCTTTCTGAATTAATTT (SEQ ID NO: 29) Human MRC1 enhancer 7 GAAGAAAACACATCCAGTTCTTGGAGGAAAATTGCAATAAATATTTTGAAGAGAGTTCCATCTCTTAT TCTCCCTCAATTTTCTGAAAGTCAGAGTAACACTTGGCTATAAAAGTGATAGGGAAACTAAGTGCCTA TCATATACCAGGCACAGTGGCATGCAATCAAGTGGGATTTCATGTATTTCCCAAGTGTGTTTTGCTGG CTGCCATGTAAGACCCTAGTGTTAATTCCAAAACTCAGAGGTCCTGGCTCTTGAATGGGTGGGGACAG GAGGTGGATTTAAAGTTTCCAGCAGAGAAGAAGTGTGGGACTGATCGTCTGCTGGAACCAGTTCTCTG AATATGATGGTTTATCTGGCAAGGTTTGATTCCCTCAAGGAAGTTCCAGGCTAAAAGAGGAGCTAAGC TTCTACAGTCTCTGAGCTTTTTGTGTTACTGATCTTGAGTCTTATTAAAAAAAAAAAAAAAAAAAAAA AAACCCTGACTTTATCTGGCCGTGCCAGGCTCCCTTCTGGGCCCTTGCTGCCGTGTGTCAGTACGCTG TAACTAGAGATGGATTATGTAAGAAATGTTTTTGTCAGAAGAGGCTGTTGCAGTATTTTATGTGCCCT GGTGCACAGCACCTCTGCTGTGAGTCCCCTCCCCGTGTGAGTTGCAGCCTCGCTGGATACACCTGCTA CTGGTCTCAAGCACCTGTGATTTATTGGTCATGAAGCAGGTACTGGGACTCCTGCTTTTATTATCTGC ACGGCCATCCCTCAGCCAGTATAGATGCCCAGGCTAGACTTGCAGAGCATGAAGAAGGAAGCAGAACC AGTTTGGGACCTTCGCCTGAGCAGCTGACTCAACTCCACTGCCAAGCAGCCAGGAATCTGGCACAGTT TCTGAGATGTCTCAGCCAACTGGTATTTCTGAAGCCATAGTTTTCCTCTGAGCTCCCCCTCAACATGG GTATAGTCATTGTGCTTGTTTTGCTTCTTCCTTGTTCTTGTTTAAAAGCCAGTTTTCTTCCTTGAAAG GCAGTGAAATCTCTGGGTTTTCCATGTGGGTAGAGAGGAGCAGGCGGAACAAGCTTAGGGAGGCCAGG CGGTAACAGGTAACCATTTCTTGAAGTTGATCCTCAGAGCATCCAAGCCAGTGGGTTCATTTGGATGA TCTTCAGCCAGGCATAGTCCCTGGTGCCAAGCCTCAGGATATCAAAGAACTACTTACAAAATATGTGT TCCTTCATAAGGAAAAGAATGGTTTAGCTCAGAGGGCGTGCCTGCCAATAAATCTCACATAGGTTAAG ACACAAACTGAAAAAATGCTCAAGGACCATGAGCCACAGTCACTGGAGAAGCCACAGTCATTCATTCT CCAGCAGTTCCCCTTAACCACCTACCA (SEQ ID NO: 30) In some embodiments, the MRC1 enhancer comprises or consists of a nucleotide sequence which is at least 70% identical to any one of SEQ ID NOs: 17-30 or a fragment thereof. Suitably, the MRC1 enhancer 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 any one of SEQ ID NOs: 17-30 or a fragment thereof. In some embodiments of the invention, the MRC1 enhancer comprises or consists of the nucleotide sequence of any one of SEQ ID NO: 17-30 or a fragment thereof. In some embodiments of the invention, the MRC1 enhancer comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 32 or a fragment thereof. Suitably, the MRC1 enhancer 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: 32 or a fragment thereof. In some embodiments of the invention, the MRC1 enhancer comprises or consists of the nucleotide sequence SEQ ID NO: 32 or a fragment thereof. Exemplary XhoI-Human.MRC1.enhancer: CTCGAGAGCCCCACCATGTTATTGATGGCCAAACAATACGCATGCTGACAGCCATTATCTGTGGCCTC TGATGCTATTAGCCAAACCATGTTATTGATGGTCAAACAATACGCATGCTGACAGCCATTATCTGGGA CTCAGAAAGTTCTGCATATTCAAGTCAGGCCAGAGGATCCGAGTTCTAATGTTAAGAGAAACCAACAC ACCAACAAGCAAATAAACAAACCTACCCTTGAACCAAAATATACATCAATACCTCCGTTGCAAATGGA TAAATGGAACTGCATTGCCCTCTGCTGTTGGGGAATCTTGGCAACCATTTCAACTCTATGGCTGGAGA TGACTTACTGCTCTGTTTATTTTCCATCCTCCTGCTTAGATTATTGCTTTCAAAGTTTCCAGAATAGA AGAAGTCAGTGGTGGCCAGTTGTCCTTTAATGGTCTCTTATCTACCAATGGCTAGTATCCTTTTTGCA TTATCGTAGCTCTACTCTTGTAGATGTTAAATT (SEQ ID NO: 32) miRNA target sequence 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. 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-II 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. 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. 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. 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). 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. 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. 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. 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. 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. 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. 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. Suitably, the one or more miRNA target sequence suppresses transgene expression in liver sinusoidal endothelial cells (LSECs) and/or hepatocytes. 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. 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. 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: 3 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: 3 or a fragment thereof. In some embodiments of the invention, the miR-126 target sequence comprises or consists of the nucleotide sequence SEQ ID NO: 3 or a fragment thereof. Exemplary miRT-126 CGCATTATTACTCACGGTACGA (SEQ ID NO: 3) 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. 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: 4 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: 4 or a fragment thereof. In some embodiments of the invention, the miR-122 target sequence comprises or consists of the nucleotide sequence SEQ ID NO: 4 or a fragment thereof. Exemplary miRT-122 ACAAACACCATTGTCACACTCCA (SEQ ID NO: 4) 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. 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. 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: 5-7 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: 5-7 or a fragment thereof. 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: 5-7 or a fragment thereof. Exemplary miRT-1224 x miRT TCTAGATAAACAAACACCATTGTCACACTCCATTCGAAACAAACACCATTGTCACACTCCAACGCGTA CAAACACCATTGTCACACTCCAATGCATACAAACACCATTGTCACACTCCACCCGGGTCGAGCTCGGT ACC (SEQ ID NO: 5) Exemplary miRT-1264 x miRT GGTACCAGCAAACGCATTATTACTCACGGTACGACCATCGCATTATTACTCACGGTACGAACTTCGCA TTATTACTCACGGTACGACGAACGCATTATTACTCACGGTACGACACGTGTCGGTACC (SEQ ID NO: 6) Exemplary miRT-122 and miR1264 x miRT GGTACCAGCGCTACAAACACCATTGTCACACTCCAACATACAAACACCATTGTCACACTCCAGATTAC AAACACCATTGTCACACTCCACAGAACAAACACCATTGTCACACTCCAGTTTAAACGCATTATTACTC ACGGTACGACCATCGCATTATTACTCACGGTACGAACTTCGCATTATTACTCACGGTACGACGAACGC ATTATTACTCACGGTACGACACGTGTCGGTACC (SEQ ID NO: 7) 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: 36 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: 36 or a fragment thereof. In some embodiments of the invention, the one or more miRNA target sequence comprises or consists of the nucleotide sequence SEQ ID NO: 36 or a fragment thereof. Exemplary AfeI-4xmiRT122-4xmiRT126-PmlI AGCGCTACAAACACCATTGTCACACTCCAACATACAAACACCATTGTCACACTCCAGATTACAAACAC CATTGTCACACTCCACAGAACAAACACCATTGTCACACTCCAGTTTAAACGCATTATTACTCACGGTA CGACCATCGCATTATTACTCACGGTACGAACTTCGCATTATTACTCACGGTACGACGAACGCATTATT ACTCACGGTACGACACGTGTC (SEQ ID NO: 36) 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. In some embodiments, the one or more miRNA target sequence suppresses transgene expression in splenic phagocytes (e.g. splenic macrophages). 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. Other expression control sequences The vector of the present invention may further comprise one or more regulatory element which may act pre- or post-transcriptionally. Suitably, the transgene is operably linked to one or more regulatory element which may act pre- or post-transcriptionally. The one or more regulatory element may facilitate expression of the transgene in phagocytes. 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. Post-transcriptional regulatory elements The vector of the present 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. The vector of the present invention may comprise a Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element (WPRE). Suitably, the transgene is operably linked to a WPRE. In some embodiments of the invention, the WPRE comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 35 or a fragment thereof. Suitably, the WPRE 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: 35 or a fragment thereof. In some embodiments of the invention, the WPRE comprises or consists of the nucleotide sequence SEQ ID NO: 35 or a fragment thereof. Exemplary SalI-WPRE GTCGACCCGACAGTTTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTT AACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTC CCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGC CCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATT GCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCAT CGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCC TTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCG GCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG GAATTCGAGCTCGGTACC (SEQ ID NO: 35) Destabilising domain The vector of the present invention may comprise a nucleotide sequence encoding a destabilising domain. Suitably, the transgene is operably linked to a destabilising domain, i.e. in frame with the transgene product, such that when the transgene is translated a fusion protein is produced comprising the destabilising domain fused to the transgene product. Destabilization domains (DDs) represent a fusion protein component that is intrinsically unstable and destabilizes other proteins upon incorporation, leading to protein degradation. A well-known example of DDs is the Shield system, which incorporates a rampamycin-binding protein (FKBP12) into proteins as a build-in destabilising domain to cause protein degradation in cells. In the absence of its specific ligand (Shield-1), the protein is degraded by the proteasome (Banaszynski, L.A., et al., 2006. Cell, 126(5), pp.995-1004). Another exemplary destabilization domain is dihydrofolate reductase (DHFR), or a variant thereof. In mammalian cells, fusion proteins containing the DHFR protein are rapidly ubiquitinated and degraded by the proteasome system. The antibiotic trimethoprim (TMP) or a TMP-based small molecule can bind to the DHFR protein and prevent the protein from being degraded, which allows the fusion protein to escape degradation (Peng, H., et al., 2019. Molecular Therapy-Methods & Clinical Development, 15, pp.27-39). The vector of the present invention may comprise a dihydrofolate reductase coding sequence, or a variant or derivative thereof. Suitably, the transgene is operably linked to a dihydrofolate reductase coding sequence (or a variant or derivative thereof), i.e. in frame with the transgene product, such that when the transgene is translated a fusion protein is produced comprising the dihydrofolate reductase coding sequence (or a variant or derivative thereof) fused to the transgene product. Polyadenylation sequence The vector of the present 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. 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. Kozak sequence The vector of the present 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. In some embodiments of the invention, the Kozak sequence comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 33 or a fragment thereof. Suitably, the Kozak 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: 33 or a fragment thereof. In some embodiments of the invention, the Kozak Sequence comprises or consists of the nucleotide sequence SEQ ID NO: 33 or a fragment thereof. Exemplary BamHI-KOZAC GGATCCGCCACC (SEQ ID NO: 33) TRANSGENE The vector of the present invention may comprise one or more transgene. Suitably, the one or more expression control sequence is operably linked to a transgene. 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. The transgene may be a therapeutic transgene. The transgene may encode a therapeutic polypeptide and/or an antigenic polypeptide. In some embodiments, the transgene comprises a nucleotide sequence encoding a signal peptide, preferably wherein the signal peptide is operably linked to the encoded polypeptide (e.g. therapeutic polypeptide and/or antigenic polypeptide). The signal peptide may, for example, be a natural signal peptide of the encoded polypeptide. In some embodiments, the transgene does not comprise a nucleotide sequence encoding a signal peptide. Therapeutic polypeptides Suitably, the transgene encodes a therapeutic polypeptide. 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. In some embodiments, the transgene encodes a cytokine, for example a cytokine that can activate immune responses, particularly anti-tumour responses. 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). For example, any cytokine which can activate immune responses, particularly anti-tumour responses can be used. Exemplary cytokines include IFNα, IFNβ, IFNɣ, IL-2, IL-12, TNFα, CXCL9, and IL-1β. Further exemplary cytokines include IL10, IL15 or IL18. Further exemplary cytokines include GMCSF, FLT3, IL7 or IL21 A variant of any of these cytokines may be used, provided that the variant retains the capacity to activate immune responses, particularly anti-tumour responses. A skilled person will be able to arrive at such variants using methods known in the art. The variant may have at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to any of the cytokines. A fragment of any of these cytokines (or variants thereof) may be used, provided that the fragment retains the capacity to activate immune responses, particularly anti-tumour responses. A skilled person will be able to arrive at such fragments using methods known in the art. For example, a fragment may retain residues or domains necessary to activate an immune response. In some embodiments, the transgene encodes a cytokine selected from IFNα, IFNβ, IFNγ, IL- 2, IL-12, TNFα, CXCL9, and IL-1β, or a variant and/or fragment thereof. In some embodiments, the transgene encodes a cytokine selected from IL10, IL15 or IL18, or a variant and/or fragment thereof. In some embodiments, the transgene encodes a cytokine selected from GMCSF, FLT3, IL7 or IL21. Interferons There are three major types of interferon (IFN). The human type I IFN genes encode a family of 17 distinct proteins (including 13 sub-types of IFNα, plus IFNβ, IFNε, IFNκ and IFNω). There is only a single type II IFN, IFNγ. The type III IFNs consist of IFNλ1, IFNλ2, IFNλ3, and IFNλ4. All IFNs have the potential to act on tumour cells to exert direct anti-tumour effects or on immune cells, exerting indirect anti-tumour effects (Parker, B.S., et al., 2016. Nature Reviews Cancer, 16(3), p.131). In some embodiments, the transgene encodes an interferon, for example a Type I interferon (e.g. IFNα, IFNβ), a Type II interferon (e.g. IFNγ), or a Type III interferon (e.g. IFNλ, IFNλ2, IFNλ3, IFNλ4). In some embodiments, the transgene encodes a Type I interferon (e.g. IFNα, IFNβ). IFNα Interferon-alpha (IFNα), a type 1 interferon, is a pleiotropic cytokine playing key role in defending the organism against viral infections. It is well established that IFNα can exert anti- tumour functions including direct tumour cell killing, activation of adaptive and innate immune functions and angiostatic activity. IFNα has been approved for clinical use for several types of tumours, including melanoma, renal cell carcinoma and Kaposi’s sarcoma. However, recombinant IFNα alone is not well tolerated when administered systemically, thus alternative therapeutic options to IFNα are currently preferred. The vector of the present invention may reduce the systemic toxic effects associated to IFNα delivery by delivering therapeutic IFNα selectively to tumours. Routes of administration and expected target cells as phagocytic cells whose physiological turnover may facilitate natural loss of the vector. In some embodiments, the transgene encodes IFNα. An exemplary human interferon-alpha (IFNα) for use in the present invention is UniProtKB P01562. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 8 or a fragment thereof. Suitably, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 8 or a fragment thereof. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of the amino acid sequence SEQ ID NO: 8 or a fragment thereof. Exemplary human interferon-alpha: MASPFALLMVLVVLSCKSSCSLGCDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDG NQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMN ADSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE (SEQ ID NO: 8) In some embodiments of the invention, the transgene comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 34 or a fragment thereof. Suitably, the transgene 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: 34 or a fragment thereof. In some embodiments of the invention, the transgene comprises or consists of the nucleotide sequence SEQ ID NO: 34 or a fragment thereof. Exemplary Human.IFNA transgene: ATGGCCTCGCCCTTTGCTTTACTGATGGTCCTGGTGGTGCTCAGCTGCAAGTCAAGCTGCTCTCTGGG CTGTGATCTCCCTGAGACCCACAGCCTGGATAACAGGAGGACCTTGATGCTCCTGGCACAAATGAGCA GAATCTCTCCTTCCTCCTGTCTGATGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGATGGC AACCAGTTCCAGAAGGCTCCAGCCATCTCTGTCCTCCATGAGCTGATCCAGCAGATCTTCAACCTCTT TACCACAAAAGATTCATCTGCTGCTTGGGATGAGGACCTCCTAGACAAATTCTGCACCGAACTCTACC AGCAGCTGAATGACTTGGAAGCCTGTGTGATGCAGGAGGAGAGGGTGGGAGAAACTCCCCTGATGAAT GCGGACTCCATCTTGGCTGTGAAGAAATACTTCCGAAGAATCACTCTCTATCTGACAGAGAAGAAATA CAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCCCTCTCTTTATCAACAAACTTGC AAGAAAGATTAAGGAGGAAGGAATAA (SEQ ID NO: 34) IFNβ In some embodiments, the transgene encodes IFNβ. An exemplary human IFNβ for use in the present invention is UniProtKB P01574. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 9 or a fragment thereof. Suitably, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 9 or a fragment thereof. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of the amino acid sequence SEQ ID NO: 9 or a fragment thereof. Exemplary human interferon-beta: MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL QQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGK LMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN (SEQ ID NO: 9) IFNɣ In some embodiments, the transgene encodes IFNɣ. An exemplary human IFNɣ for use in the present invention is UniProtKB P01579. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 10 or a fragment thereof. Suitably, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 10 or a fragment thereof. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of the polypeptide sequence SEQ ID NO: 10 or a fragment thereof. Exemplary human interferon-gamma: MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIM QSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHEL IQVMAELSPAAKTGKRKRSQMLFRGRRASQ (SEQ ID NO: 10) Other cytokines IL-2 Interleukin-2 (IL-2), as well as other members of the IL-2-related family of T cell growth factors (e.g. IL-4, IL-7, IL-9, IL-15, and IL-21), utilize a common receptor signalling system that results in the activation and expansion of CD4+ and CD8+ T cells (Lee, S. and Margolin, K., 2011. Cancers, 3(4), pp.3856-3893). In some embodiments, the transgene encodes IL-2 or an IL-2 related-cytokine (e.g. IL-7, IL- 15, IL-21). An exemplary human IL-2 for use in the present invention is UniProtKB P60568. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 11 or a fragment thereof. Suitably, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 11 or a fragment thereof. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of the amino acid sequence SEQ ID NO: 11 or a fragment thereof. Exemplary human Interleukin-2: MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPK KATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVE FLNRWITFCQSIISTLT (SEQ ID NO: 11) IL-12 In some embodiments, the transgene encodes IL-12. Exemplary human IL-12 alpha and beta subunits for use in the present invention are UniProtKBs P29459 and P29460. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 12 or a fragment thereof. Suitably, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 12 or a fragment thereof. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of the amino acid sequence SEQ ID NO: 12 or a fragment thereof. Exemplary human Interleukin-12 subunit alpha: MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEE IDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQV EFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRI RAVTIDRVMSYLNAS (SEQ ID NO: 12) In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 13 or a fragment thereof. Suitably, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 13 or a fragment thereof. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of the amino acid sequence SEQ ID NO: 13 or a fragment thereof. Exemplary human Interleukin-12 subunit beta: MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEV LGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNY SGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEE SLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLT FCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 13) In some embodiments, the transgene encodes a single chain IL12. The single chain IL 12 may comprise IL12 subunit beta (e.g. the amino acid sequence SEQ ID NO: 13 or a fragment thereof, or a sequence that is at least 70%, at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 13 or a fragment thereof) and IL12 subunit alpha (the amino acid sequence SEQ ID NO: 12 or a fragment thereof, or a sequence that is at least 70%, at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 12 or a fragment thereof). The single chain IL12 may be a fusion protein comprising the IL12 subunit beta and the IL12 subunit alpha. The IL12 subunit beta and IL12 subunit alpha may be joined by a linker sequence. The linker sequence may comprise or consist of the amino acid sequence SEQ ID NO: 42 or a fragment thereof, or a sequence that is at least 70%, at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 42 or a fragment thereof. RRAGGGGSGGGGSGGGGSRT (SEQ ID NO: 42) In some embodiments, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 37 or 46 or a fragment thereof. Suitably, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 37 or 46 or a fragment thereof. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of the amino acid sequence SEQ ID NO: 37 or 46 or a fragment thereof. Exemplary single chain human Interleukin-12 sequences: MCPQKLTISWFAIVLLVSPLMAIAGQLMWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLD QSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRC EAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSAC PAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHS YFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSRAGGGGS GGGGSGGGGSRTRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDK TSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLL MDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMS YLNAS (SEQ ID NO: 37) WELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCH KGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSS RGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSF FIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTS ATVICRKNASISVRAQDRYYSSSWSEWASVPCSRAGGGGSGGGGSGGGGSRTRNLPVATPDPGMFPCL HHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFIT NGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNS ETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 46) In some embodiments of the invention, the transgene comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 40 or a fragment thereof. Suitably, the transgene 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: 40 or a fragment thereof. In some embodiments of the invention, the transgene comprises or consists of the nucleotide sequence SEQ ID NO: 40 or a fragment thereof. atgtgtcctcagaagctaaccatctcctggtttgccatcgttttgctggtgtctccactcatggccat cgccgggcaattgatgtgggagctggagaaagacgtttatgttgtagaggtggactggactcccgatg cccctggagaaacagtgaacctcacctgtgacacgcctgaagaagatgacatcacctggacctcagac cagagacatggagtcataggctctggaaagaccctgaccatcactgtcaaagagtttctagatgctgg ccagtacacctgccacaaaggaggcgagactctgagccactcacatctgctgctccacaagaaggaaa atggaatttggtccactgaaattttaaaaaatttcaaaaacaagactttcctgaagtgtgaagcacca aattactccggacggttcacgtgctcatggctggtgcaaagaaacatggacttgaagttcaacatcaa gagcagtagcagttcccctgactctcgggcagtgacatgtggaatggcgtctctgtctgcagagaagg tcacactggaccaaagggactatgagaagtattcagtgtcctgccaggaggatgtcacctgcccaact gccgaggagaccctgcccattgaactggcgttggaagcacggcagcagaataaatatgagaactacag caccagcttcttcatcagggacatcatcaaaccagacccgcccaagaacttgcagatgaagcctttga agaactcacaggtggaggtcagctgggagtaccctgactcctggagcactccccattcctacttctcc ctcaagttctttgttcgaatccagcgcaagaaagaaaagatgaaggagacagaggaggggtgtaacca gaaaggtgcgttcctcgtagagaagacatctaccgaagtccaatgcaaaggcgggaatgtctgcgtgc aagctcaggatcgctattacaattcctcatgcagcaagtgggcatgtgttccctgcagggtccgatcc cggcgcgccggcggcggcggcagcggcggcggcggcagcggcggcggcggcagccgtacgagggtcat tccagtctctggacctgccaggtgtcttagccagtcccgaaacctgctgaagaccacagatgacatgg tgaagacggccagagaaaaactgaaacattattcctgcactgctgaagacatcgaccatgaagacatc acacgggaccaaaccagcacattgaagacctgtttaccactggaactacacaagaacgagagttgcct ggctactagagagacttcttccacaacaagagggagctgcctgcccccacagaagacgtctttgatga tgaccctgtgccttggtagcatctatgaggacttgaagatgtaccagacagagttccaggccatcaac gcagcacttcagaatcacaaccatcagcagatcattctagacaagggcatgctggtggccatcgacga gctgatgcagtctctgaatcataatggcgagactctgcgccagaaacctcctgtgggagaagcagacc cttacagagtgaaaatgaagctctgcatcctgcttcacgccttcagcacccgcgtcgtgaccatcaac agggtgatgggctatctgagctccgccacgcgtgctagctga (SEQ ID NO: 40) IL10 In some embodiments, the transgene encodes IL-10. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 38 or a fragment thereof. Suitably, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 38 or a fragment thereof. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of the amino acid sequence SEQ ID NO: 38 or a fragment thereof. Exemplary human Interleukin-10: MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKE SLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKS KAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 38) In some embodiments of the invention, the transgene comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 39 or a fragment thereof. Suitably, the transgene 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: 39 or a fragment thereof. In some embodiments of the invention, the transgene comprises or consists of the nucleotide sequence SEQ ID NO: 39 or a fragment thereof. ATGCCAGGCTCCGCCCTGCTGTGCTGTCTGCTGCTGCTGACCGGCATGAGGATCAGCAGAGGACAGTA CTCCCGGGAGGACAACAATTGCACCCACTTCCCTGTGGGACAGTCCCACATGCTGCTGGAGCTGCGCA CAGCTTTTTCTCAGGTGAAGACCTTCTTTCAGACAAAGGACCAGCTGGATAACATCCTGCTGACCGAC AGCCTGATGCAGGATTTCAAGGGCTACCTGGGATGTCAGGCCCTGTCCGAGATGATCCAGTTTTATCT GGTGGAGGTGATGCCTCAGGCTGAGAAGCACGGCCCCGAGATCAAGGAGCACCTGAATTCTCTGGGAG AGAAGCTGAAGACACTGCGGATGCGCCTGAGGAGATGCCACAGGTTCCTGCCTTGTGAGAACAAGTCT AAGGCCGTGGAGCAGGTGAAGAGCGACTTTAATAAGCTGCAGGATCAGGGCGTGTACAAGGCCATGAA CGAGTTCGATATCTTTATCAATTGCATCGAGGCTTATATGATGATCAAGATGAAGAGCTGA (SEQ ID NO: 39) IL15 In some embodiments, the transgene encodes IL-15. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 44 or a fragment thereof. Suitably, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 44 or a fragment thereof. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of the amino acid sequence SEQ ID NO: 44 or a fragment thereof. Exemplary human Interleukin-15: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLII LANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 44) IL18 In some embodiments, the transgene encodes IL-18. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 45 or 47 or a fragment thereof. Suitably, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 45 or 47 or a fragment thereof. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of the amino acid sequence SEQ ID NO: 45 or 47 or a fragment thereof. Exemplary human Interleukin-18 sequences: YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKC EKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKL ILKKEDELGDRSIMFTVQNED (SEQ ID NO: 45) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISAYGDSRARGKAVTISVKC EKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKL ILKKEDELGDRSIMFTVQNED (SEQ ID NO: 47) TNFα In some embodiments, the transgene encodes Tumour necrosis factor alpha (TNFα). An exemplary human TNFα for use in the present invention is UniProtKB P01375. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 14 or a fragment thereof. Suitably, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 14 or a fragment thereof. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of the polypeptide sequence SEQ ID NO: 14 or a fragment thereof. Exemplary human TNFα: MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLS LISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYS QVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEK GDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ ID NO: 14) CXCL9 In some embodiments, the transgene encodes C-X-C motif chemokine 9 (CXCL9). An exemplary human CXCL9 for use in the present invention is UniProtKB Q07325. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 15 or a fragment thereof. Suitably, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 80%, at least 90% at least 95% identical to SEQ ID NO: 15 or a fragment thereof. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of the amino acid sequence SEQ ID NO: 15 or a fragment thereof. Exemplary human CXCL9: MKKSGVLFLLGIILLVLIGVQGTPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLK NGVQTCLNPDSADVKELIKKWEKQVSQKKKQKNGKKHQKKKVLKVRKSQRSRQKKTT (SEQ ID NO: 15) IL-1β In some embodiments, the transgene encodes interleukin-1 beta (IL-1β). An exemplary human IL-1β for use in the present invention is UniProtKB P01584. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 16 or a fragment thereof. Suitably, the transgene encodes a polypeptide which comprises or consists of an amino acid sequence which is at least 80%, at least 90% or at least 95% identical to SEQ ID NO: 16 or a fragment thereof. In some embodiments of the invention, the transgene encodes a polypeptide which comprises or consists of the polypeptide sequence SEQ ID NO: 16 or a fragment thereof. Exemplary human IL-1β: MAEVPELASEMMAYYSGNEDDLFFEADGPKQMKCSFQDLDLCPLDGGIQLRISDHHYSKGFRQAASVV VAMDKLRKMLVPCPQTFQENDLSTFFPFIFEEEPIFFDTWDNEAYVHDAPVRSLNCTLRDSQQKSLVM SGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPK NYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSS (SEQ ID NO: 16) Antigenic polypeptides 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. The antigen may be patient-specific. Tumour antigen In some embodiments, the transgene encodes a tumour antigen, for example a tumour- specific antigen or a tumour-associated antigen. 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. 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. Tumour antigens are described in, for example, Lu et al. (2021) Hepatology 73: 821-832 and Wu et al. (2022) Medicine in Drug Discovery 16: 100144. Certain tumours have certain tumours antigens in abundance. Certain tumours antigens are thus used as tumours markers and can also be used in cancer therapy as tumour antigen vaccines. Similar to vaccines against pathogens, tumour vaccines consist in the delivery of inactivated cancer cells or tumour antigens (TA) in combination with adjuvants. Tumour vaccines also include DCs challenged ex vivo with TAs. Despite several years of experimentation, tumour vaccines have mostly delivered disappointing results, leading to only one tumour vaccine approved for clinical use. Identifying new vaccine delivery systems that bypass the barriers to effective cancer vaccines should enable their therapeutic applicability. The vector of the present invention may represent a valid strategy to design tumour vaccines. In some embodiments, the transgene encodes a tumour antigen which is abundant on liver metastases. In some embodiments, the transgene encodes a tumour antigen selected from carcinoembryonic antigen (CEA), TRP2, melanoma associated antigen (MAGE) family, cancer germline (CAGE) family, B melanoma antigen (BAGE-1), synovial sarcoma x breakpoint 20 (SSX-2), Sarcoma antigen (SAGE) family, LAGE1, NY-ESO-1, HER2, EGFR, MUC-1, and GAST. In some embodiments, the transgene encodes OVA. In some embodiments of the invention, the transgene comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 41 or 43 or a fragment thereof. Suitably, the transgene 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: 41 or 43 or a fragment thereof. In some embodiments of the invention, the transgene comprises or consists of the nucleotide sequence SEQ ID NO: 41 or 43 or a fragment thereof. In some embodiments, the transgene encodes TRP2. In some embodiments of the invention, the transgene comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 48 or 49 or a fragment thereof. Suitably, the transgene 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: 48 or 49 or a fragment thereof. In some embodiments of the invention, the transgene comprises or consists of the nucleotide sequence SEQ ID NO: 48 or 49 or a fragment thereof. The invention contemplates the combined use of the cytokine gene therapy of the invention and the tumour vaccine of the invention. In another aspect, the invention provides a product (e.g. a composition or kit) comprising a first vector of the invention comprising a transgene encoding a cytokine (preferably IL12) and a second vector of the invention comprising a transgene encoding a tumour antigen. In another aspect, the invention provides a product (e.g. a composition or kit) comprising a cell comprising a first vector of the invention comprising a transgene encoding a cytokine (preferably IL12); and a second vector of the invention comprising a transgene encoding a tumour antigen. In another aspect, the invention provides a product (e.g. a composition or kit) comprising a first vector of the invention comprising a transgene encoding a cytokine (preferably IL12) and a cell comprising a second vector of the invention comprising a transgene encoding a tumour antigen. In another aspect, the invention provides a product (e.g. a composition or kit) comprising a first cell comprising a first vector of the invention comprising a transgene encoding a cytokine (preferably IL12) and a second cell comprising a second vector of the invention comprising a transgene encoding a tumour antigen. The composition may be a pharmaceutical composition as disclosed herein. In another aspect, the invention provides a first vector of the invention comprising a transgene encoding a cytokine (preferably IL12) for use in therapy, wherein the first vector is administered to a subject simultaneously, sequentially or separately in combination with a second vector of the invention comprising a transgene encoding a tumour antigen. In another aspect, the invention provides a second vector of the invention comprising a transgene encoding a tumour antigen for use in therapy, wherein the second vector is administered to a subject simultaneously, sequentially or separately in combination with a first vector of the invention comprising a transgene encoding a cytokine (preferably IL12). In another aspect, the invention provides use of a first vector of the invention comprising a transgene encoding a cytokine (preferably IL12) for the manufacture of a medicament, wherein the first vector is administered to a subject simultaneously, sequentially or separately in combination with a second vector of the invention comprising a transgene encoding a tumour antigen. In another aspect, the invention provides use of a second vector of the invention comprising a transgene encoding a tumour antigen for the manufacture of a medicament, wherein the second vector is administered to a subject simultaneously, sequentially or separately in combination with a first vector of the invention comprising a transgene encoding a cytokine (preferably IL12). In preferred embodiments, the use in therapy is treatment or prevention of cancer. In another aspect, the invention provides a method of treating or preventing cancer comprising administering a first vector of the invention comprising a transgene encoding a cytokine (preferably IL12) and a second vector of the invention comprising a transgene encoding a tumour antigen to a subject in need thereof. The first vector and the second vector may be administered, for example, simultaneously, sequentially or separately. In some embodiments, the first vector and/or the second vector is administered by intravenous injection, intraportal injection or intrahepatic artery injection. In another aspect, the invention provides a cell comprising a first vector of the invention comprising a transgene encoding a cytokine (preferably IL12) and/or a second vector of the invention comprising a transgene encoding a tumour antigen. EXEMPLARY VECTORS In preferred embodiments, the vector comprises from 5’ to 3’: an MRC1 promoter, a transgene, and one or more miRNA target sequence as defined herein. In other preferred embodiments, the vector comprises from 5’ to 3’: a MRC1 enhancer, an MRC1 promoter, a transgene, and one or more miRNA target sequence as defined herein. In some embodiments, the vector comprises from 5’ to 3’: an MRC1 promoter, a transgene encoding IFNalpha, and one or more miRNA target sequence that suppresses transgene expression in hepatocytes and/or liver sinusoidal endothelial cells and/or splenic phagocytes. In some embodiments, the vector comprises from 5’ to 3’: a MRC1 enhancer, an MRC1 promoter, a transgene encoding IFNalpha, and one or more miRNA target sequence that suppresses transgene expression in hepatocytes and/or liver sinusoidal endothelial cells and/or splenic phagocytes. In some embodiments, the vector comprises from 5’ to 3’: a MRC1 enhancer, an MRC1 promoter, a Kozak sequence, a transgene encoding IFNalpha, a WPRE, and one or more miRNA target sequence that suppresses transgene expression in hepatocytes and/or liver sinusoidal endothelial cells and/or splenic phagocytes. In some embodiments, the vector comprises from 5’ to 3’: a nucleotide sequence which is at least 70% identical to SEQ ID NO: 31 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 34 or a fragment thereof; and a nucleotide sequence which is at least 70% identical to SEQ ID NO: 36 or a fragment thereof. In some embodiments, the vector comprises from 5’ to 3’: a nucleotide sequence which is at least 70% identical to SEQ ID NO: 32 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 31 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 34 or a fragment thereof; and a nucleotide sequence which is at least 70% identical to SEQ ID NO: 36 or a fragment thereof. In some embodiments, the vector comprises from 5’ to 3’: a nucleotide sequence which is at least 70% identical to SEQ ID NO: 32 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 31 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 33 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 34 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 35 or a fragment thereof; and a nucleotide sequence which is at least 70% identical to SEQ ID NO: 36 or a fragment thereof. In some embodiments, the vector comprises from 5’ to 3’: an MRC1 promoter, a transgene encoding IL10, and one or more miRNA target sequence that suppresses transgene expression in hepatocytes and/or liver sinusoidal endothelial cells and/or splenic phagocytes. In some embodiments, the vector comprises from 5’ to 3’: a MRC1 enhancer, an MRC1 promoter, a transgene encoding IL10, and one or more miRNA target sequence that suppresses transgene expression in hepatocytes and/or liver sinusoidal endothelial cells and/or splenic phagocytes. In some embodiments, the vector comprises from 5’ to 3’: a MRC1 enhancer, an MRC1 promoter, a Kozak sequence, a transgene encoding IL10, a WPRE, and one or more miRNA target sequence that suppresses transgene expression in hepatocytes and/or liver sinusoidal endothelial cells and/or splenic phagocytes. In some embodiments, the vector comprises from 5’ to 3’: a nucleotide sequence which is at least 70% identical to SEQ ID NO: 31 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 39 or a fragment thereof; and a nucleotide sequence which is at least 70% identical to SEQ ID NO: 36 or a fragment thereof. In some embodiments, the vector comprises from 5’ to 3’: a nucleotide sequence which is at least 70% identical to SEQ ID NO: 32 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 31 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 39 or a fragment thereof; and a nucleotide sequence which is at least 70% identical to SEQ ID NO: 36 or a fragment thereof. In some embodiments, the vector comprises from 5’ to 3’: a nucleotide sequence which is at least 70% identical to SEQ ID NO: 32 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 31 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 33 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 39 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 35 or a fragment thereof; and a nucleotide sequence which is at least 70% identical to SEQ ID NO: 36 or a fragment thereof. In some embodiments, the vector comprises from 5’ to 3’: an MRC1 promoter, a transgene encoding IL12, and one or more miRNA target sequence that suppresses transgene expression in hepatocytes and/or liver sinusoidal endothelial cells and/or splenic phagocytes. In some embodiments, the vector comprises from 5’ to 3’: a MRC1 enhancer, an MRC1 promoter, a transgene encoding IL12, and one or more miRNA target sequence that suppresses transgene expression in hepatocytes and/or liver sinusoidal endothelial cells and/or splenic phagocytes. In some embodiments, the vector comprises from 5’ to 3’: a MRC1 enhancer, an MRC1 promoter, a Kozak sequence, a transgene encoding IL12, a WPRE, and one or more miRNA target sequence that suppresses transgene expression in hepatocytes and/or liver sinusoidal endothelial cells and/or splenic phagocytes. In some embodiments, the vector comprises from 5’ to 3’: a nucleotide sequence which is at least 70% identical to SEQ ID NO: 31 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 40 or a fragment thereof; and a nucleotide sequence which is at least 70% identical to SEQ ID NO: 36 or a fragment thereof. In some embodiments, the vector comprises from 5’ to 3’: a nucleotide sequence which is at least 70% identical to SEQ ID NO: 32 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 31 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 40 or a fragment thereof; and a nucleotide sequence which is at least 70% identical to SEQ ID NO: 36 or a fragment thereof. In some embodiments, the vector comprises from 5’ to 3’: a nucleotide sequence which is at least 70% identical to SEQ ID NO: 32 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 31 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 33 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 40 or a fragment thereof; a nucleotide sequence which is at least 70% identical to SEQ ID NO: 35 or a fragment thereof; and a nucleotide sequence which is at least 70% identical to SEQ ID NO: 36 or a fragment thereof. IMMUNE CHECKPOINT INHIBITOR As used herein the term immune checkpoint inhibitor refers to a molecule, compound, antibody or drug that inhibits, blocks, prevents, reduces or downregulates the expression of, or is otherwise antagonistic to, an inhibitory checkpoint molecule. When expressed on the cell surface, an inhibitory checkpoint molecule inhibits or dampens the T-cell-mediated immune response to said cell. For example, expression of inhibitory checkpoint molecules may prevent a cell from being killed by a T-cell response. This mechanism is particularly deleterious where a cancer cell expresses inhibitory checkpoint molecules as this may allow the cancer cell to evade the host T-cell response. Accordingly, when inhibitory checkpoint molecules on tumor cells are inhibited by an immune checkpoint inhibitor, an enhanced host T-cell response against the tumor cell should occur. In some embodiments, the immune checkpoint inhibitor inhibits an inhibitory checkpoint molecule selected from the group consisting of CTLA-4 (Cytotoxic T-Lymphocyte-Associated protein 4; CD152), A2AR (Adenosine A2A receptor), B7-H3 (CD276), B7-H4 (VTCN1), BTLA (B and T Lymphocyte Attenuator; CD272), HVEM (Herpesvirus Entry Mediator), IDO (Indoleamine 2,3-dioxygenase), TDO (tryptophan 2,3-dioxygenase), KIR (Killer-cell Immunoglobulin-like Receptor), LAG3 (Lymphocyte Activation Gene-3), PD-1 (Programmed Death 1 receptor), PD-L1 (PD-1 ligand 1), PD-L2 (PD-1 ligand 2), TIM-3 (T- cell Immunoglobulin domain and Mucin domain 3), VISTA (V-domain Ig Suppressor of T cell Activation), B7-1 (CD80), B7-2 (CD86), a TGFB (Transforming growth factor beta) pathway- associated protein, Il13 (interleukin-13), IL4 (interleukin-4), FGL (Fibrinogen Like 1), TIGIT (T cell immunoreceptor with Ig and ITIM domains), CD96 (TACT protein), Ceacam-1 (Carcinoembryonic antigen related cell adhesion molecule 1), CD155 (PVR protein), CD112 (PVR-related protein 2 (PVRL2)), LGALS3 (Galectin 3) and CD47 (integrin associated protein). Combinations of check point inhibitors may also be used. In some embodiments, the TGFB pathway-associated protein is selected from the group consisting of TGFB1 (Transforming growth factor beta-1), TGFB2 (Transforming growth factor beta-2), TGFB3 (Transforming growth factor beta-3), LTBP1 (Latent Transforming Growth Factor Beta Binding Protein 1), TGFBR1 (Transforming growth factor beta receptor 1), TGFBR2 (Transforming growth factor beta receptor 2), Integrin αv, Integrin β5, Integrin β6, Integrin β8, and LRRC32 (Leucine Rich Repeat Containing 32). In some embodiments, the immune checkpoint inhibitor is an antibody. In some embodiments, the immune checkpoint inhibitor antibody is selected from the group consisting of an anti- CTLA4 antibody, an anti-PD1 antibody, an anti-PDL1 antibody, an anti-PDL2 antibody and an anti-LAG-3 antibody. In some embodiments, the immune checkpoint inhibitor is a CTLA4 inhibitor, preferably the CTLA4 inhibitor is an anti-CTLA4 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA4 antibody. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor; preferably the PD- 1 inhibitor is an anti-PD-1 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody. In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor; preferably the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the immune checkpoint inhibitor is a PD-L2 inhibitor, preferably the PD-L2 inhibitor is an anti-PD-L2 antibody. In some embodiments, the immune checkpoint inhibitor is a LAG-3 inhibitor; preferably the LAG-3 inhibitor is an anti-LAG-3 antibody. As used herein, the term "antibody" is understood as a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically bind and recognize an antigen (e.g. a cell surface marker). As used herein, the term "antibody" refers to a whole or intact antibody molecule (e.g., IgM, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA, IgD, or IgE) or any antigen-binding fragment thereof. An antibody may be a polyclonal antibody or a monoclonal antibody. Monoclonal antibodies are produced by identical immune cells (e.g. hybridomas that may be generated from the fusion of an antibody producing B-cell line and a cancerous B-cell line). A monoclonal antibody directed to a particular antigen will recognise a single specific epitope on said antigen. In contrast, polyclonal antibodies are produced from multiple non-identical cell lines and therefore recognise several different epitopes on a particular antigen. Antigen-binding fragments of an antibody include, e.g. a single chain antibody, a single chain Fv fragment (scFv), an Fd fragment, an Fab fragment, an Fab' fragment, or an F(ab')2 fragment. An scFv fragment is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived. In addition, intrabodies, minibodies, triabodies, and diabodies (see, e.g., Todorovska et al. (2001) J Immunol Methods 248(1):47-66; Hudson and Kortt (1999) J Immunol Methods 231(1):177- 189; Poljak 25 (1994) Structure 2(12): 1121-1123; Rondon and Marasco (1997) Annual Review of Microbiology 21:257-283, are also included in the definition of antibody and are compatible for use in the methods described herein. The term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant methods. Suitable methods for producing an antibody or antigen binding fragments thereof directed to a particular antigen are known in the art (see, e.g., Greenfield (2014) Antibodies: A Laboratory Manual, Second Edition 201-221). TR1 CELL INHIBITOR Type 1 regulatory (Tr1) cells are a class of regulatory T cells that participate in peripheral immunity. Tr1 cells are a subset of CD4+ T cells. Tr1 cells may regulate tolerance, and may be self or non-self antigen specific. An important natural role of Tr1 cells is to suppress tissue inflammation in autoimmunity and graft versus host disease. In some embodiments, the Tr1 cell inhibitor inhibits a molecule selected from the group consisting of Cd4, Eomes, Gzmk, Lag3, Pdcd1, Ahr, Maf, Prdm1, Ctla4 and Il10ra. In some embodiments, the Tr1 cell inhibitor is an antibody. COMBINATION The term “combination”, or terms “in combination”, “used in combination with” or “combined preparation” as used herein may refer to the combined administration of two or more agents simultaneously, sequentially or separately. The term “simultaneous” as used herein means that the agents are administered concurrently, i.e. at the same time. The term “sequential” as used herein means that the agents are administered one after the other. The term “separate” as used herein means that the agents are administered independently of each other but within a time interval that allows the agents to show a combined, preferably synergistic, effect. Thus, administration “separately” may permit one agent to be administered, for example, within 1 minute, 5 minutes or 10 minutes after the other. VARIANTS, DERIVATIVES, ANALOGUES, AND FRAGMENTS In addition to the specific proteins and nucleotides mentioned herein, the invention also encompasses variants, derivatives, and fragments thereof. 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. 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. 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. 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: ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar - charged D E K R H AROMATIC F W Y Typically, a variant may have a certain identity with the wild type amino acid sequence or the wild type nucleotide sequence. 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. 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. Sequence identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent identity between two or more sequences. Percent identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues. Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the amino acid or nucleotide sequence may cause the following residues or codons to be put out of alignment, thus potentially resulting in a large reduction in percent identity when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall identity score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local identity. However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will 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. 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). 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. Once the software has produced an optimal alignment, it is possible to calculate percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The percent sequence identity may be calculated as the number of identical residues as a percentage of the total residues in the SEQ ID NO referred to. “Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full- length polypeptide or polynucleotide. Such variants, derivatives, 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. CELLS In one aspect, the present invention provides a cell comprising the product of the invention. In some embodiments of the product of the invention, the vector(s) is comprised in a cell. In one aspect, the present invention provides a cell comprising the vector of the invention. 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. The cell may comprise the first and/or second vector (and/or, optionally, the third vector) of the invention. Method of making a cell The vector of the present invention may be introduced into cells using a variety of techniques known in the art, such as transfection, transduction and transformation. Suitably, the vector of the present invention is introduced into the cell by transfection or transduction. In one aspect, the present invention provides a method of making the cell of the invention. The method may comprise introducing the vector of the invention into the cell, for example by transfection or transduction. Suitably, the cell may be from a sample (e.g. peripheral blood, bone marrow or umbilical cord blood) isolated from a subject. The cell may be further separated from the sample by any suitable method. The cell of the present invention may be generated by a method comprising the following steps: (i) isolation of a cell-containing sample from a subject or provision of a cell-containing sample; and (ii) transduction or transfection of the cell-containing sample with the vector of the invention, to provide a population of engineered cells. The cells may be cultured prior to, or after, introducing the vector of the invention. The steps may be performed in a closed and sterile cell culture system. Hematopoietic stem/progenitor cells and differentiated cells Suitably, the cell may be a hematopoietic stem cell (HSC), a hematopoietic progenitor cell (HPC) (e.g. a myeloid/monocyte-committed progenitor cell) or a differentiated cell (e.g. a macrophage or a monocyte). Suitably, the cell may be autologous and/or allogenic to a subject. Haematopoietic stem cells (HSCs) are multipotent stem cells that may be found, for example, in peripheral blood, bone marrow and umbilical cord blood. HSCs are capable of self-renewal and differentiation into any blood cell lineage. They are capable of recolonising the entire immune system, and the erythroid and myeloid lineages in all the haematopoietic tissues (such as bone marrow, spleen and thymus). They provide for life-long production of all lineages of haematopoietic cells. Haematopoietic progenitor cells (HPCs) have the capacity to differentiate into a specific type of cell. In contrast to stem cells however, they are already far more specific: they are pushed to differentiate into their "target" cell. A difference between HSCs and HPCs is that HSCs can replicate indefinitely, whereas HPCs can only divide a limited number of times. Differentiated cells have become more specialised in comparison to a stem cell or progenitor cell. Differentiated cells includes differentiated cells of the haematopoietic lineage such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells, T-cells, B-cells and NK-cells. For example, differentiated cells of the haematopoietic lineage can be distinguished from HSCs and HPCs by detection of cell surface molecules which are not expressed or are expressed to a lesser degree on undifferentiated cells (HSCs and HPCs). Examples of suitable human lineage markers include CD33, CD13, CD14, CD15 (myeloid), CD19, CD20, CD22, CD79a (B), CD36, CD71 , CD235a (erythroid), CD2, CD3, CD4, CD8 (T), CD56 (NK). The cell of the present invention may be used for adoptive cell transfer. As used herein the term “adoptive cell transfer” refers to the administration of a cell population to a patient. The cell may be isolated from a subject, the vector of the invention may be introduced by a method described herein before the cell is administered to the patient. Adoptive cell transfer may be allogenic or autologous. By “autologous cell transfer” it is to be understood that the starting population of cells is obtained from the same subject as that to which the transduced cell population is administered. Autologous transfer is advantageous as it avoids problems associated with immunological incompatibility and is available to subjects irrespective of the availability of a genetically matched donor. By “allogeneic cell transfer” it is to be understood that the starting population of cells is obtained from a different subject as that to which the transduced cell population is administered. Optionally, the donor will be genetically matched to the subject to which the cells are administered to minimise the risk of immunological incompatibility. Alternatively, the donor may be mismatched and unrelated to the patient. Suitable doses of transduced cell populations are such as to be therapeutically and/or prophylactically effective. The dose to be administered may depend on the subject and condition to be treated, and may be readily determined by a skilled person. Producer cells and packaging cells Suitably, the cell may be a producer cell. The term “producer cell” includes a cell that produces viral particles, after transient transfection, stable transfection or vector transduction of all the elements necessary to produce the viral particles or any cell engineered to stably comprise the elements necessary to produce the viral particles. Suitable producer cells will be well known to those of skill in the art. Suitable producer cell lines include HEK 293 (e.g. HEK 293T), HeLa, and A549 cell lines. Suitably, the cell may be a packaging cell. The term “packaging cell” includes a cell which contains some or all of the elements necessary for packaging an infectious recombinant virus. The packaging cell may lack a recombinant viral vector genome. Typically, such packaging cells contain one or more vectors which are capable of expressing viral structural proteins. 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”. Suitable packaging cells will be well known to those of skill in the art. In some embodiments, the cell is genetically engineered to decrease expression of CD47 and/or HLA on the surface of the cell. In some embodiments, the cell comprises a genetically engineered disruption of a gene encoding CD47, and/or a gene encoding β2-microglobulin, and/or one or more genes encoding an MHC-I α chain. The cell may comprise genetically engineered disruptions in all copies of the gene encoding CD47. The expression of CD47 and/or HLA on the surface of the cell may be decreased such that the cell is substantially devoid of surface-exposed CD47 and/or HLA molecules. In some embodiments, the cell does not comprise any surface-exposed CD47 and/or HLA molecules. In one aspect, the present invention provides a method of making the viral vector particle of the invention. The method may comprise culturing a viral particle producer or packaging cell comprising the vector of the invention under conditions suitable for the production of the viral particles. The method may comprise: (a) introducing the vector of the invention into a viral particle producer or packaging cell, for example by transfection or transduction; and (b) culturing the cell under conditions suitable for the production of the viral particles. Such conditions will be well known to those of skill in the art. PHARMACEUTICAL COMPOSITIONS A pharmaceutical composition is a composition that comprises or consists of a therapeutically effective amount of a pharmaceutically active agent. It preferably includes a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof). In some embodiments the pharmaceutical composition is a cancer vaccine. A “cancer vaccine” is a vaccine that either treats existing cancer or prevents development of cancer. By “pharmaceutically acceptable” is included that the formulation is sterile and pyrogen free. The carrier, diluent, and/or excipient must be “acceptable” in the sense of being compatible with the vector and not deleterious to the recipients thereof. Typically, the carriers, diluents, and excipients will be saline or infusion media which will be sterile and pyrogen free, however, other acceptable carriers, diluents, and excipients may be used. Acceptable carriers, diluents, and excipients for therapeutic use are well known in the pharmaceutical art. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilising agent(s). Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like. The product, vector, inhibitor, cell, or pharmaceutical composition according to the present invention may be administered in a manner appropriate for treating and/or preventing the diseases described herein. The quantity and frequency of administration will be determined by such factors as the condition of the subject, and the type and severity of the subject's disease, although appropriate dosages may be determined by clinical trials. The pharmaceutical composition may be formulated accordingly. The product, vector, inhibitor, cell, or pharmaceutical composition according to the present invention may be administered parenterally, for example, intravenously, or by infusion techniques. The product, vector, inhibitor, cell, or pharmaceutical composition may be administered in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solution may be suitably buffered (preferably to a pH of from 3 to 9). The pharmaceutical composition may be formulated accordingly. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art. The product, vector, inhibitor, cell, or pharmaceutical composition according to the present invention may be administered systemically, for example by intravenous injection. The product, vector, inhibitor, cell, or pharmaceutical composition according to the present invention may be administered locally, for example by targeting administration to the liver. Suitably, the product, vector, inhibitor, cell, or pharmaceutical composition may be administered by intraportal injection or by intrahepatic artery injection. The pharmaceutical compositions may comprise products, vectors, inhibitors or cells of the invention in infusion media, for example sterile isotonic solution. The pharmaceutical composition may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The product, vector, inhibitor, cell, or pharmaceutical composition may be administered in a single or in multiple doses. Particularly, the product, vector, inhibitor, cell, or pharmaceutical composition may be administered in a single, one off dose. The pharmaceutical composition may be formulated accordingly. The product, vector, inhibitor, cell, or pharmaceutical composition may be administered at varying doses (e.g. measured in vector genomes (vg) per kg). The physician in any event will determine the actual dosage which will be most suitable for any individual subject and it will vary with the age, weight and response of the particular subject. The pharmaceutical composition may further comprise one or more other therapeutic agents. The product, vector, inhibitor, cell, or pharmaceutical composition may be administered in combination with one or more other therapeutic agent. The invention further includes the use of kits comprising the product, vector, inhibitor, cell, and/or pharmaceutical composition of the present invention. Preferably said kits are for use in the methods and used as described herein, e.g., the therapeutic methods as described herein. Preferably said kits comprise instructions for use of the kit components. METHODS FOR TREATING AND/OR PREVENTING DISEASE In one aspect, the present invention provides the product, vector, inhibitor, cell, or pharmaceutical composition according to the present invention for use as a medicament. In a related aspect, the present invention provides use of the product, vector, inhibitor, cell, or pharmaceutical composition composition according to the present invention in the manufacture of a medicament. In a related aspect, the present invention provides a method of administering the product, vector, inhibitor, cell, or pharmaceutical composition according to the present invention to a subject in need thereof. Suitably, the subject is a human subject. Cancer The product, vector, inhibitor, cell, or pharmaceutical composition according to the present invention may be used to prevent or treat cancer in a subject. Suitably, the subject is a human subject. In one aspect, the present invention provides the product, vector, inhibitor, cell, or pharmaceutical composition according to the present invention for use in preventing or treating cancer. In a related aspect, the present invention provides use of the product, vector, inhibitor, cell, or pharmaceutical composition according to the present invention for the manufacture of a medicament for preventing or treating cancer. In a related aspect, the present invention provides a method of preventing or treating cancer comprising administering the product, vector, inhibitor, cell, or pharmaceutical composition according to the present invention to a subject in need thereof. The subject may be afflicted with a cancer. Alternatively, the subject may be at risk of developing a cancer. The subject may have been previously determined to be at risk of developing a cancer. The increased risk may have been determined by genetic screening and/or by reviewing the subject’s family history. The subject may have been determined to express one or more genetic markers indicative of an increased risk of developing a cancer. Suitably, a person skilled in the art will be aware of genetic risk factors (e.g. genetic markers) associated with increased risk of developing a cancer. The skilled person may use any suitable method or technique known in the art to determine whether the subject has an increased risk of developing a cancer. The subject may have previously received treatment for the cancer. The subject may be in remission from the cancer. The subject may be resistant to chemotherapy. Liver metastases In some embodiments the cancer is liver cancer, for example secondary liver cancer (e.g. liver metastases). In some embodiments the subject has or is at risk of developing a secondary liver cancer (e.g. liver metastases) and the product, vector, inhibitor, cell, or pharmaceutical composition is used to prevent or treat the secondary liver cancer. In some embodiments the subject has a primary cancer (e.g. of colorectal, pancreatic or breast origin) and the product, vector, inhibitor, cell, or pharmaceutical composition is used to prevent or treat a secondary liver cancer (e.g. liver metastases). Metastasis is the development of secondary malignant growths at a distance from a primary site of cancer. Metastases most commonly develop when cancer cells break away from the main tumour and enter the bloodstream or lymphatic system. The liver is one of the most common sites for cancer metastasis, accounting for nearly 25% of all cases. The high frequency of liver involvement in metastatic disease can be explained by the different hypotheses of metastatic spread. The double blood supply of the liver by the portal vein and the hepatic artery facilitates entrapment of circulating cancer cells, according to the “mechanical or hemodynamic hypothesis”, which explains the high incidence of liver metastases in patients with gastrointestinal carcinomas. On the other hand, some primary tumours selectively target the liver as a metastatic location, according to the “seed-and-soil” hypothesis, examples are patients with uveal melanoma with a loss of chromosome 3, and patients with breast cancer with the human growth factor receptor 2 (HER-2) positivity in combination with estrogen (ER) and progesterone receptor (PR) positivity (de Ridder, J., et al., 2016. Oncotarget, 7(34), p.55368). The majority of liver metastases are carcinomas, particularly adenocarcinoma. The primary tumour may be any primary tumour, and the primary tumour may be unknown. However, most common primary tumours in patients with adenocarcinoma are from colorectal, pancreatic or breast origin (de Ridder, J., et al., 2016. Oncotarget, 7(34), p.55368). Subjects may be diagnosed with liver metastases by any suitable method known to those of skill in the art. For example, subjects may be diagnosed by CT imaging with a hepatic protocol, colonoscopy, and EGD. The product, vector, inhibitor, cell, or pharmaceutical composition of the present invention may be used for treating or preventing liver metastases in combination with any other suitable therapy. For example, in combination with surgical resection of hepatic metastases and/or chemotherapy. The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed. Preferred features and embodiments of the invention will now be described by way of non- limiting examples. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch.9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O’D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference. EXAMPLES EXAMPLE 1 RESULTS Generation of a LV platform enabling in vivo liver macrophage engineering We first generated LVs containing a putative 1.8 Kb promoter sequence obtained from the mouse mannose receptor C-type 1 (Mrc1) gene (Figure 8A). MRC1 is expressed by most macrophage subsets, including KCs, and is upregulated by alternatively activated macrophages, such as tumor-associated macrophages (TAMs). We then cloned a GFP coding sequence downstream to the Mrc1 promoter sequence (originating the Mrc1.GFP LV) and produced VSV G-pseudotyped Mrc1.GFP LV stocks (Figure 1A). Mrc1.GFP LV drove robust transgene expression in IL4-exposed (M2-like) bone marrow-derived macrophages (BMDMs) but not in LPS/IFNγ-exposed (M1-like) BMDMs (Figures S1B-E). Intravenous injection (i.v.) of Mrc1.GFP LV to immunocompromised mice resulted in GFP expression selectively in liver cells (KCs and liver sinusoidal endothelial cells, LSECs) and in some splenic cells (Mrc1-positive macrophages). We did not observe GFP expression or integrated LV copies in blood cells, bone marrow or other organs such as lung, lymph nodes, small intestine and brain (Figures 1B-1D). To further fine tune gene expression to KCs, we leveraged on microRNA (miRNA) regulation. We first used a bidirectional LV containing selected miRNA target sequences (miRT) downstream to the GFP to verify suppression of transgene expression in the off-target cell types. Four tandem copies of mirT-122-5p fully prevented GFP expression in hepatocytes while preserving it in KCs (Figures 8F-9I), whereas 4 copies of miRT-126-3p prevented GFP expression in LSECs but not in Kupffer cells (Figures 8J and 8K). Building on these results, we incorporated 4 copies each of miRT-122-5p and miRT-126- 3p downstream to GFP in the Mrc1.GFP LV generating the Mrc1.GFP.miRT LV (Figure 1A). To investigate in vivo expression of the new Mrc1.GFP.miRT LV in the presence of liver tumors, we generated experimental liver metastases by implanting either mCherry (a red fluorescent protein)-expressing MC38 colorectal cancer cells, or colorectal epithelial cancer cells derived from APCΔ716; KrasG12D; Tgfbr2-/-; Trp53R270H; Fbxw7-/- mice, here on referred as AKTPF cancer cells. We injected i.v. the Mrc1.GFP or the Mrc1.GFP.miRT LV to mice challenged with liver metastases. In agreement with our findings in tumor-free mice, the Mrc1.GFP drove GFP expression selectively in KCs, LSECs and splenic MRC1-positive macrophages, whereas in the presence of miRNA regulation (Mrc1.GFP.miRT LV), GFP expression in LSECs was virtually completely abated (Figure 1E). In both MC38- and AKTPF- derived metastatic lesions we found enhanced GFP expression in liver peri metastatic areas, indicating enrichment of transduced KCs, including bona fide monocyte-derived tumor- associated macrophages, in these areas and/or upregulation of Mrc1 promoter activity (Figures 1F and 8L). We did not observe GFP expression in other organs such as brain, small intestine, lung and lymph nodes (Figure 8M). In summary, the selective biodistribution and expression in KC of the newly developed Mrc1.GFP.miRT LV, together with its enriched expression in areas surrounding tumor lesions, support the feasibility of in vivo genetically engineering of KCs (including liver metastasis-associated macrophages) for delivery of therapeutic molecules to liver metastatic lesions. In vivo LV-engineered KCs enable rapid, sustained and well-tolerated IFNα production We then exploited engineered KCs to deliver IFNα to liver metastases. To this aim, we replaced the GFP with an IFNα DNA coding sequence in the Mrc1.GFP.miRT LV, originating the herein termed IFNα LV. In order to efficiently transduce KCs, we produced LVs based on a manufacturing process aimed to yield high-titer LV stocks with low levels of contaminants, such as plasmids and endotoxin, which could lead to bystander innate immune activation or adverse systemic effects. We then engineered KCs in vivo by injecting i.v. either the IFNα LV or an LV with the same regulatory elements but lacking transgene (herein, Control LV), to immunocompetent mice at dose ranges previously reported to target liver cells at high efficiency (Figure 2A). In mice hosting IFNα LV-engineered KCs, we observed rapid transgene output reflected by the detection of increasing concentrations of IFNα in the plasma, peaking after 3 weeks at 700-1,000 pg/ml and stabilizing thereafter between 200-700 pg/ml. These IFNα levels remained stable compared to the Control LV cohort for up to 240 days and eventually dropped to virtually undetectable levels by day 360 (Figure 2B). Integrated LV copies in the liver of IFNα LV-treated mice were lower than found in Control LV-treated mice, suggesting long-term counterselection of IFNα LV-transduced liver cells, including KCs (Figure 2C). IFNα expression by KCs decreased numbers of circulating B cells and eosinophils over time compared to Control LV-treated mice. CD4 and CD8 T lymphocytes, inflammatory and resident monocytes, neutrophils, platelets, red blood cells and hemoglobin levels were not altered compared to Control LV-treated mice (Figures 2D and 9A). To investigate if the drop in B cells may be associated to B cell activation and autoantibody production, we measured the presence of autoantibodies in the plasma of either saline (PBS), Control LV or IFNα LV-treated mice at days 52 and 366. We found no differences in the levels of autoantibodies among all the analyzed groups (Figure 9B). Furthermore, we did not observe altered levels of indicators of liver or tissue damage (i.e. alanine aminotransferase, ALT, and aspartate aminotransferase, AST) suggesting absence of hepatotoxicity in IFNα LV-treated mice (Figure 2E). To further investigate whether exogenous IFNα expression by KCs induced inflammation, tissue damage or other alterations, we performed a histopathological analysis of most relevant organs at the end of the experiment. No treatment-related abnormalities were observed in any of the analyzed compartments (Figures 2F and 9C). Taken together, these results indicate that KC-driven IFNα expression leads to robust and long-term levels of plasmatic IFNα, which at least in mice were safe and well tolerated. Gene-based enforced IFNα expression by KCs unleashes T cell activation and impairs liver metastasis growth We delivered systemically 2 different doses (1.5*10⁹ or 1.5*10¹⁰ TU/kg) of IFNα LV to engineer KCs in mice previously challenged with MC38-based experimental liver metastases (Figure 3A). In agreement with previous results, we found dose-dependent sustained levels of IFNα in the plasma (Figure 3B) as well as dose-dependent LV copies integrated in the liver (Figure 10A), and negative association between IFNα levels in the plasma and number of circulating B cells in IFNα LV-treated mice (Figure 10B). We monitored liver metastasis growth by magnetic resonance and found that both IFNα LV doses delayed tumor progression and, in 3 mice (1 for the lower and 2 for the higher dose), enabled complete response (CR) and long- term survival (Figures 3C and 3D). CR mice re-challenged with MC38 tumors displayed impaired growth suggesting induction of adaptive immune memory against tumor-associated antigens (Figure 10C). To better investigate the induction of tumor responsive T cells upon KC engineering, we delivered systemically the Control LV or the IFNα LV to syngeneic immunocompetent mice previously challenged with experimental liver metastases of MC38 cells expressing chicken ovalbumin (OVA, used as surrogate tumor antigen). In agreement with previous results, IFNα expression by KCs delayed liver metastasis growth (Figures 3E and 10D). We found that tumor specific T cells, identified by staining with a pentamer MHCI complexed with an OVA immunogenic peptide (SIINFEKL), were enriched in tumors from IFNα LV-treated compared to Control LV-treated mice (Figure 3F). Moreover, TAMs from liver metastases exposed to KC-derived IFNα showed an increased proportion of cells expressing CD11c, a marker associated with an activated or inflammatory phenotype and a lower proportion of TAMs expressing a putative M2-like phenotype (Figure 3G). These data suggest that IFNα from engineered KCs may delay tumor progression by skewing TAMs towards inflammatory phenotypes and enabling T cell activation and expansion of tumor-reactive T cell clones. To further investigate the effects of enforced IFNα expression on the tumor microenvironment we employed AKTPF CRC cells to generate experimental liver metastases. AKTPF liver metastases recapitulate some of the histopathological features of human CRC liver metastases, such as epithelial gland structures formed by CRC cells, dirty necrosis zones, fibrosis, angiogenesis and immune infiltration (Figures 10E and 10F). IFNα-expressing KCs delayed tumor progression and led to CR in 5 out of 10 treated mice in two independent experiments (Figures 3I, 3J and 10G-10I). According to our previous findings with MC38 experimental metastasis model, we observed that KC-derived IFNα skewed TAMs towards an M1-like phenotype and increased the number of tumor-infiltrating CD8 T lymphocytes (Figures 3K-3M). To further investigate the effects of exogenous IFNα expression from KCs on liver metastasis from a different origin than CRC, we challenged syngeneic mice by intrahepatic injection of KrasG12DTrp53R172H pancreatic ductal adenocarcinoma (PDAC) cells (K8484) and treated them with either Control or IFNα LV. We found strong inhibition of tumor growth, including 6 CR out of 8 treated mice in the IFNα LV group compared to the Control group (Figures 3N and 10J). Altogether, these experiments indicate that KC engineering through systemic delivery of IFNα LV results in IFNα expression from KCs, which in turn, robustly inhibits tumor growth at least in part by skewing TAM phenotypes and promoting CD8 T cell recruitment. Engineering of KCs by IFNα LV enables preferential IFNα signaling in peri metastatic areas To investigate the mechanism underlying the observed tumor response, we performed comprehensive transcriptomic analyses of AKTPF liver metastases of Control LV or IFNα LV- treated mice. We observed increased expression of interferon-stimulated genes indicating IFNα activity in the metastatic lesions of the IFNα LV cohort (Figure 11A). We then employed spatial transcriptomics to investigate, within the metastatic liver, if there were areas of preferential IFNα signaling. To this purpose, we assigned mice to three distinct cohorts: (1) Control: Control LV-treated mice, (2) Responder: IFNα LV-treated mice with reduced metastasis volume as compared to Control and, (3) Resistant: IFNα LV-treated mice with metastasis volume similar to Control (Figure 11B). Note that we could not analyze mice achieving a complete response because of the lack of tumor at the time of analysis. We then performed spatial transcriptomic on 36 mm² sections of liver containing metastatic lesions. Unsupervised clustering analysis was then performed to cluster spatial spots based on similar transcriptomics profile (Figure 11C). Spatial spots belonging to cluster 1 and 6 displayed high expression of genes associated to adenocarcinoma and were putatively assigned to liver metastasis areas. On the contrary, cluster 0, 2-5 and 7 displayed high expression of genes associated to normal liver and were putatively assigned to liver tissue (Figure 11D). We then used an unsupervised method, which weighs the nature of surrounding spatial spots (i.e. liver or metastatic lesion), to infer the putative distance from the metastatic lesion interface of each spatial spot. We grouped spatial spots, according to their relative distance from the metastasis/liver parenchyma boundary, into distinct spatial compartments comprising inner metastatic (spatial compartments A - C), front metastatic (spatial compartment D), peri metastatic (spatial compartments E - G) and intact liver areas (spatial compartment H) (Figures 4A and 11E). As expected, we found that, in all cohorts, genes belonging to biological processes or pathways related to cancer (e.g. angiogenesis, p53 pathway, epithelial to mesenchymal transition) were enriched in the metastatic areas (inner and front areas) compared to areas outside the liver metastases (peri metastatic and intact liver). In agreement with this observation, epithelial cell-associated genes such as epithelial cell adhesion molecule (Epcam), cadherin 1 (Cdh1) and villin 1 (Vil1) were highly expressed in inner and front metastatic areas. In contrast, hepatocyte-associated genes (e.g. albumin, Alb; apolipoprotein 2, Apoa2; and cytochrome p450 family 27a1, Cyp27a1) as well as gene sets belonging to liver-associated pathways (e.g. adipogenesis or bile acid metabolism) were upregulated in intact liver areas. In agreement with enhanced transgene expression from engineered KCs in areas surrounding liver metastases upon systemic Mrc1.GFP.miRT LV delivery, we found that genes associated with response to type I interferon (e.g. suppress of cytokine signaling 1, Socs1; signal transducer and activator of transcription 1, Stat1; and NLR family CARD domain containing 5, Nlrc5) were enriched in liver metastasis and peri metastatic areas of the IFNα LV cohort (responder and resistant), in agreement with the capacity of the LV platform to preferentially engineer KCs in proximity to liver metastases. Moreover, upregulation of genes related to type I interferon activity and belonging to gene ontology (GO) categories such as response to interferon gamma, response to virus, positive regulation of cytokine production and T cell activation were associated with areas of type I interferon signaling. Furthermore, in responder compared to resistant or control cohorts, genes belonging to adaptive immune activation GO categories, such as adaptive immune response (e.g. CD3 gamma subunit of TCR complex, Cd3g; CD8, Cd8a; and TCR alpha subunit, Trac) and regulation of immune effector process were upregulated in inner, front and peri metastatic areas, corresponding to sites of enhanced IFNα activity. Interestingly genes associated with antigen presentation were also highly expressed in metastatic lesions of both responder and resistant cohorts compared to control. Importantly, in the resistant cohort, but not in the control or responder, we found increased IL10 signaling in front and peri metastatic areas, suggesting that IL10 might play a role in counteracting IFNα effect in resistant mice. Of note, markers associated with exhaustion and tolerogenic phenotype of T cells, such as transforming growth factor beta 1 (Tgfb1), eomesodermin (Eomes) and granzyme k (Gzmk) were also upregulated in the resistant group in inner, front and peri metastatic areas (Figures 4B and Figures 4C). Altogether, IFNα expression by KCs was associated with selective immune activation in liver metastasis and peri metastatic areas of responder mice. However, in resistant mice, immune activation appeared dampened compared to responders and was associated with an enrichment of IL10 signaling in the metastasis/liver parenchyma boundary area. IFNα promotes APC immune activation and enhanced MHCII-restricted antigen presentation in responder mice We performed single cell transcriptomics on live cells from the same metastatic lesions assayed by spatial transcriptomics (Figure 11A). We employed an unsupervised clustering method to identify distinct cell types, such as: (1) APCs, (2) T and NK cells, (3) B cells, (4) neutrophils, (5) endothelial cells, (6) hepatocytes, (7) cancer cells, which were manually annotated based on their transcriptomics profile (Figures 12A-12D). We then focused on cells belonging to the APC cluster. We found that genes belonging to GO categories related to IFNα, IFNγ or LPS signaling were relatively enriched in all IFNα-LV treated cohorts. On the other hand, genes linked to IL10, PGE2 and IL4 signaling were upregulated in the resistant compared to the responder cohort, suggesting that these genes might play a role in inducing resistance to gene-based IFNα therapeutic activity. Genes linked to antigen presentation, i.e. MHC protein complex and antigen processing and presentation, were upregulated in the partial responder compared to resistant or control cohorts and showed the lowest expression in the control cohort (Figure 5A). We then performed a sub clustering analysis to better define cell populations and differentially expressed genes within the APC cluster. Within APCs, we found overlapping cell clusters in all three experimental cohorts, with exception of the TAM cluster, which was reshaped by IFNα treatment, indicating gene expression reprograming upon exposure to gene-based IFNα delivery. Building on this observation and considering the predominant effect of IFNα on TAM genetic program, we termed all TAMs belonging to the cluster present in IFNα LV-treated tumors IFNα-TAMs, while those present in the control LV cohort were termed TAMs. All the other cellular clusters were manually annotated based on their gene expression profile (Figures 5B and 12E). By employing differential gene expression analysis between TAM subsets, we found that genes upregulated in IFNα-TAMs compared to TAMs, for all three cohorts, were enriched in biological processes related to IFNα/IFNγ response, such as Stat1, Socs1 and Nlrc5; TNFα signaling; LPS activation; and antigen processing and presentation, such as MHC subunits (H2-D1 and H2-Ab1, Cd74), TNF- receptor superfamily 5 (Cd40), transporter-associated with antigen processing 1 (Tap1) in line with a role of IFNα-TAMs in positively regulating immune activation. Of note, Il10 and Tgfb1, both genes associated with immunosuppression, were upregulated in the IFNα LV cohort, hinting to a key role of these genes in resistant mice. On the other hand, protumoral genes commonly associated with TAM protumoral activities, such as matrix metallopeptidase 8, (Mmp8), transmembrane protein 176B (Tmem176B), triggering receptor expressed on myeloid cells 2 (Trem2) and fibronectin 1 (Fn1) were upregulated in TAMs vs IFNα-TAMs (Figures 5C and 5D). Of note, professional APCs, i.e. classical dendritic cells (cDCs) and monocyte-derived DCs (Mo DCs) were enriched in the responder cohort compared to resistant and control cohorts (Figure 5E). In agreement with this observation, we found that, in APCs from responder mice, genes associated with MHCII-restricted antigen presentation such as genes encoding for MHCII subunits (H2-Aa, H2-Ab1, H2-Eb1, H2-DMb1 and H2-Oa), MHCII transactivator (Ciita), Cd74 and Cd40 were upregulated compared to resistant or control mice. Of note, MHCI-restricted genes, such as genes encoding for MHCI subunits (H2-T22, H2-T23, H2-D1 and B2m), Tap1, Tap2, Tap binding protein (Tapbp), and proteasome S20 subunit beta 8 and 9 (Psmb8 and Psmb9) were upregulated in all IFNα LV-treated (resistant and responder) cohorts. In agreement with IL10 playing a putative role in resistant to KC-derived IFNα expression, IL10-associated genes, such as Tgfb, CCAAT enhancer binding protein beta (Cebpb), IL4 receptor (Il4r), Socs3 and C-C motif chemokine ligand 24 (Ccl24) were upregulated in APCs of the resistant compared to the responder and control cohorts (Figure 5F). Out of all the APC populations, Ccr7-expressing DCs, cDCs and KCs and Mo DCs expressed the highest levels of genes related to MHCII-restricted antigen presentation in all cohorts. Therefore, differences in expression level of genes related to MHCII-restricted antigen presentation in APCs may be, at least in part, attributed to enhanced infiltration of professional APCs, such as Mo DCs and cDCs in the responder cohort. On the other hand, upregulation of genes related to MHCI-restricted antigen presentation may be upregulated because of a direct effect of IFNα on cells (Figure 5G). In summary, IFNα released from KCs promoted APC reshaping toward an immunostimulatory phenotype through boosting antigen presenting functions. However, MHCII-restricted functions and DC infiltration appeared reduced in resistant compared to responder mice. Concomitantly, IL10 signaling was enhanced in APCs from resistant mice, supporting an association between lack of response, IL10 upregulation and impaired MHCII-restricted antigen presentation. Therapeutic response to IFNα is associated with T cell activation and is counteracted by Eomes CD4 T cell infiltration We then performed differential expression analysis in the T and NK cell compartment among the three experimental cohorts. Similar to TAMs, genes belonging to IFNα and IFNγ signaling were enriched in all IFNα-LV treated cohorts. Conversely, genes belonging to immune activation (i.e. T cell mediated cytotoxicity, natural killer cell activation or regulation of cell killing) were upregulated exclusively in the partial responder cohort (Figure 6A). We then performed an unsupervised sub clustering analysis to identify distinct cell populations within the T and NK cell compartment and manually annotated the resulting clusters. We found overlapping cell clusters in all three experimental cohorts (Figures 6B and S6A). Selectively in resistant mice, we observed a population of regulatory CD4 T cells, which transcriptionally resembled previously described Tr1 cells (here on termed Eomes CD4 T cells) (Bonnal et al. (2021) Nature immunology 22: 735-745; Roncarolo et al. (2018) Immunity 49: 1004-1019) expressing markers of CD4 T cell exhaustion, such as Ctla4, granzyme k (Gzmk), Lag3 and PD1 (Pdcd1), as well as genes associated with immune suppression such as IL10 receptor (Il10ra), Il10 and the transcription factor Eomes, and lacking expression of the transcription factor Foxp3 (Figures 6C and 13B). On the other hand, selectively enriched in the responder cohort, we observed a population of CD8 T effector 1 cells (Figure 6D). The latter displayed a transcriptomic signature resembling tissue resident effector memory T cells, which were previously associated with response to immunotherapy (Figures 6E and 13B) (Kim et al. (2021) Liver international : official journal of the International Association for the Study of the Liver 41: 764-776). Moreover, we found that IFNα released by KCs increased IFNα and IFNγ signaling on all CD8 T cell populations. Of note, genes belonging to T cell exhaustion, such as Pdcd1, Lag3, TIM3 (Havcr2), Ctla4, Eomes, thymocytes selection associated high mobility group box protein (Tox), Ccl3, Ccl4 and caspase 3 (Casp3), were downregulated in responder compared to control or resistant mice. In contrast, genes associated with adaptive immune response and T cell-mediated immunity and cytotoxicity, such as transcription factor 7 (Tcf7), T-box transcription factor 21 (Tbx21), Cd69, integrin subunit alpha e (Itgae), integrin subunit alpha 1 (Itga1), Cd7, Il2, tumor necrosis factor alpha (Tnf), and Il12a, were more upregulated in responder than in resistant mice (Figures 6E and 13C). Altogether, these data indicate that IFNα released by engineered KCs promoted adaptive immunity in responder mice by reshaping the T cell infiltrate enriching for effector phenotypes associated with response to immunotherapy while dampening T cell exhaustion. Conversely, in resistant mice, infiltrating Eomes CD4 T cells and enhanced exhaustion of CD8 T cells may prevent anti-tumor effect. IFNα from engineered KCs in combination with functional inhibition of regulatory T cells eradicates liver metastases We then investigated whether, like in mice, in human CRC liver metastases IFNα signaling was positively associated with the presence of Eomes CD4 T cells in the tumor microenvironment. To this aim, we employed bulk RNA sequencing data from human CRC liver metastases collected from our center and found that patients with high IFNα signaling score displayed higher levels of Eomes CD4 signature score (Figures 7A, 7B and 14A). We then performed immunostaining on CRC liver metastasis samples from two patients in this cohort, one with high and the other with low IFNα signaling score. We found that most CD4 T cells expressed detectable levels of LAG3 in the IFNα high group whereas CD4 T cells in the IFNα low group did not display detectable LAG3 expression (Figures 7C and 14B). Of note, LAG3 was previously reported as a marker of T cell exhaustion and also of human Tr1 cells. This observation suggests that Eomes CD4 T cells positively associate with endogenous IFNα signaling and may, at least in part, counteract immune activation in the tumor microenvironment. In mice resistant to IFNα LV, we found, concomitantly increased IL10 signaling, impaired MHCII-restricted antigen presentation, enhanced Eomes CD4 T cell infiltration and enhanced CD8 T cell exhaustion. This observation is in agreement with previous studies that indicate that IL10 may play a role in the differentiation, accumulation and effector function of Eomes CD4 T cells, which have been described to suppress antigen presentation functions, through perforin-mediated direct killing of DCs, and T cell activities in CRC liver metastases, through IL10 secretion. Building on these observations, we inhibited IL10 signaling by using a monoclonal antibody blocking IL10 receptor (a-IL10R). Mice challenged with AKTPF liver metastases and treated with IFNα or Control LVs, were treated with either aIL10R or an unrelated IgG. Anti-IL10R blocked IFNα-induced accumulation of EOMES CD4 T cells (Figure 7D), indicating that IL10 signaling is necessary for IFNα-induced accumulation of these cells in liver metastases. However, the combination of IFNα and a-IL10R achieved lower therapeutic effect than either IFNα LV or a-IL10R alone (Figure 7E), suggesting that IL10 signaling may also be necessary for the deployment of IFNα therapeutic activity. Indeed, we found that the combination of a-IL10R and IFNα LV induced the highest increase in PD1 expression on CD4 and CD8 T cells circulating in the peripheral blood (Figures 14C and 14D), a result in agreement with a role of IL10 in reinvigorating T cells and preventing their exhaustion, which may be especially necessary in case of IFNα T cell exposure. We observed that Ctla4 was expressed in Eomes CD4 T cells, exhausted CD4 and CD8 T cells and Foxp3 T regulatory (Treg) cells (Figure 13B). Furthermore, Ctla4 was strongly upregulated in CD8 T cells in the resistant mice. Of note, CTLA4 in Tr1 cells may play a key role in suppressing T cell functions as well as in attenuating antigen presentation by sequestering the costimulatory molecules CD80/CD86 in APCs. Building on this observation we combined KC-based IFNα delivery with an anti-CTLA4 blocking monoclonal antibody (a- CTLA4, Figure 7F). The combination of IFNα by KCs and a-CTLA4 strongly inhibited liver metastasis growth as compared to either treatment alone in two distinct experimental models of CRC liver metastases, the MC38 (Figures 7G and 14E) and AKTPF (Figures 7H and 14F). Remarkably, in mice hosting AKTPF liver metastases we observed up to 70% of the mice displaying complete response upon IFNα and CTLA4 combination. This result indicates that potentiating antigen presenting functions in APCs through inhibition of CTLA4 function in regulatory Eomes CD4 T cells, exhausted CD4/CD8 T cells and CD4 Treg cells strongly enhanced the therapeutic activity of IFNα LV, uncovering a major contribution of CTLA4 to development of therapy resistance. Overall, these findings demonstrate a powerful synergy between our strategy of gene-based IFNα delivery through KCs from within the tumor bed and checkpoint blockade targeting regulatory T cell functions. DISCUSSION We developed a novel LV platform to engineer KCs in proximity to liver metastases and leveraged on this strategy to deliver IFNα to CRC and PDAC liver metastasis models. KC- released IFNα led to: i) reprogramming of TAMs and infiltrating DC towards immune activation and antigen presentation; ii) increased recruitment, activation and reduced exhaustion of CD8 T cells; iii) enrichment of a CD8 T cell subpopulation with features of tissue-resident effector memory cells, previously associated with positive response to immunotherapy. This immune cell reshaping resulted in inhibition of metastases in most mice. In depth analysis of resistant mice uncovered emergence of an Eomes-expressing CD4 T cell population transcriptionally resembling Tr1 cells, which are associated with immunosuppressive and tolerogenic functions. Furthermore, APCs from resistant mice displayed increased IL10 signaling and reduced MHCII-restricted antigen presentation, whereas CD8 T cells displayed increased markers of exhaustion. Co-administration of CTLA4 blockade with IFNα LV overcame these resistance mechanisms allowing nearly complete therapeutic responses, providing the proof- of-principle of a new therapeutic strategy with potential for translation in cancer patients with severe unmet medical need. Efficient KC engineering was achieved through the preferential biodistribution of i.v. administered LV to the liver and the Mrc1 promoter and miRNA target sites incorporated in the vector, which enable selective transgene expression in KCs, especially in areas proximal to metastatic lesions. LV-based KC engineering was enriched in peri metastatic areas, possibly due to tumor-driven changes in the local vasculature undergoing remodeling and in KC phagocytic activity. Moreover, previous reports have shown that MRC1 expression by macrophages is increased in the presence of tumors. Selective exogenous expression of cytokines in KCs may limit hepatic toxicity from direct expression in hepatocytes or LSECs. Furthermore, it has been proposed that macrophages, including KCs, in the presence of tumors rewire their genetic programs to promote tumor growth and immune evasion. Therefore, expression of IFNα directly in these cells may reshape their protumoral genetic programs, leading to higher therapeutic benefit. On the other hand, KC-sourced IFNα also reached the systemic circulation, establishing sustained plasma levels. It is possible that this systemic exposure may contribute to the therapeutic benefit observed here, although previous studies only reported a prophylactic activity against metastatic seeding of recombinant type I interferon administered through an implanted mini- osmotic pump. Expression from within the tissue, through KC engineering, bypasses the biodistribution and vascular barriers of systemic administration, likely achieving more effective concentration on the target cells within the TME. Whereas systemic IFNα administration has been associated with significant toxicity in preclinical models and in humans, we did not collect evidence of tissue damage or autoimmunity in our study. This may be due to: (i) improved therapeutic index of IFNα locally produced in the liver stroma and preferential IFNα signaling in the liver areas harboring liver metastases, (ii) stable expression of IFNα compared to peak and trough dynamics in plasma from systemically delivered cytokines, which is often associated to desensitization and toxicity, (iii) plasma levels of IFNα within the physiological range and similar to those observed upon viral infections. Of note, LV-based cell engineering results in integration of the vector and sustained transgene expression. Importantly, our strategy eventually reached virtual extinction upon 1 year. Termination of expression was likely due to turnover of the engineered KCs, which appeared faster for cells expressing exogenous IFNα than for those transduced with control LV, suggesting some counter selection of the former ones. Alternatively, it might be possible to employ integrase defective (ID) LVs, which persist in the nucleus as episomal forms driving lower and more transient transgene expression. Whether employing LVs or IDLVs may thus depend on the desired transgene output in terms of level and duration. We observed a therapeutic benefit of IFNα LV in all murine models of liver metastasis tested, with superior responses in the AKPTF CRC, which better recapitulates the human disease in terms of genetic mutations and histopathological features. It is possible that, in the AKPTF model, the presence of multifocal gland-like structures, which enable closer interaction between engineered macrophages and the TME, as well as a slower tumor growth, which extends the therapeutic window, favored the therapeutic activity of IFNα LV. Despite the therapeutic activity observed upon KC engineering through IFNα LV as a single dose treatment, a population of Eomes CD4 T cells, displaying a Tr1-like gene signature, counteracted IFNα action in a fraction of resistant mice. In agreement with previous reports, we showed that Eomes CD4 T cell development depends on type I IFN and IL10 stimulation. This observation highlights the complex and sometimes opposite effects of IFNα, which in some circumstances can promote tumor growth and immune evasion. For example, in mouse models of chronic viral infection, IFNα exposure promoted myeloid-derived suppressor cell differentiation, which in turn inhibited CD8 T cell responses or promoted cancer stem cell phenotypes in mouse models of fibrosarcoma. On the other hand, enforced expression of IFNαR1 in CD8 T cells enhanced cytotoxic activity in subcutaneous MC38 mouse tumors, or restoring IFNα signaling in cancer cells led to CD8-dependent therapeutic activity in distinct syngeneic and xenograft tumor mouse models. In a similar way, depending on its target and the presence of other stimuli, IL10 can either promote or inhibit tumor immunity. For example, by acting on DCs, IL10 impairs activation and antigen presentation, whereas at the same time, by impairing DC functions, IL10 prevents DC-induced CD8 T cell apoptosis. By acting on CD8 T cells, IL10 prevents T cell exhaustion and promotes T cell invigoration in renal cell carcinoma patients and tumor mouse models. In line with these observations, we found that either IFNα LV or IL10 blockade alone promoted tumor immunity and delayed liver metastasis growth. Conversely, combination of IFNα LV and IL10 blockade did not affect liver metastasis growth. We found that exogenous IFNα released by KCs strongly upregulated genes involved in MHCI-restricted antigen presentation in distinct population of liver metastasis-infiltrating APCs, including TAMs and DCs, from both responder and resistant mice, suggesting that, at least in part, IFNα may exert its therapeutic activity through activation of antigen presentation. Interestingly, MHCII-restricted antigen presentation was inhibited in liver metastasis from resistant mice, in part due to lower infiltration of distinct populations of DCs and downregulation of genes related to MHCII-restricted antigen presentation. In agreement with this observation, MHCII-restricted antigen presentation may be necessary to maintain functional T cells in tumors, and to enable response to immunotherapy. Whether reduced MHCII-restricted antigen presentation in resistant mice is upstream or downstream to enhanced IL10 signaling as well as Eomes CD4 T cell differentiation needs to be further investigated. It has been described that Tr1 cells, as well as Tregs suppress immunity through expression of IL10 and CTLA4. CTLA4 may play a key role in reducing antigen presentation and T cell priming trough CD80/CD86 sequestering, which in turn leads to defective T cell activation. Of note, CTLA4 was highly upregulated in resistant mice in CD8 T cells as well as in Eomes CD4 T cells, suggesting that it may play an important role in preventing immune activation in the presence of high IFNα signaling. In agreement with this concept, dual intervention with IFNα LV delivery and CTLA4 blockade resulted in strong therapeutic effect, achieving complete regression of liver metastases in most mice. Our findings have clinical correlates supporting their relevance. These include the differential prognostic value of gene signatures associated with specific subpopulations of innate or adaptive immune cells described in our study, such as protumoral macrophages, CD8 tissue- resident effector memory cells, exhausted T cells, and Eomes CD4 T cells. We present evidence of a correlation between the extent of IFNα signaling and Tr1 signature score in clinical samples of liver metastatic CRC. Furthermore, we found expression of the Tr1 cell marker LAG3 in CD4 T cells infiltrating human CRC liver metastases with a high IFNα- signaling score. Of note, Eomes CD4 T cells were associated to a subgroup of patients displaying high IFNα signaling in liver metastasis and might be present at low levels in patients displaying low IFNα signaling. Therefore, although previous studies have dissected and highlighted the complexity of CD4 T cell in CRC liver metastases, only a fraction of studies have detected Tr1-like Eomes CD4 T cells infiltrating liver metastases (Bonnal et al. (2021) Nature immunology 22: 735-745). Overall, we developed a new off-the-shelf gene therapy tool that upon a single and well tolerated systemic administration engineers KCs, which in turn rapidly deliver IFNα to liver metastases from within the liver and unleash tumor immunity against liver metastases in relevant mouse models. METHODS Plasmid design To originate the Mrc1.GFP lentiviral vector (LV), we inserted a putative Mrc1 promoter sequence encompassing a 1883 bp DNA sequence (MM39 assembly: CHR2:14232425- 14234307) into a previously described PGK.GFP LV by replacing the PGK promoter sequence by using the restriction enzyme sites XhoI and AgeI. The bidirectional miRT LVs were generated by inserting four tandem copies with perfect complementarity to miR-122-5p (miRT- 122-5p: 5’- ACAAACACCATTGTCACACTCCA -3’) or to miR-126-3p (miRT-126-3p: 5’- CGCATTATTACTCACGGTACGA -3’) with randomized 4 bp DNA linker sequences separating the miRT sites. The 4 copies of the miRT sequences were then inserted downstream to the WPRE sequence of a bidirectional LV containing a minimal cytomegalovirus (mCMV) and a human phosphoglycerate kinase 1 (PGK) promoter located in opposite direction and driving the expression of a truncated low affinity nerve growth factor receptor (dlNGFR) and GFP respectively. The miRTs were inserted by employing the restriction enzyme site KpnI. The Mrc1.GFP.miRT LV was originated by inserting 4 copies of the miRT-122-5p and 4 of the miRT-126-3p downstream to the WPRE of the Mrc1.GFP LV transfer vector plasmid by using the restriction enzyme site KpnI. The IFNα LV transfer vector plasmid was created by replacing the GFP sequence of the Mrc1.GFP.miRT LV transfer vector plasmid with a cDNA encoding for the murine IFNα1 protein by using the restriction enzyme sites SalI and ScaI. The Control LV was generated by depleting the GFP sequence of the Mrc1.GFP LV transfer vector plasmid by digesting with the restriction enzymes AgeI and SalI, followed by insertion of 4 copies of miRT-122-5p and 4 copies of miRT-126-3p downstream to the WPRE by using the restriction enzyme site KpnI. Cell culture HEK293T, MC38 and K8484 cells were cultured in adherent cell culture plates in Iscove’s Modification of Dulbecco’s Modified Eagle Medium (IMDM, Corning) supplemented with 10 % fetal bovine serum (FBS; HyCloneTM), penicillin (100 IU/mL) and streptomycin (100 µg/mL). For generation of the MC38-mCherry cells expressing mCherry in virtually all cells (99.97 % of all cells), MC38 cells were transduced with a LV driving the expression of a chimeric protein formed by mCherry fused to the C terminus of the CD81 transmembrane domain from a constitutively expressed human phosphoglycerate kinase 1 (PGK) promoter. For generation of and MC38-OVA cells, MC38 cells were transduced with an LV driving the expression of full length chicken ovalbumin (OVA) from a hPGK promoter and a VCN of 2.86 was detected in MC38-OVA cells. AKTPF-organoids were cultured at 37 °C in 30 µL of phenol-red free and growth factor reduced matrigel (BD Biosciences) in a 48-well surrounded by 300 µL Advanced Dulbecco’s Modified Eagle Medium (DMEM)/F-12 medium (Thermo Fisher Scientific) supplemented with 2 % GlutaMAX™ supplement (Gibco), penicillin (100 IU/mL), streptomycin (100 µg/mL), 1 % hepes buffer solution (Gibco), 1 % N-2 supplement (Gibco), 2 % B-27 supplement (Gibco), 1 mM N- acetylcysteine- (Sigma-Aldrich) and 50 ng/mL murine epidermal growth factor (rmEGF; Gibco). To convert the AKTPF organoids into an adherent 2D cell culture, AKTPF organoids were passed two times through NSG mice and once through c57Bl6 mice. For that purpose, AKTPF organoids were transplanted into NSG mice through intrasplenic injection and recovered after four weeks. Single cells were obtained by cutting the tumor into small pieces and filtering through a 45 µm cell strainer. Then, 1,000,000 single cells were transplanted into NSG mice by intrasplenic injection. After recovery of the tumor cells, 4 weeks after tumor transplant, tumor cells were cultured in cell culture-treated plates using DMEM/F-12 medium supplemented with 10 % FBS, 2 % GlutaMAX™, penicillin (100 IU/mL) and streptomycin (100 µg/mL). After in vitro culture, 1,000,000 tumor cells were transplanted into c57Bl6 mice. Four weeks after transplant tumor cells were retrieved and put in culture as described previously. Resulting cells were cultured as described above and used in experiments employing AKTPF cells. For retrieval of bone marrow-derived macrophages (BMDMs), bone marrow was harvested from C57Bl6 mice by flushing the femur and tibia with 10 mL MACS buffer (Miltenyi Biotec). For red blood cell lysis, 1 mL of desalt water was added to the cell pellet and immediately afterwards 50 mL MACS buffer were added. Cells were cultured in macrophage culture medium composed of RPMI medium (Corning) supplemented with 10 % FBS, 2 % GlutaMAX™ Supplement (Gibco), penicillin (100 IU/mL), streptomycin (100 µg/mL) and 100 ng/mL of mouse M-CSF (Miltenyi Biotec). After seven days, 1,000,000 BMDMs were seeded into a 24 well plate and transduced with LVs at an MOI of 10. The following day macrophage culture medium was added to the cells. We added to the cell culture medium either, for M2-like polarization, 50 ng/mL of mouse IL-4 (Miltenyi) or, for M1-like polarization, 100 ng of lipopolysaccharides (LPS) from Escherichia coli O55:B5 (Sigma-Aldrich) and 5 ng/mL of mouse IFNγ (Miltenyi). Six days after induction of polarization, flow cytometry (FC) analysis was performed. LV-Production In this study, third generation VSV-G pseudotyped LVs were used. LV stocks were either produced in laboratory grade or Process development (PDL) grade. For the biodistribution study in presence of AKTPF derived LMS, CD47 depleted LVs were produced in CD47- negative HEK 293T cells. The titer of the LV stocks was measured in HEK 293T cell- transducing units (TU/mL). LV copy number determination by ddPCR From cell culture samples genomic DNA was extracted by using the Maxwell® 16 instrument (Promega) with Maxwell® 16 DNA purification kits (Promega). Genomic DNA from whole tissue samples was extracted by using the DNeasy Blood and Tissue Kit (Qiagen). LV copy number was determined using a QX200 Droplet Digital PCR System (Biorad) apparatus. The digital droplet PCR was performed according to the manufacturer’s instructions; briefly 5-20 ng of genomic DNA was added to the reaction, primers were used at a concentration of 900 nM and the detection probes at 250 nM. Droplet quantification was acquired using the BioRad QX200 Droplet Reader and analysed by using the QuantaSoft software (Biorad). For the detection of HIV genomes, the following primer and probe set was used: forward primer: 5’- TACTGACGCTCTCGACC -3’; reverse primer: 5’-TCTCGACGCAGGACTCG -3’; probe in the FAM detection channel: 5’-(FAM)-ATCTCTCTCCTTCTAGCCTC-(MGB)-3’. As normalizer for murine samples the Sema3a gene was used: forward primer: 5’- ACCGATTCCAGATGATTGGC -3’; reverse primer: 5’-TCCATATTAATGCAGTGCTTG -3’; detection probe in Hex channel: 5’-(HEX)-AGAGGCCTGTCCTGCAGCTCATGG –(BHQ-1)- 3’. As normalizer for human samples, a commercially available GAPDH expression assay was used (TaqMan™ Gene Expression Assay, Invitrogen; Hs00894322_cn). LV copies per cell were calculated by the formula: LV copies per cell =((concentration(HIV))/concentration(Normalizer)) *2 Gene expression by ddPCR For Gene expression analysis, RNA was extracted from frozen tissue using the RNeasy® Plus Mini Kit (Qiagen). Retrotranscription was performed according to the manufacturer’s instruction by using the SuperScriptTM IV VILO (Invitrogen). Five-20 ng of cDNA were used as input for the gene expression analysis. The following TaqMan™ Gene Expression Assay from Invitrogen were used: Table 1: TaqMan^TM Gene Expression Assays used in this project. Target Detection channel Reference Catalogue number number Hprt 4448491 4331182 4331182 Oas1a FAM 4331182 Data acquisition using ddPCR and analysis were performed as described for LV copy number determination (above). FC analysis and fluorescence activated cell sorting Viability of cells was assessed by using either 7AAD nuclear staining or LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (Invitrogen) according to the manufacturer’s recommendation for fixed samples. Upon single cell dissociation (see below), to prevent unspecific staining through binding of the FC receptor, we added to the cells Fc Block (BD Pharmagen). 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 the manufacturer’s recommendation. For the staining of TCRs specific for the SIINFEKL peptide loaded on MHC class I (H2kb), samples were stained with an SIINFEKL-loaded MHC class I pentamer (ProImmune) according to the manufacturer’s instruction. We used the following gating strategy to define cell populations by flow cytometry: B cells (CD45+ B220+), CD4 T cells (CD45+ CD4+), CD45- cells in the bone marrow (CD45-), CD8 T cells in the tumor (CD45+ TCRb+ CD8+), CD8 T cells in the blood (CD45+ B220- CD8+), CD86+ TAMs (CD45+ CD11b+ F4/80+ CD86+), DCs in the liver (CD45+ F4/80- CD11chigh), EOMES+ CD4 T cells in the tumor (CD45 + B220- CD11b- CD4+ EOMES+), granulocytes (CD45+ B220- Ly6g+), inflammatory monocytes in the blood (CD45+ CD11b+ Ly6c+ Gr1-), KCs in the liver (CD45+ F4/80+), lineage (Lin)- cells in the bone marrow (CD45+ B220- Ly6g- CD11b- MRC1-), LSECs in the liver (CD45- CD31+), M1-like TAMs (CD11b+ CD11c+ Ly6c+ F4-80+), M2-like TAMs (CD11b+ CD11c- Ly6g- F4-80+), monocytes in the blood in Nude mice (CD45+ Ly6g- CD11b+ MRC1-), monocytes in the blood (CD45+ CD11b+ Gr1-), monocytes in the bone marrow (CD45+ CD11b+), monocytes in the liver (CD45+ CD11b+ F4/80- CD11c-), monocytes in the lung (CD45 Ly6g- CD11b+ MRC1-), monocytes in the spleen (CD45+ Ly6g- CD11b+ MRC1-), MRC1 macrophages in the spleen (CD45+ Ly6g- CD11b- MRC1+), MRC1 monocytes in the spleen (CD45+ Ly6g- CD11b+ MRC1+), MRC1 macrophages in the spleen (CD45+ Ly6g- CD11b- MRC1+), MRC1 monocytes in the blood (CD45+ Ly6g- CD11b+ MRC1+), MRC1 macrophages in the lung (CD45+ Ly6g- CD11b+ MRC1+), neutrophils in the blood (CD45+ CD11b+ Ly6c+ Gr1+), parenchymal cells in the lung (CD45-), parenchymal cells in the liver (CD45- CD31/MRC1-), Pentamer CD8 T cells in the tumor (CD45+ CD8+ Pentamer+), resident monocytes in the blood (CD45+ CD11b+ Ly6c- Gr1-), and T cells in the blood (CD45+ CD11b- CD3+). For FC analysis, we used the following antibodies: Table 2: Antibodies used for flowcytometry. Target Provider Clone Target localisation CD11b Biolegend Rat Membrane CD11c Biolegend N418 Hamster Membrane MRC1 BD Pharmagen Rat Membrane dlNGFR BD Pharmagen C40-1457 Mouse Membrane P^DL1 Biolegend Rat Membrane PD1 Biolegend 29F.1A12 Rat Membrane CD4 BD Horizon Rat Membrane C^D45 Biolegend 30-F11 Rat Membrane B220 Biolegend Rat Membrane B220 BD Pharmagen RA3-6B2 Rat Membrane C^D86 Biolegend Rat Membrane CD8a BD Pharmagen Rat Membrane F4/80 Biolegend Rat Membrane Ly6c eBioscience HK1.4 Rat Membrane Ly6c Biolegend Rat Membrane Ly6g Biolegend 1A8 Rat Membrane L^y6g Biosciences Rat Membrane T_{CR ^} Biosciences H57-597 Hamster Membrane C^D3 Biolegend Rat Membrane EOMES Biolegend W17001A Rat Intracellular Samples were acquired by using either a FACSCanto II or a FACSymphony™ A5 Cell Analyzer (BD Biosciences). For fluorescence activated cell sorting a BD FACSAria Fusion was used. Mouse strains C57Bl/6N mice (in all experiments performed using IFNα LV or Control LV), NUDE mice (if not indicated differently, in all experiments using Mrc1.GFP LV or Mrc1.GFP.miRT LV) or NSG 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: 1007, 1098, 1108 and 1227) and authorized by the Italian Ministry of Health and local authorities according to the Italian law. For endpoint analysis, mice were euthanized by cervical dislocation. The liver was perfused by injecting 10 mL of PBS containing 5 mM EDTA (Invitrogen) through the inferior vena cava and cutting the portal vein to allow exiting of the solution containing most circulating blood cells from the liver. When FC analysis but not immunofluorescence (IF) analysis was performed, 10 mL of IMDM (Corning) containing 0.35 mg/mL collagenase (Sigma-Aldrich) was injected through the inferior vena cava. LV intravenous injection 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 300 µL. For intravenous injection (i.v.), mice were warmed under an infrared/red-light lamp and the LVs were delivered in the tail vein. If not indicated otherwise, LVs were delivered 7 days after tumor placement. In experiments employing K8484, LVs were delivered 3 days after tumor engraftment. If not indicated differently, IFNα LV and Control LV were used at 5*10⁹ TU/kg; If not indicated differently, bidirectional LVs (i.e. miRT LVs), Mrc1.GFP LV and Mrc1.GFP.miRT LV were used at 1.5*10¹⁰ TU/kg. In experiments involving MC38.OVA cells, IFNα LV and Control LV were delivered 3 days after tumor engraftment at 1.5*10¹⁰ TU/kg. In all experiments untransduced (UT) mice were injected with PBS ranging from 200 to 300 µL. Monoclonal antibody injection Monoclonal antibodies were injected by an intraperitoneal injection in 100 µL diluted in PBS. The doses were: In-vivoMAB anti-mouse IL-10R (a-IL10R, BioXCell) as well as the isotype control In-vivoMAB rat IgG1κ anti-horseradish peroxidase (BioExcell) 1 mg/mouse in the first injection followed by injection of 0.5 mg/mouse every 4 days; In-vivoMAB anti-mouse CTLA4 (a-CTLA4, BioXCell) and the isotype control In-vivoMAB polyclonal serum hamster (BioXCell) were injected at 0.1 mg/mouse 3 times per week. Monoclonal antibody treatment regimens were started concomitantly with LV delivery. Mouse experimental liver metastasis models We delivered AKTPF organoids or AKTPF cells by intrasplenic injection. Briefly, mouse fur was removed at the left upper flank of the mice by shaving followed by application of hair removal cream (Balea). Immediately prior to surgery, mice were injected with 50 µL carprofen (2.5 mg/mL) for pain management. Isoflurane (Iso-Vet) at a concentration of 3 % in flow of oxygen at 1.5 L/min was used to anesthetize the mice during surgery. AKTPF cells were resuspended in 50 µL of either Matrigel (BD Biosciences) for the AKTPF organoids or Geltrex (Thermo Scientific) and carefully injected into the spleen using a precooled syringe. For the injection of AKTPF cells from organoids, the latter were split two days prior to the injection. AKTPF cells from organoids or from 2D cultured AKTPF cells were dissociated into single cells and 30,000 cells per mouse were injected. The peritoneum wall was sutured by using adsorbable stitches while the skin was closed by applying stainless steel wound clips. 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. For intrahepatic transplantation of MC38 and K8484 cells, the fur in the abdominal area of the mice was removed as described above. Pain management, anesthesia and surgery procedures were conducted as above. Single cells were obtained from cultured cancer cells and washed in PBS. We then injected 500,000 cells for MC38-mCherry and MC38-OVA cells, and 100,000 cells for MC38 and K8484 cells. Cells were injected in 5 µL PBS preferentially in the left liver lobe. Following surgery, mice were given antibiotic therapy as described above. Liver metastasis growth was measured by using magnetic resonance imaging (MRI), as described below, or 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). Magnetic resonance imaging analysis for liver metastasis volume assessment A 7-Tesla preclinical scanner (Bruker, BioSpec 70/30 USR, Paravision 6.0.1), equipped with 450/675 mT/m gradients (slew-rate: 3400-4500T/m/s; rise-time 140µs) and a circular polarized mouse body volume coil with an inner diameter of 40 mm was used. During acquisition, mice were kept in anesthesia by inhaling isoflurane (Iso-Vet) at a concentration of 3 % in flow of oxygen at 1.5 L/min under a dedicated temperature control apparatus to prevent hypothermia. The breathing rate and the body temperature were continuously monitored (SA Instruments, Inc., Stony Brook, NY, USA). To aid liver lesion visualization, we used a hepatocyte-specific contrast agent, the Gd-EOB-DTPA (Bayer Schering Pharma), at 0.05 μmol/g of body weight. Axial fat-saturated T2-weighted images (RARE-T2, Rapid Acquisition with Relaxation Enhancement, TR = 3000 ms, TE = 40 ms, voxel-size = 0.125 × 0.100 × 0.8 mm, averages = 4,) and axial fat-saturated T1-weighted sequences (RARE-T1: TR = 540 ms, TE = 7.2 ms, voxel size = 0.125 × 0.100 × 0.8 mm, averages = 4) were acquired during the hepatobiliary phase of Gd- EOB-DTPA enhancement (10 minutes after administration). Volume measurement was performed by using the Medical Image Processing, Analysis, and Visualization (MIPAV) software. Subcutaneous injection of MC38 cells One-million MC38 cells were injected subcutaneously into the flank of mice in a volume of 100 µL of 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: Volume=3/4*π*(0.5*diameter(x))^2*0.5*diameter(y)/2 Blood collection and analysis Blood was withdrawn either from the tail vein or the retroorbital vein plexus. Hemocytometer analysis was performed on whole blood by using the ProCyte DXTM (IDEXX). For FC analysis, red blood cell lysis was performed using the Red Blood Cell Lysis Buffer Hybri-MaxTM (Sigma). To retrieve absolute numbers of hematopoietic cell populations, the percentage of cells out of CD45+ cells, identified by using FC, was multiplied by the absolute count of white blood cells detected by hemocytometer analysis. For the collection of plasma, blood collected in Microvette® (Sarstedt) tubes was centrifuged at 3,000 rpm for 10 minutes at room temperature and precipitated red and white blood cells were discarded. For the collection of blood serum, blood collected in a conventional Eppendorf tube was incubated at room temperature for 40 minutes, and then centrifuged at 3,000 rpm for 10 minutes at room temperature. The fraction of containing platelets, red and white blood cells was discarded. Quantification of IFNα content in the blood was performed on plasma using the Mouse IFN Alpha All Subtypes ELISA KIT High Sensitivity (pbl Assay Science) according to the manufacturer’s instructions. For assessment of transaminases in the serum, ALT (Instrumentation Laboratory) and AST (Instrumentation Laboratory) quantification kits were used with an International Federation of Clinical Chemistry and Laboratory Medicine– optimized kinetic ultraviolet (UV) method in an ILab Aries chemical analyzer (Instrumentation Laboratory). In parallel, SeraChem Control Level 1 and Level 2 (#0018162412 and #0018162512) were analyzed as quality control. The quantification of autoreactive antibodies was performed on blood serum. Autoantigen microarrays were manufactured in the Microarray & Immune Phenotyping core Facility of University of Texas Southwestern Medical Center, Dallas, TX, USA. A selection of 120 autoantigens was made based on published literature, prior known autoantibodies in various immune related disease, cancer, allergic disease etc.8 positive control proteins (Ig control 1:2, Ig control 1:4, Ig control 1:8, Ig control 1:16, anti-Ig control 1:2, anti-Ig control 1:4, anti-Ig control 1:8, anti-Ig control 1:16) were also imprinted on the arrays as positive controls. Mouse serum samples were first treated with DNAse I to remove free-DNA and then applied onto autoantigen arrays with 1:50 dilution. The autoantibodies binding to the antigens on the array was detected with cy3-labeled anti-mouse IgG and cy5-labeled anti-mouse IgM, and the array slides were scanned with Genepix 4400A scanner with laser wavelengths 532nm for cy3 and 635nm for cy5 to generate Tiff images. Genepix Pro 7.0 software is used to analyze the images and generate the genepix report (GPR) files (Molecular Devices, Sunnyvale, California, USA). The net fluorescent intensity (NFI) of each antigen was generated by subtracting the local background and negative control (Phosphate buffered saline or simplified as PBS) signal. The NFI were normalized by the absolute amount of IgGs detected in each sample (based on the 1:2 anti-Ig control). Then we normalized each individual value by the average detected across all experimental mice included in this study (excluding the positive control) resulting in a value describing the fold change compared to the average. Processing of organs for FC analysis For FC analysis, organs were cut into small pieces and incubated with a tissue digestion solution composed of 1 mL IMDM (Corning) supplemented with 0.35 mg/mL collagenase type IV (from Clostridium histolyticum, Sigma-Aldrich), 1 mg/ml Dispase II (Gibco) and 0.2 mg/ml DNAse (Roche) were added. Tissue digestion solution was then incubated at 37 °C under agitation at 350 rpm for 10 min. The tissue was then further dissociated by pipetting and filtered using 0.4 µm cell strainers (Corning). Processing of organs for imaging For IF, tissues were incubated in a paraformaldehyde solution 4 % in PBS (PFA; ChemCruz®) for 4-12 hours (according to tissue size) at 4 °C. Afterwards, the PFA was exchanged for a solution of 10% sucrose (Sigma-Aldrich) and 0.02% NaN₃ in H₂O. After 8 - 15 h incubation at room temperature, sucrose solution was increased to 20%, and to 30% after additional 8 - 15 h. The organ was then embedded into Killik, O.C.T. Compound embedding medium for cryostat (Bio-Optica). Sections of 20 mm thickness were prepared and placed on glass slides using a cryostat. Sections were dried for 30 minutes at room temperature. For antigen retrieval, slides were incubated for 20 minutes in a 95 °C preheated water bath in the following solutions: (1) low pH antigen retrieval: 10 mM citric acid in H2O, pH adjusted to pH 6; (2) high pH antigen retrieval: 10 mM Tris base and 1mM EDTA plus 0.05 % tween in H2O, pH adjusted to pH 9. Slides were then cooled down in the indicated solution for 15 minutes at room temperature and then slides were washed with PBS 3 times. Blocking was performed by using a blocking buffer composed of 5 % normal donkey serum, 1 % BSA (Sigma-Aldrich) and 0.3 % TritonTM X-100 (Sigma) in PBS. For staining with mouse primary antibodies, mouse on mouse IgG blocking solution (Vector Laboratories) was added to the blocking buffer according to the manufacturer’s instructions. After 1h of blocking at RT, the blocking buffer was replaced by blocking buffer containing the indicated concentrations of primary antibodies and incubated overnight at 4 °C. The sections were then washed with washing buffer (PBS containing 0.3 % TritonTM X-100) for 5 times. Sections were stained with the secondary antibodies in blocking buffer at the indicated concentrations. An incubation for 1 h at room temperature in the dark was performed followed by 6 washing steps with washing buffer. For staining of the nuclei, sections were covered with a 1/2000 dilution of Hoechst 33342 solution (life technolog) in PBS for 2 min. Slides were washed additional 3 times with PBS and mounted using Fluoromount- G® (SouthernBiotech). Images were acquired using an SP8 lightning confocal microscope (Leica Microsystems) at a 10x or 20x magnification. The antibody combinations of primary and secondary antibodies and antigen retrieval protocols are indicated in the table below. For the histopathologic evaluation of side effects, the indicated organs were collected from mice after euthanasia and fixed in 10% buffered formalin, embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin (H&E) following OECD Good Laboratory Practices principles, principles of data integrity and applicable GLP SR-TIGET SOPs. Histopathological changes were evaluated by an experienced pathologist and graded on a scale of 1 to 5 as minimal (1), mild (2), moderate (3), marked (4), or severe (5); minimal referred to the least extent discernible and severe the greatest extent possible. The slides were independently reviewed by an experienced pathologists and a consensus reached on the findings and scores. IF analysis of human livers containing CRC-LMS was performed on formalin-fixed and paraffin-embedded (FFPE) tumor specimens with antibodies against human antigenes LAG3 (Biotechne; clone: 874512; Host: Mouse) and CD4 (Roche-Ventana; clone: SP35; Host: Rabbit), followed by donkey anti-mouse Alexa 555 and goat anti-rabbit Alexa 488 secondary antibodies as described above. Representative images were captured with a Nikon 80i Eclipse fluorescence microscope at a 500x magnification. For preparation of H&E staining of human and murine livers containing metastases, samples were fixed in 10% buffered formalin, embedded in paraffin wax, sectioned, and stained with H&E. Slides were analyzed by an experienced pathologist and were digitalized with scanner Leika Aperio Scanscope XT at 20x magnification. Table 3: Antibodies and antibody combinations used for IF staining. Single-cell RNA (scRNA) sequencing Immediately after perfusing the livers, liver metastases were isolated and dissociated into single cells as described above. Single cells were resuspended in MACS buffer containing 7AAD (BioLegend). Viable cells were sorted by gating on 7AAD negative cells. Sorted cells were further processed for scRNA sequencing. ScRNA sequencing was performed using the Next GEM Single Cell 3' GEM Kit v3.1 from Chromium 10X according to the manufacturer’s recommendation (User Guide Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1). We loaded 10,000 cells belonging to the same sample per reaction. We sequenced 8 samples, 100 bp paired-end reads in a NovaSeq 6000 Illumina apparatus, 4.75*10⁹ reads total. Base call files obtained as result from the Illumina sequencing were converted into FASTQ files and processed with the Cell Ranger Single-Cell Software Suite (10X Chromium v3.1.0) using default setting. In details, the demultiplexed samples were aligned against the murine mm10 reference genome employing the STAR aligner (producing alignment files in BAM format) and a UMI-count gene quantification was performed (based on the reference annotation). This latter gene-by-cell matrix was then imported into R and processed with the Seurat package (http://satijalab.org/seurat v4.0.3). As a first step of the analyses, doublets were assessed using the DoubletFinder (v3) software. More precisely, following the 'Best-Practices' suggested by the authors for scRNA-seq processing, the following parameters were selected to annotate doublets in each sample: Table 4: Parameters used for the DoubletFinder v3. Sample Treatment cohort nExp pK Sample 3 Control 0.07 0.005 Sample 4 Resistant 0.09 0.005 Sample 7 Control 0.07 0.005 Sample 10 Partial Responder 0.09 0.01 Sample 11 Partial Responder 0.07 0.005 S^{ample 14} Resistant 0.09 0.005 Sample 19 Control 0.05 0.005 Sample 22 Partial Responder 0.09 0.2 Samples were merged into a single Seurat dataset, using Seurat package (http://satijalab.org/seurat v4.0.3), keeping the information about the original sample as well as the corresponding treatment cohort. Then, the pre-processing step on the produced data started by removing cells with a low sequencing quality, those with a feature count below 1,000 and above 6,000, as well as cells with a fraction of mitochondrial genes higher than 10 %. Afterwards, cells annotated as doublets with the DoubletFinder (v3) were excluded from the analysis with Seurat. RNA UMI-counts were normalized using a global-scaling normalization method and the Variance Stabilizing Transformations (SCTransform) was performed to scale based on the percentage of mitochondrial genes, the absolute count of RNAs in each cell, and the difference between S and G2/M cell cycle scores computed for each cell. A principal component analysis with 50 principal components (PCs) was performed for dimensional reduction, and a UMAP-representation as well as clusters (with a resolution of 1.2) were computed on those reductions. Marker genes for each cluster were obtained using the FindAllMarkers Seurat function, and consequently clusters were annotated and manually curated, including a small population of undefined cells which was then removed from the dataset. Analysis of the subclusters “T and NK cells” and “APCs” was performed accordingly. First, T and NK cells were isolated using the subset function, then SCTransform based on the RNA-count matrix was performed, followed by a principal component analysis with 35 PCs, and cluster identification with a resolution of 0.8. At this resolution, CD4 T cells, CD8 T cells, γδ T cells, NK cells, ILCs and NKT cells were identified, as well as a population of undefined cells. CD4, CD8 and NKT cells were further refined by a sub-clustering. The number of PCs used for sub-clustering in NKT cells, CD8 T cells and CD4 T cells were 30, 35, and 35, while the resolution was 0.6, 0.3 and 0.3, respectively. Similarly, a specific analysis was performed in the APC compartment with 35 PCs and a resolution of 1.2. Cluster annotations were reintegrated into the full dataset and undefined cells were removed. SCTransform was repeated on the RNA slot and PC analysis was repeated on the full dataset as well as the subsets T and NK cells and APCs with the same parameters depicted before. Top upregulated markers of each population were calculated based on the FindAllMarkers function and a heatmap was generated based on the top 20 upregulated genes in each cluster to represent them. For the calculation of differentially expressed genes within individual clusters comparing the different treatment cohorts, namely control, partial responders and resistant, the FindMarker function was utilized. For GSEA the gene sets from https://www.gsea- msigdb.org/gsea/msigdb/genesets.jsp were used. For the gene sets extracted from Cilenti et al., (Cilenti et al. (2021) Immunity 54: 1665-1682), the genes upregulated in BMDMs stimulated with the indicated cytokine in vitro compared to unstimulated BMDMs were included in the gene set term. Furthermore, the CD8 T cell exhaustion signature, termed Exhaution_T_cells was retrieved from a previous publication (Wherry et al. (2007) Immunity 27: 670-684). Only GO terms containing a minimum of 7 and maximum of 500 overlapping genes between the GO term and genes in the data set were considered in the analysis. Codes and data for scRNA sequencing analysis are available at http://www.bioinfotiget.it/gitlab/custom/squadrito_livertumor2022 and at NCBI’s Gene Expression Omnibus https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE221360 respectively. Spatial transcriptomic analysis Immediately after perfusion of the liver, small pieces of the liver containing metastasis were shock-frozen in isopentane. Afterwards, the samples were embedded in Killik, O.C.T. Compound embedding medium for cryostat (Bio-Optica). To process the samples for Visium analysis, 10 µm sections were prepared using a cryostat and placed on the Visium slides. For that purpose, the cryostat was cooled to –16 °C. Methanol fixation and H&E staining was performed according to the manual provided by 10x Genomics under the name Methanol Fixation, H&E Staining & Imaging for Visium Spatial Protocols (10x Genomics) using a Aperio ePathology digital scanner (Leica Biosystems) for image acquisition. Afterwards, the samples were processed for Visium analysis according to the manufacturer’s instructions (10X Genomics). We sequenced 8 samples, 100 bp paired-end sequencing in a NovaSeq 6000 Illumina apparatus, 1.5*10⁹ reads total. Illumina results were analyzed by using the Space Ranger software v1.2.2. More precisely, samples were demultiplexed using the mkfastq utility (which exploits the Illumina’s bcl2fastq program) to produce initial FASTQ files. Then, starting from these latter input reads and the corresponding microscope slide image, the count step was run on each sample to perform alignment (exploiting STAR), tissue detection, fiducial detection, and barcode/UMI counting. This results in a spot-by-gene matrix, which was imported (with the corresponding tissue slide image) and analyzed with Seurat. For all samples separately a SCTransform and normalization was performed, and variable features were determined. Sample data were integrated into one object by applying the IntegrateData function, in which the anchor set was previously determined by using the FindIntegrationAnchors function with anchor features being defined by the SelectIntegrationFeatures function. Data scaling was performed on the whole dataset followed by a principal component analysis. For the generation of a UMAP plots containing cluster determination, 25 PCs were employed and a resolution of 0.1 was used. Clusters were further manually merged towards the 8 clusters based on their marker genes. Differentially expressed genes in each cluster were determined using the FindMarker function. Individual spots belonging to clusters 1 and 6 were annotated as tumor, while all the remaining ones as liver. Based on a moving average function, spots annotated as liver and tumor were divided into four zones each, leading to a classification of each spot dependent on the distance to the tumor-liver interface. For that purpose, the geographic spot matrix was converted to a binary form based on tumor and liver annotations. To define the closeness to the tumor-liver interface, the moving average for each spot assigned as tumor and liver was determined separately for the tumor and liver area with the function ma.matrix (package OLIN) using the following formular: moving average=ma.matrix[delta=3]+2*ma.matrix[delta=2] For the determination of the zones, the following thresholds were set: zone A: moving average (tumor) > 2.96; zone B: moving average (tumor) ≤ 2.96 and > 2.7; zone C: moving average (tumor) ≤ 2.7 and > 2.3; zone D: moving average (tumor) ≤ 2.3; zone E: moving average (liver) ≤ 1.7; zone F: moving average (liver) ≤ 1.91 and > 1.7; zone G: moving average (liver) ≤ 2.095 and > 1.91; zone H: moving average (liver) > 2.095. Differentially expressed genes comparing the different zones in the different treatment cohorts were calculated using the FindMarker function. GSEA determination was performed as described for scRNA sequencing as described above. Codes and data for spatial transcriptomics analysis are available at http://www.bioinfotiget.it/gitlab/custom/squadrito_livertumor2022 and at NCBI’s Gene Expression Omnibus https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE221360 respectively. Bulk RNA sequencing of samples from human LMS Total RNA was extracted from OCT-embedded samples of CRC liver metastases using AllPrep DNA/RNA Mini kit (Qiagen). RNA samples were quantified with Qubit RNA HS Assay (Life Technologies) and their integrity was assessed using High Sensitivity RNA ScreenTape Assay on 4200 TapeStation System (Agilent Technologies). Libraries were prepared using TruSeq Stranded mRNA kit (Illumina) and then sequenced 1x100bp on the Illumina NovaSeq 6000 platform. Reads were trimmed using Trimmomatic, version 0.32, in order to remove adapters and to exclude low-quality reads from the analysis. The remaining reads were then aligned to the reference human genome hg38, Gencode version 31, using the STAR aligner, version 2.5.3a. The FeatureCounts tool was used to assign exonic reads to the corresponding genes. Expression data were imported in the R statistical environment (R version 3.1.1) to be analysed. Only genes showing a counts per million (CPM) value higher than 1 in at least one sample were defined as expressed and used for the analysis. Data were shown as reads per kilobase of transcript per Million reads mapped (RPKM) and log2-transformed. The gene signatures score for TR1-up and IFNα were evaluated: for each sample, the scores of the considered signatures were defined as the average expression of the corresponding genes. Pairwise correlations of the signatures were calculated using the Pearson’s index. Correlations between individual genes were also assessed. Moreover, linear regressions were fitted to describe the relationship between each pair of signatures. Clinical trial protocol, data collection and study design was deposited in ClinicalTrials.gov with the identifier number NCT04622423 and can be retrieved at https://clinicaltrials.gov/ct2/show/NCT04622423?cond=NCT04622423&draw=2&rank=1. The sequencing data have been deposited at NCBI’s Gene Expression Omnibus repository and are accessible through accession number GSE200133. Statistical methods Comparisons between two independent groups were performed with Mann-Whitney test and, when needed, p-values were adjusted for multiple testing with Bonferroni’s correction. Comparisons between paired groups were performed, in general, with the paired Wilcoxon test. Since for n=5, the minimum achievable two-sided p-value of the test is 0.0625, a corresponding nonparametric test based on bootstrap sampling was employed for that sample size (function boot.t.test in the MKinfer R package). When comparing more than two groups with the aim of performing all pairwise comparisons, Kruskal-Wallis test was employed followed by post-hoc analysis through Dunn’s test and p-values adjusted with Bonferroni’s correction. Instead, when the aim was comparing only prespecified pairs of groups, the Mann- Whitney test was used and p-values were adjusted with Bonferroni’s correction. In case it was necessary to account in the analysis for the order of the mice in the experiment, the comparison among groups was performed with a linear model. The terms of the model corresponded to the groups and to a variable representing the order in which the mice were taken. The response variable was transformed by using the transformation log(x+0.01) in order to meet the normality assumptions of the model. The correlation among two variables was performed with Spearman’s correlation coefficient. In all analyses, the significant level was set at 0.05. All statistical analyses were performed using R 3.6.2 (http://www.R-project.org/). Figures were created using Prism 9 Version 9.3.1. FC analyses were performed using FlowJo version 10.8.1 or FCS Express Version 7.12.0007. EXAMPLE 2 RESULTS In vivo IFNα LV delivery to tumor mouse models, in combination with immune checkpoint inhibitor, results in a strong therapeutic response, enhanced antigen presentation and infiltration of CD8 T cells. Building on these observations, we decided to combine IFNα LV delivery with liver-directed tumor antigen vaccination and IL12 expression. Tumor antigen delivery to antigen presenting cells promote the expansion of T cells clones harboring tumor reactive TCRs. As tumor antigen, we employed a surrogate tumor antigen, chicken ovalbumin (OVA, amino acids 242-353), fused to mouse invariant chain, CD74, resulting in Ii.OVA. To drive IFNα, IL12 and Ii.OVA, expression from macrophages from within the liver, we employed a VSV-G pseudotyped LV, based on the Mrc1 promoter and on miRT-122 and miRT-126 regulatory sequences. We inoculated MC38 cancer cells expressing OVA (MC38.OVA) intra- liver to syngeneic mice. Three days after tumor implantation, we delivered the LVs by intravenous injection (Fig 15a). At the end of the experiment, at day 22, we sacrificed the treated mice and found that systemic delivery of either Ii.OVA + IL12 LV or Combo 3x (i.e. combination of IFNα LV + IL12 LV + Ii.OVA LV) impaired liver metastases growth compared to Ii.OVA LV alone (Fig.15b). Of note, in tumors from the Ii.OVA + IL12 LV and Combo 3x groups, we observed reduced expression of OVA mRNA compared to the Ii.OVA LV group, indicating clearance of cancer cell clones expressing OVA (Fig. 15c). Blood analysis, performed at day 15, showed expansion of anti-OVA tetramer CD8 T cells in the Ii.OVA + IL12 LV and Combo 3x groups compared to Ii.OVA LV group (Fig 15d). In MC38.OVA liver metastases, we observed enhanced infiltration of CD8 T cells, specially of tetramer-positive progenitor exhausted (PEX) CD8 T cells, which have been associated with enhanced antitumor activity and response to a-PD1, in the Combo 3x group (Fig 15 e and f). In the liver, Ii.OVA + IL12 LV and Combo 3x treatment reduced the number of tetramer-positive terminally exhausted (TEX) T cells, increased the number of tetramer-positive PEX T cells and reduced the detection of PD1 on tetramer-positive CD8 T cells (Fig 15 g – i). Building on the fact that previous studies indicate that PEX CD8 T cells efficiently respond to immune checkpoint inhibitors, we combined Combo 3x treatment with immune checkpoint inhibitors. As control group we used a Control LV, containing the same regulatory elements as the IL12, IFNα and Ii.OVA LV, but lacking a fully functional DNA coding sequence downstream of the Mrc1 promoter. Upon engraftment of MC38.OVA liver metastases in syngeneic mice, we delivered systemically, at day 7, Control LV or Combo 3x, and at days 10 and 17, a-PD1. At the end of the experiment at day 22, we sacrificed the treated mice and found that either a-PD1, Combo 3x or Combo 3x + a-PD1 impaired liver metastases growth (Fig.16b). Of note, in tumors from the treated group we observed reduced expression of OVA mRNA compared to Control LV group, indicating clearance of cancer cells expressing OVA (Fig.16c). We found that in the Combo 3x + a-PD1 group, 7 out of 9 mice completely cleared liver metastases (Fig.16d). In agreement with an effect of Ii.OVA expression in combination with IL12 LV and IFNα LV, blood analysis, performed at day 14, showed expansion of anti- OVA tetramer positive CD8 T cells in the Combo 3x and Combo 3x + aPD1 groups compared to Control LV (Fig 16e). In liver metastases, we observed enhanced infiltration of CD8 T cells, in particular tetramer-positive progenitor exhausted T cells, which have been associated with enhanced antitumor activity and response to a-PD1 (Fig 16 f and g). In the liver, in agreement with a positive regulation of aPD1 on PEX T cells, adding aPD1 to the Combo x3 group reduced the number of tetramer-positive TEX T cells, increased the number of tetramer- positive PEX T cells and reduced the detection of PD1 on tetramer-positive CD8 T cells (Fig 16 h – j). We then employed a naturally occurring tumor-associated antigen, TRP2, which is a protein naturally expressed by melanocytes, and hence, often expressed in melanoma cells, including B16 mouse melanoma cells. We originated a LV driving TRP2 (amino acids 25 - 472) fused with Ii, originating the Ii.TRP2 LV. We then engrafted B16 cells, at day 0, in the liver of syngeneic mice to simulate melanoma liver metastases, by using intraliver injection and, at day, 5 we either delivered the LVs or treated with PBS (Control group) mice as indicated. Magnetic resonance imaging (MRI) at days 13 and 19 showed reduced tumor growth in mice from the IL12 + IFNα LV group and in mice treated with IL12 + IFNα + Ii.TRP2 LV (Trp2.Combo group) compared to the Control group (Fig 17b - d). Tumor analysis showed enhanced infiltration of CD8 and CD4 T cells in IL12 + IFNα LV and Trp2.Combo LV groups (Fig 17e and f) compared to the Control group. In the liver, IL12 + IFNα LV and Trp2.Combo treatment increased PD1 expression in CD4 T cells indicating immune activation of CD4 T cells (Fig 17g). MATERIAL AND METHODS Mouse experimental liver metastasis models We delivered MC38-OVA cells or B16- by intrasplenic injection. Briefly, mouse fur was removed on the abdominal area by shaving followed by application of hair removal cream (Balea). Immediately prior to surgery, mice were injected with 50 mL carprofen (2.5 mg/mL) for pain management. Isoflurane (Iso-Vet) at a concentration of 3 % in flow of oxygen at 1.5 L/min was used to anesthetize the mice during surgery. MC38-OVA or B16-F10 cells were resuspended in 5 mL of PBS. For the injection of MC38-OVA cells, the latter were split two days prior to the injection and injected at a dose of 100,000 cells/mouse. B16-F10 cells were split two days prior to the injection and injected at a dose of 20,000 cells/mouse. The peritoneum wall was sutured by using adsorbable stitches while the skin was closed by applying stainless steel wound clips. 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 using magnetic resonance imaging (MRI), or 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). Plasmid design The following cDNA sequences were all cloned in the MRC1.miRT backbone using Bam-HI and Sal-I cloning sites. li.OVA LV: Invariant chain: Atggatgaccaacgcgacctcatctctaaccatgaacagttgcccatactgggcaaccgccctagaga gccagaaaggtgcagccgtggagctctgtacaccggtgtctctgtcctggtggctctgctcttggctg ggcaggccaccactgcttacttcctgtaccagcaacagggccgcctagacaagctgaccatcacctcc cagaacctgcaactggagagccttcgcatgaagcttccgaaatctgccaaacctgtgagccagatgcg gatggctactcccttgctgatgcgtccaatgtccatggataacatgctccttgggcctgtgaagaacg ttaccaagtacggcaacatgacccaggaccatgtgatgcatctgctcacgaggtctggacccctggag tacccgcagctgaaggggaccttcccagagaatctgaagcatcttaagaactccatggatggcgtgaa ctggaagatcttcgagagctggatgaagcagtggctcttgtttgagatgagcaagaactccctggagg agaagaagcccaccgaggctccacctaaagagccactggacatggaagacctatcttctggcctggga gtgaccaggcaggaactgggtcaagtcaccctg (SEQ ID NO: 41) OVA (242-353): Tgcatgttggtgctgttgcctgatgaagtctcaggccttgagcagcttgagagtataatcaactttga aaaactgactgaatggaccagttctaacgttatggaagagaggaagatcaaagtgtacttacctcgca tgaagatggaggaaaaatacaacctcacatctgtcttaatggctatgggcattactgacgtgtttagc tcttcagccaatctgtctggcatctcctcagcagagagcctgaagatatctcaagctgtccatgcagc acatgcagaaatcaatgaagcaggcagagaggtggtagggtcagcagaggctggagtggatgctgcca gc (SEQ ID NO: 43) li.TRP2.LV: Invariant chain: Atggatgaccaacgcgacctcatctctaaccatgaacagttgcccatactgggcaaccgccctagaga gccagaaaggtgcagccgtggagctctgtacaccggtgtctctgtcctggtggctctgctcttggctg ggcaggccaccactgcttacttcctgtaccagcaacagggccgcctagacaagctgaccatcacctcc cagaacctgcaactggagagccttcgcatgaagcttccgaaatctgccaaacctgtgagccagatgcg gatggctactcccttgctgatgcgtccaatgtccatggataacatgctccttgggcctgtgaagaacg ttaccaagtacggcaacatgacccaggaccatgtgatgcatctgctcacgaggtctggacccctggag tacccgcagctgaaggggaccttcccagagaatctgaagcatcttaagaactccatggatggcgtgaa ctggaagatcttcgagagctggatgaagcagtggctcttgtttgagatgagcaagaactccctggagg agaagaagcccaccgaggctccacctaaagagccactggacatggaagacctatcttctggcctggga gtgaccaggcaggaactgggtcaagtcaccctg (SEQ ID NO: 48) TRP2 (24-472): tttccccgagtctgcatgaccttggatggcgtgctgaacaaggaatgctgcccgcctctgggtcccga ggcaaccaacatctgtggatttctagagggcagggggcagtgcgcagaggtgcaaacagacaccagac cctggagtggcccttatatccttcgaaaccaggatgaccgtgagcaatggccgagaaaattcttcaac cggacatgcaaatgcacaggaaactttgctggttataattgtggaggctgcaagttcggctggaccgg ccccgactgtaatcggaagaagccggccatcctaagacggaatatccattccctgactgcccaggaga gggagcagttcttgggcgccttagacctggccaagaagagtatccatccagactacgtgatcaccacg caacactggctggggctgctcggacccaacgggacccagccccagatcgccaactgcagcgtgtatga cttttttgtgtggctccattattattctgttcgagacacattattaggtccaggacgcccctataagg ccattgatttctctcaccaagggcctgcctttgtcacgtggcacaggtaccatctgttgtggctggaa agagaactccagagactcactggcaatgagtcctttgcgttgccctactggaactttgcaaccgggaa gaacgagtgtgacgtgtgcacagacgagctgcttggagcagcaagacaagatgacccaacgctgatta gtcggaactcgagattctctacctgggagattgtgtgcgacagcttggatgactacaaccgccgggtc acactgtgtaatggaacctatgaaggtttgctgagaagaaacaaagtaggcagaaataatgagaaact gccaaccttaaaaaatgtgcaagattgcctgtctctccagaagtttgacagccctcccttcttccaga actctaccttcagcttcaggaatgcactggaagggtttgataaagcagacggaacactggactctcaa gtcatgaaccttcataacttggctcactccttcctgaatgggaccaatgccttgccacactcagcagc caacgaccctgtgtttgtggtcctccactcttttacagacgccatctttgatgagtggctgaagagaa acaacccttccacagatgcctggcctcaggaactggcacccattggtcacaaccgaatgtataacatg gtccccttcttcccaccggtgactaatgaggagctcttcctaaccgcagagcaacttggctacaatta cgccgttgatctgtcagaggaagaagctccagtttggtccacaactctc (SEQ ID NO: 49) IL12 single chain: atgtgtcctcagaagctaaccatctcctggtttgccatcgttttgctggtgtctccactcatggccat cgccgggcaattgatgtgggagctggagaaagacgtttatgttgtagaggtggactggactcccgatg cccctggagaaacagtgaacctcacctgtgacacgcctgaagaagatgacatcacctggacctcagac cagagacatggagtcataggctctggaaagaccctgaccatcactgtcaaagagtttctagatgctgg ccagtacacctgccacaaaggaggcgagactctgagccactcacatctgctgctccacaagaaggaaa atggaatttggtccactgaaattttaaaaaatttcaaaaacaagactttcctgaagtgtgaagcacca aattactccggacggttcacgtgctcatggctggtgcaaagaaacatggacttgaagttcaacatcaa gagcagtagcagttcccctgactctcgggcagtgacatgtggaatggcgtctctgtctgcagagaagg tcacactggaccaaagggactatgagaagtattcagtgtcctgccaggaggatgtcacctgcccaact gccgaggagaccctgcccattgaactggcgttggaagcacggcagcagaataaatatgagaactacag caccagcttcttcatcagggacatcatcaaaccagacccgcccaagaacttgcagatgaagcctttga agaactcacaggtggaggtcagctgggagtaccctgactcctggagcactccccattcctacttctcc ctcaagttctttgttcgaatccagcgcaagaaagaaaagatgaaggagacagaggaggggtgtaacca gaaaggtgcgttcctcgtagagaagacatctaccgaagtccaatgcaaaggcgggaatgtctgcgtgc aagctcaggatcgctattacaattcctcatgcagcaagtgggcatgtgttccctgcagggtccgatcc cggcgcgccggcggcggcggcagcggcggcggcggcagcggcggcggcggcagccgtacgagggtcat tccagtctctggacctgccaggtgtcttagccagtcccgaaacctgctgaagaccacagatgacatgg tgaagacggccagagaaaaactgaaacattattcctgcactgctgaagacatcgaccatgaagacatc acacgggaccaaaccagcacattgaagacctgtttaccactggaactacacaagaacgagagttgcct ggctactagagagacttcttccacaacaagagggagctgcctgcccccacagaagacgtctttgatga tgaccctgtgccttggtagcatctatgaggacttgaagatgtaccagacagagttccaggccatcaac gcagcacttcagaatcacaaccatcagcagatcattctagacaagggcatgctggtggccatcgacga gctgatgcagtctctgaatcataatggcgagactctgcgccagaaacctcctgtgggagaagcagacc cttacagagtgaaaatgaagctctgcatcctgcttcacgccttcagcacccgcgtcgtgaccatcaac agggtgatgggctatctgagctccgccacgcgtgctagctga (SEQ ID NO: 40) LV intravenous injection For systemic LV injection, LVs were diluted in PBS to obtain the desired transducing unit (TU) to be injected per mouse in a volume ranging from 200 to 300 uL. For intravenous injection (i.v.), mice were warmed under an infrared/red-light lamp and the LVs were delivered in the tail vein. IFNa LV was injected at a dose of 1*10⁸ TU/mouse while IL12 LV and li.OVA LV at a dose of 1*10⁷ TU/mouse. Flow cytometry analysis Viability of cells was assessed using LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (Invitrogen) according to the manufacturer’s recommendation for fixed samples. Upon single cell dissociation, to prevent unspecific staining through binding of the FC receptor, we added to the cells Fc Block (BD Pharmagen). 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 the manufacturer’s recommendation. For the staining of TCRs specific for the SIINFEKL peptide loaded on MHC class I (H2kb), samples were stained with an SIINFEKL-loaded MHC class I tetramer (provided by the NIH tetramer core facility). We used the following gating strategy to define cell populations by flow cytometry: B cells (LIVE/DEAD- CD45+ B220+), CD4 T cells (LIVE/DEAD- CD45+ Cd11b- B220- CD4+), CD8 T cells (LIVE/DEAD- CD45+ Cd11b- B220- CD8+), Tetramer + (Tetramer+, CD45+ CD4+ Cd11b- B220- LIVE/DEAD-), Terminally exhausted tetramer+ (EOMES+, PD1high , tetramer+, CD45+ CD4+ Cd11b- B220- LIVE/DEAD-), Progenitor exhausted tetramer+ (Tbet+, PD1int , tetramer+, CD45+ CD4+ Cd11b- B220- LIVE/DEAD-). Gene expression by ddPCR For Gene expression analysis, RNA was extracted from frozen tissue using the RNeasy® Plus Mini Kit (Qiagen). Retrotranscription was performed according to the manufacturer’s instructions by using the SuperScriptTM IV VILO (Invitrogen).5 to 20 ng of cDNA were used as input for the gene expression analysis. The following TaqMan™ Gene Expression Assay from Invitrogen were used: Table 5: TaqMan^TM Gene Expression Assays used in this project. Target Detection channel Reference Catalogue number number Hprt 4448491 OVAL 4331182 Monoclonal antibody injection Monoclonal antibodies were injected by an intraperitoneal injection in 100 mL diluted in PBS. The doses were: InVivoMAB anti-mouse PD1 (a-PD1, BioXCell) as well as the isotype control InVivoMAb Armenian hamster IgG isotype control (BioExcell) 0.2 mg/mouse at day 10 and 17 post tumor injection. EXAMPLE 3 RESULTS Combined delivery of TA, IL-12 and IFNα promotes TA presentation by APCs in liver and LM To investigate the phenotype of immune cells upon combinatorial treatment, we performed single cell RNA sequencing (scRNA-seq) on CD45⁺ cells isolated from healthy liver parenchyma and matched LMs of treated mice. For both liver and LM, we employed an unsupervised clustering method to group similar cell types, such as APCs, T and NK cells, B cells. All cells were manually annotated based on their transcriptomic profile (Figures 18A, 18B, 19A and 19B). Within the APC compartment, we identified KCs, macrophages, monocytes, granulocytes (neutrophils and basophils) and different subsets of DCs (Figures 18C and 18D). Analyzing together all APC cell types, we observed in both liver and LM that OVA.Ifna increased the expression of genes involved in MHC-I antigen presentation, such as MHC-I subunits (H2-Q7, H2-T22, H2-Q4, H2-K1, H2-M3, H2-D1) and peptide transporters (Tap1, Tap2), and of interferon stimulated genes (Ifi44, Irf7, Isg20, Oas1a, Oas1g) compared to the IiOVA group (Figures 18E and 18F). On the other hand, OVA.Il12 enhanced the expression of genes associated with MHC-II antigen presentation, such as the MHC-II subunits (H2-Eb1, H2-Ab1, H2-Aa, H2-DMb1, H2-Dma) and the MHC-II transactivator Ciita, as well as genes associated to IFNγ stimulation (Ccl5, Upp1, Slamf7, Gbp4, Cd74), compared to the IiOVA group. In agreement with a superior therapeutic effect, OVA.Combo treatment increased the expression of genes involved in both MHC-I and MHC-II antigen presentation, as well as IFNα and IFNγ signaling. Moreover, in APCs from OVA.Combo-treated animals we observed a decreased expression of genes associated with immunosuppressive and pro- tumoral functions of APCs (Vav2, Tgfb1, Il10, Ccl24, Mmp8, Tmem176B, Trem2, Fn1) in both liver and LM. We then investigated how distinct cell types among those identified in the APC compartment were affected by the treatment. On DCs, OVA.Combo and OVA.Ifna preferentially upregulated genes associated with MHC-I presentation and IFNα signaling (Figure 18G). On KCs, macrophages and monocytes, OVA.Combo and OVA.Il12 preferentially upregulated genes related to MHC-II presentation and IFNγ signaling. Conversely, in the same cell types, OVA.Combo and OVA.Il12 downregulated the expression of pro-tumoral and immunosuppressive genes. In agreement with these findings, gene-set enrichment analysis (GSEA) showed that categories related to immune activation, such as antigen processing and presentation, antigen binding, MHC-II presentation, and positive regulation of cell killing were enriched in KCs, macrophages and monocytes in the OVA.Combo group compared to IiOVA in the liver and LM (Figures 19C and 19D). In summary, simultaneous delivery of liOVA, IFNα and IL-12 promoted reprogramming of liver and tumor APCs, including KCs, macrophages and monocytes boosting their antigen presenting functions both in the context of MHC-I and II. Combined delivery of TA, IL-12 and IFNα improves the fitness of CD4+ and TA specific CD8+ T cells Within the T and NK cluster, we identified and manually annotated distinct populations of CD4⁺, CD8⁺, NK and innate lymphoid cells in liver and matched LM based on their expression profile (Figures 20A). By performing GSEA, we found that IFNα increased the expression of genes associated with negative regulation of viral replication as well as IFNα and IFNγ signaling on T and NK cells compared to the IiOVA group (Figure 20B). On the other hand, IL-12 increased the expression of genes associated with enhanced cell division and proliferation. The combination of IL-12 and IFNα in the OVA.Combo group resulted in an additive effect leading to upregulation of all these gene ontology (GO) terms. Interestingly, focusing on liver CD8⁺ T cells, OVA.Ifna increased the expression of genes associated with IFNα signaling, such as Stat1, Ly6c2, Oas1a, Irf8, Ifitm3, and PEX phenotype such as Tbx21, Klrg1, Cx3cr1, Tcf7, Klf2 and Sell, while it reduced the expression of genes associated with T cell exhaustion, such as immune checkpoint molecules (Pdcd1, Lag3, Ctla4, Havcr2, Tigit), IL-10 receptor Il10ra and the transcriptional regulator of exhaustion, Tox. On the other hand, OVA.Il12 increased the expression of genes associated with IFNγ stimulation, such as cytokine receptors (Il18rap, Il18r1, Il12rb1) and IFNγ response genes (Ifngas1, Gbp2), as well as genes associated with effector functions such as granzymes (Gzma, Gzmb), perforin (Prf1), inflammatory cytokines (Ifng, Tnf) and death-inducing signaling FasL. Genes associated with T cell exhaustion were upregulated by OVA.Il12 treatment but combination of IL-12 with IFNα in the OVA.Combo group reduced exhaustion compared to OVA.Il12 and increased the expression of genes associated with effector functions and PEX T cells compared to IiOVA alone, suggesting additive effects of these two cytokines (Figures 20C and 20D). To identify TA specific CD8⁺ T cells among CD45⁺ cells in the scRNA-seq dataset, we stained OVA specific CD8⁺ T cells with DNA-barcoded antibodies. DNA-barcoded CD8⁺ T cells were isolated independently and pooled with all CD45⁺ cells. We also employed TCR sequencing to track distinct T cell clonotypes, including OVA specific and bystander CD8⁺ T cell clones, across liver and matched LM. Most OVA specific clones were shared across the liver and the LM in all groups (Figure 21A) and enriched in the cluster of CD8⁺ Teff3, indicating that virtually all OVA specific CD8⁺ T clones were activated (Figure 20A and 20E). However, genes associated with IFNα, IFNγ, IL-15 signaling, and immune activation were upregulated in OVA specific CD8⁺ T cells in the OVA.Combo group compared to the IiOVA group (Figure 20F). In addition to this, we observed upregulation of PEX genes and downregulation of the exhaustion signature in the OVA.Combo groups compared to the liOVA group (Figures 20G and 21B). OVA.combo effects appeared additive when compared to individual cytokine delivery. These findings suggest that liOVA treatment alone can promote OVA specific CD8⁺ T cell expansion in the liver as well as their infiltration into LM. Cytokine co-delivery additionally reprograms their phenotype and favors their effector function. Interestingly, clonotype tracking of CD4⁺ T cells revealed TCR sharing between clonally expanded CD4⁺ T cells in the liver and LM only in the mice treated with either OVA.Il12 or OVA.Combo (Figure 20H). These cells were enriched in the IFNγ CD4 cluster indicating a Th1 skewed state (Figure 20A and 20I), which has been previously associated with response to immunotherapy and immune activation. They also upregulated genes associated with IFNγ stimulation (Il18rap, IL18r1, Il12rb1 and Ifng) and effector functions (Tnf, Il21, Gzmk, Fasl and Slamf1), while downregulated genes associated with T cell exhaustion and immune suppression (Figure 21C). In agreement with this observation, OVA.Il12 and OVA.Combo treatments increased the fraction of Ifng CD4⁺ T cells, while reduced the fraction of Th2- skewed Il4 CD4⁺ T cells (Figure 20J). Moreover, OVA.Il12 and OVA.Combo treatment increased the number of small, large and hyperexpanded CD4⁺ T cell clones compared to the other groups (Figure 20K). In summary, OVA.Combo therapeutic activity was associated with enriched PEX phenotype and reduced exhaustion of OVA specific CD8⁺ T cells, and increased CD4⁺ T cell clonal expansion and Th1 skewing. The higher activation and expansion of CD4⁺ T cells observed in the liver is in agreement with the enhanced MHC-II restricted antigen presentation observed in the OVA.Il12 and OVA.Combo groups compared to the other groups. Co-delivery of naturally occurring TAs with IFNα and IL-12 inhibits CRC LM growth by inducing TA specific CD8+ T cells To investigate the efficacy of our platform in a translational setting in which TAs have not been previously identified, we employed AKTPF CRC cells derived from APC^D716; Kras^G12D; Tgfbr2^– ^/–; Trp53^R270H; Fbxw7^–/– mice. To identify putative immunogenic peptides in this cell line, we performed whole exome sequencing (WES) and RNA sequencing of cultured AKTPF, AKTPF LM and reference control heathy tissues. By integrating two antigen prediction methods, antigen garnish and Pvac tools, we identified 33 putative peptides with predicted high binding affinity for the C57BL/6 H2-Kb MHC-I (Figure 22A). Of note, building on the fact that our previous results suggest that MHC-II-restricted peptides may not be necessary in the presence of IFNα + IL-12 to drive effective immune activation, we focused our pipeline on the identification of MHC-I-restricted peptides. The 33 peptides identified in our analyses were combined into a single chimeric protein fused downstream to the CD74 moiety and incorporated in the liver macrophage-targeting LV, as we did to express IiOVA and TRP-2 (Figure 22B). To reduce the possibility of generating immunogenic peptides from the junctions between different peptides, we arranged peptide order and incorporated linker sequences. We then employed the resulting LV (referred to as “TA33”) alone or in combination with LVs driving the expression of IFNα and IL-12 (“TA33.Combo”) to treat mice bearing established AKPTF LM (Figure 22C). After LV delivery we detected levels of cytokines in the plasma of TA33.Combo-treated mice in line with our previous experiments (Figure 23A). TA33.Combo treatment impaired LM growth compared to untreated control or TA33-treated mice, leading to 6 out of 9 mice completely eradicating LM (Figure 22D). TA33.Combo increased PD1 expression in circulating CD8⁺ T cells compared to control and led to a trend towards increased fraction of Ly6c⁺ CD44⁺ CD8⁺ T cells, suggesting superior activation of CD8⁺ T cells compared to TA33 treatment (Figure 23B and 23C). Compared to controls, in the livers of the TA33.Combo-treated mice we found higher infiltration of CD8⁺ T cells, which accounted for more than 20% of all CD45⁺ cells in the liver. Of note, both TA33 and TA33.Combo increased the fraction of TEX CD8⁺ T cells compared to controls. Conversely, as in the MC38.OVA and in the B16-F10 melanoma LM models, only TA33.Combo increased the fraction of CD8⁺ T cells displaying features of PEX T cells (Figure 22E). Moreover, in accordance with our previous findings, we observed that TA33.Combo increased the expression of PD-1 in CD4⁺ T cells compared to TA33 or control mice, suggesting increased activation of CD4⁺ T cells (Figure 22F). To confirm that the therapeutic efficacy observed was associated with generation of tumor specific memory CD8⁺ T cells, we performed an IFNγ ELISPOT assay on CD8⁺ T cells purified from the spleen of either control untreated or TA33.Combo-treated mice. Notably, CD8⁺ T cells isolated from all the TA33.Combo-treated mice responded to several pools of the predicted immunogenic peptides (Figure 22G). Conversely, in the control group only one of the mice analyzed showed a limited response against one of the predicted immunogenic peptides (Figure 22H). Overall, these data confirm that delivery of TA, both naturally occurring tumor- associated antigens and neoantigens, in combination with IL-12 and IFNα can induce the proliferation and expansion of TA specific CD8⁺ T cells with anti-tumor activity. Combining PD1 blockade with TA, IFNα and IL-12 LVs increases TA-specific PEX CD8+ T cells, enhancing tumor control We then investigated whether a-PD1 amplified the effects of Combo treatment in tumors that normally do not respond to a-PD1, such as B16-F10 melanoma LM. To this aim, we inoculated B16-F10 cells intrahepatically to C57BL/6 mice to create experimental LMs (Figure 24A) and then treated the mice with a-PD1 or Trp2.Combo alone or in combination. Compared to control untreated or a-PD1-treated mice, Trp2.Combo + a-PD1 treatment increased the fraction of PD1⁺ CD8⁺ T cells, effector CD62L^– CD44⁺ CD8⁺ T cells and activated Ly6c⁺ CD44⁺ CD8⁺ T cells in the blood (Figure 25A). Of note, Ly6c⁺ CD44⁺ CD8⁺ T cells negatively correlated with the tumor volume, suggesting that these cells comprise putative tumor specific CD8⁺ T cells (Figure 25B). In agreement with enhanced immune activation, both Trp2.Combo alone and Trp2.Combo + a-PD1 treatments delayed LM growth (Figures 24B and 24C). Of note, Trp2.Combo + a-PD1 treatment led to near complete tumor eradication in 3 out of 7 mice. On the contrary, a-PD1 alone did not exert a clear therapeutic effect in this model, in agreement with the scarce fraction of immune cells infiltrating these tumors in the absence of Combo treatment. The addition of a-PD1 to the Trp2.Combo treatment resulted in a further increase in both the overall fraction of CD8+ T cells and the fraction of CD8+ T cells exhibiting characteristics of PEX in the liver (Figures 24D). Moreover, both Combo treatments increased PD1 expression on CD4⁺ T cells in the liver, as previously observed in the MC38.OVA model (Figure 24E). Analysis of LM showed enhanced infiltration of CD8⁺ T cells in both Trp2.Combo and Trp2.Combo + a-PD1 groups compared to controls (Figure 24F). Both treatments also increased the fraction of CD8⁺ T cells displaying a PEX phenotype, with stronger effect by the a-PD1 addition. Moreover, CD4⁺ T cells infiltrating LM displayed a Th1-like phenotype in both Trp2.Combo groups, again with a stronger effect with a-PD1 addition (Figure 24G). Interestingly, the Trp2.Combo + a-PD1 resulted in a reduced fraction of CD4⁺ Tregs compared to a-PD1 alone. In summary, these results indicate that a-PD1 can enhance the phenotype and functionality of TA specific T cells induced by TA and cytokine LV delivery, providing proof- of-concept of a powerful new therapeutic option to cancer patients with unmet clinical need. METHODS IFNγ ELISpot Multiscreen filter plates (Millipore-Merck) were coated overnight 4°C with purified anti-mouse IFNγ mAb (clone R46A25µg/mL 50µL/well, BD-Pharmingen) and blocked with PBS 1% BSA for 2 hours at 37°C. Plates were equilibrated with culture medium for 10 minutes at room temperature before seeding cells. Splenic CD8+ T cell were negatively selected by magnetic beads sorting kit according to manufacturer’s recommendation (Miltenyi), plated (1x10⁵ cells/well) at least in duplicates in RPMI 1640 (Lonza) supplemented with 10% fetal bovine serum (FBS) (Euroclone), 100 U/mL penicillin/streptomycin (Lonza), 2 mM L-glutamine (Lonza), 0.1mM Minimum Essential Medium Non-Essential Amino Acids (MEM NEAA) (Gibco), 1 mM Sodium Pyruvate (Gibco), 50 nM 2-Mercaptoethanol (Gibco). Antigenic stimulation was provided by co-culture at ratio 1:1 with syngeneic irradiated (60Gy) EL4 cell line pulsed with the indicated peptide or peptide-pool, or not with wt irradiated EL4 cell line in the presence of 50U/mL IL2 (Proleukin, Chiron) for 42 hours at 37°C, 5% CO2. As positive control of IFNγ release, splenic CD8⁺ T cells were polyclonally stimulated with 10ng/ml of phorbol myristate acetate (PMA) and 1µM calcium ionomycin (Sigma-Aldrich). At the end of the culture detection biotinylated anti-mouse IFNγ mAb (XMG1.2 1µg/mL 50µL/well, BD- Pharmingen) was added and incubated for 2 hours at room temperature. Avidin-POD solution (Roche, 1:5,000, 50µL/well) was then added and incubated for 1 hours at room temperature. IFNγ-spots were developed by AEC solution (Sigma-Aldrich) at room temperature for 15 minutes in the dark. Plate images were acquired, and spots were counted by Immunospot S6- Ultra (Cellular Technology Limited). Data are reported as number of IFNγ-spot forming unit (SFU) in 1x10⁶ CD8⁺ T cells. Lyophilized peptides (Sigma-Merck) were dissolved in DMSO at 10mM and tested as single peptides (Table 6) or pooled at 1mM each as shown (Table 7). EL4 cells at 10⁷cell /mL were pulsed with single or pooled peptides at 5µM each for 2 hours at 37°C, 5% CO2. Table 6 Peptides employed Gene name nmer Chd4 ATVECAQL Emp3 VSGIVYIHL Table 7 Peptide list and pooling strategy employed n of peptide Gene name nmer 1 Abcb10 LSIPYGSV 2 B3gnt3 WSKYFIPTL 3 Babam1 QTHSSYSLL 4 Bcam SAPEELFVFL 5 Cdt1 (1) TVYPMSYRF 6 Cdt1 (2) MSYRFRQE 7 Cdt1 (3) VEMFHSMDTI 8 Cdh1 TQEVFEGSV 9 Cox11 RTVVYAEL 10 Rnaseh1 AAVVSKDTF 11 Gale KSLVFSSSA 12 Gpi1 INYTEDRAV 13 Greb1 CSSSLFTPL 14 Heatr5a KSLVFAALEL 15 Hmgcs1 IGVFSYGSGL 16 Lars2 VMAMSMLTL 17 Nomo1 VVLLDSTL 18 Nup62 VSFGLGSSTL 19 Romo1 TFGTFTAI 20 Scpep1 VQLWLLLL 21 Serinc3 YNYSFFHL 22 Stt3b LSAVAFSNV 23 Sugp1 STGSFPAL 24 Syne2 VLHHFALSV 25 Tm7sf3 VGFIFLGFF 26 Tm7sf3 KVFSTLFALL 27 Ufd1 VTYSKFQPQ 28 Xylt2 TAYTAFARLG 29 Ggh LIYKVYPI 30 Gsap NAIAFLNL pool 1 pool 2 pool 3 pool 4 pool 5 pool 7 1 7 13 19 25 pool 8 2 8 14 20 26 pool 9 3 9 15 21 27 pool 10 4 10 16 22 28 pool 11 5 11 17 23 29 pool 12 6 12 18 24 30 RNA sequencing RNA was extracted from pelleted cells or tissues using the miRNeasy plus mini Kit (Qiagen/74134). RNA sample from cultured AKTPF, liver metastases, healthy liver, and intestine of 3 mice were sent to Azenta for library preparation and sequencing with Illumina NovaSeq 2x150bp sequencing. Pre-processing of the input sequences was done with FastQC (v0.11.6) to assess reads quality and trimmomatic to get rid of low-quality sequences. Then, reads were aligned to the mouse genome assembly (GRCm38) using the STAR software (v2.7.6a) with standard parameters, and abundances were calculated using the Subread featureCounts function (v2.0.1). Differential Gene Expression (DGE) analysis was performed using the R/Bioconductor package DESeq2 (v1.30.0), normalizing for library size using DESeq2’s median of ratios. p-values were corrected using false discovery rate (FDR), and genes having FDR < 0.05 were considered as differentially expressed. Variant calling on RNA-Seq base editing data was performed exploiting alignments to the mouse genome assembly (GRCm38) using the STAR (v2.7.6a). Then, following the GATK "Best Practice Workflows" duplicates were marked using Picard (v2.25.6) MarkDuplicates and GATK (v4.2.0) SplitNCigarReads was used to split reads containing Ns. Variants were then called using HaplotypeCaller (with options --min-base-quality-score 20, --dont-use-soft- clipped-bases, and –standard-min-confidence-threshold-for-calling 20). Resulting variants were filtered using VariantFiltration based on their ‘QualityByDepth’ (i.e., - filter 'QD < 2.0'), mapping quality (i.e., -filter 'MQ < 40.0'), genotype quality (i.e., -filter 'GQ < 80.0'), and overall coverage ‘DP’ (i.e., -filter 'DP < 10). The final list of variants was then merged with those resulting from the WES experiment. WES Genewiz (Azenta) performed WES using the Agilent Sure Select Mouse All Exon V7 kit and Illumina NovaSeq 2x150bp sequencing, yielding approximately 10 gigabytes (100X) per sample. Data analysis was performed in accordance to GATK "Best Practice Workflows" for variant identification. Initially, FastQC (v0.11.9) was used to assess read quality and with trim- galore (v0.6.6) to trim low-quality bases. Alignment to the mouse genome assembly (GRCm38) was done employing BWA (v0.7.17). This was followed by duplicate marking using Picard (v2.25.6) MarkDuplicates. GATK (v4.2.0) BaseRecalibrator + ApplyBQSR was used to recalibrate base quality scores on dbSNP known sites. HaplotypeCaller in GVCF mode was used to call variants in each sample, and variants were merged using CombineGVCFs and genotyped with GenotypeGVCFs. Variant filtering was performed using VariantFiltration based on 'QualityByDepth' (i.e., --filter-expression 'QD < 2.0') and overall coverage 'DP' (i.e., --filter-expression 'DP < 500'). To identify sample-specific private variants, additional filters were applied, removing variants with low genotype quality (i.e., GQ < 80) and low coverage (i.e., DP < 50). The "control" sample served as a germline reference, and its variants were excluded from other samples. Remaining variants were annotated using SnpEff (v5.0) on the canonical isoform from the GRCm38.99 reference database and intersected with those from the RNA-seq experiment. Antigen prediction pipeline WES and RNA variant calling analysis were merged. Only missense mutations identified in at least 3 out of 4 datasets (WES and RNA from in vivo and in vitro samples) were considered as input for two different epitope prediction pipelines: antigen garnish and Pvac tools. The output of these two pipelines was integrated with the information about the expression of the mutated genes, in transcripts per million (TPM), obtained by the RNA sequencing. The following parameters were then used to select the best scoring candidates: (i) predicted affinity for MHC-I, (ii) expression in TPM, (iii) agretopicity and (iv) foreignness score. The top scoring candidates were then manually confirmed on RNA reads using integrative genomics viewer (IGV). Single cell RNA-seq Immediately after sacrificing the mice, livers were collected and dissociated into single cells. Single cells were divided into 2 tubes, one was stained only with CD45, while the second with tetramer, anti-CD3, anti-CD8, anti-Cd11b, anti F4/80, anti-B220 and Hashtag antibodies, then resuspended in MACS buffer containing Dapi. For each mouse, 30,000 CD45+ cells were sorted together with up to 2,000 barcoded tetramer+ CD8+ T cells. Sorted cells were further processed for scRNA sequencing. ScRNA sequencing was performed using the Next GEM Single Cell 5’ GEM Kit v2 from Chromium 10X according to manufacturer’s recommendation (User Guide Chromium Next GEM Single Cell 5ʹ Reagent Kits v2). We loaded 20,000 cells belonging to the same sample per reaction. We sequenced 4 samples, 150 bp paired-end reads in a NovaSeq 6000 Illumina apparatus. Base call files obtained as result from the Illumina sequencing were converted into FASTQ files, aligned to the mouse reference genome, and quantified with the 10x Genomics Cell Ranger Software (v7.2.0) using default parameters. Resulting data was imported into R and processed with the Seurat package (v5.0.1). All samples were merged into a single object and processed to remove cells with a low sequencing quality, those with a feature count below 600 and above 9,000, as well as cells with a fraction of mitochondrial genes higher than 10%. Samples were demultiplexed using the hashtag information using the Seurat function HTODemux, and cells classified as HTO doublets were filtered out. At the same time, the scDblFinder (v1.16.0) package was employed to annotate doublets of cells and exclude them from the following analyses. UMI-counts were then normalized and scaled using the Seurat functions NormalizeData (normalization method “LogNormalize”) and ScaleData. During this scaling step, unwanted sources of variation were regressed out. These sources included the number of detected transcripts per cell, the percentage of transcripts originating from mitochondria, and the difference between the scores of the cell cycle phases S and G2/M calculated for each cell. A principal component analysis with 100 principal components (PCs) was performed for dimensional reduction, and afterwards, we computed a UMAP representation using the top- 35 PCs. Additionally, cell clusters were identified based on these top 35 PCs using a resolution parameter of either 0.6 or 1.2. Marker genes for each cluster were obtained using the FindAllMarkers Seurat function, and finally, clusters were manually annotated and curated. Following GSEA analysis and visualization were performed on R using packages fgsea and enrichplot, respectively. For GSEA the gene sets from https://www.gsea- msigdb.org/gsea/msigdb/genesets.jsp were used. The CD8+ T cell exhaustion signature, termed Exhaustion, and the IFN ^ signature were retrieved from published sources. TCR sequencing was performed using Next GEM Single Cell 5’ GEM Kit v2 in combination with the Single Cell Mouse TCR Amplification Kit. The downstream analysis was performed as described above. TCR analysis was done with the R/Bioconductor package scRepertoire (v1.12.0) to identify clonotypes in each sample and compute their frequency (considering the amino acid sequence of the CDR3 region of the beta chain). TCR clonotypes were classified as unique (1 cell containing a specific TCR clone), small (2 to 5 cells containing the same TCR large (6 to 30 cells containing the same TCR), and hyperexpanded (more than 30 cells containing the same TCR), according to their numerosity. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the disclosed product, vector, inhibitor, use or method of the invention will be apparent to the skilled person without departing from the scope and spirit of the invention. Although the invention has been disclosed in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the disclosed modes for carrying out the invention, which are obvious to the skilled person are intended to be within the scope of the following claims.

Claims

CLAIMS 1. A product comprising: (a) a vector for liver and/or splenic phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence; and (b) an immune checkpoint inhibitor or a Tr1 cell inhibitor.

2. A vector for use in therapy, wherein the vector is for liver and/or splenic phagocyte- specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence, and wherein the vector is used in combination with an immune checkpoint inhibitor or a Tr1 cell inhibitor.

3. An immune checkpoint inhibitor or a Tr1 cell inhibitor for use in therapy, wherein the immune checkpoint inhibitor or Tr1 cell inhibitor is used in combination with a vector for liver and/or splenic phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence.

4. The product of, or vector, immune checkpoint inhibitor or Tr1 cell inhibitor for use according to any preceding claim, wherein the one or more expression control sequence comprises: (a) a phagocyte-specific promoter and/or enhancer; and/or (b) one or more miRNA target sequence, optionally wherein the one or more miRNA target sequence suppresses expression in cells other than liver phagocytes.

5. The product of, or vector, immune checkpoint inhibitor or Tr1 cell inhibitor for use according to any preceding claim, wherein the phagocyte is a Kupffer cell.

6. The product of, or vector, immune checkpoint inhibitor or Tr1 cell inhibitor for use according to claim 4 or 5, wherein the phagocyte-specific promoter and/or enhancer is a MRC1 promoter and/or enhancer or a fragment thereof, optionally wherein the MRC1 promoter and/or enhancer or fragment thereof comprises a nucleotide sequence having at least 70% identity to SEQ ID NO: 1, or a fragment thereof.

7. The product of, or vector, immune checkpoint inhibitor or Tr1 cell inhibitor for use according to any one of claims 4-6, wherein the one or more miRNA target sequence comprises: (a) one or more miR-126 target sequence; and/or (b) one or more miR-122 target sequence, optionally wherein the one or more miRNA target sequence comprises four miR- 126 target sequences and/or four miR-122 target sequences.

8. The product of, or vector, immune checkpoint inhibitor or Tr1 cell inhibitor for use according to any preceding claim, wherein the transgene encodes a cytokine, optionally wherein the cytokine is interferon-alpha, interferon-beta, interferon-gamma, IL2, IL12, TNF- alpha, CXCL9, IL1-beta, IL15, IL18, IL10, GMCSF, FLT3, IL7 or IL21.

9. The product of, or vector, immune checkpoint inhibitor or Tr1 cell inhibitor for use according to any preceding claim, wherein the transgene encodes a tumour antigen, optionally wherein the tumour antigen is carcinoembryonic antigen (CEA), TRP2, melanoma associated antigen (MAGE) family, cancer germline (CAGE) family, B melanoma antigen (BAGE-1), synovial sarcoma x breakpoint 20 (SSX-2), Sarcoma antigen (SAGE) family, LAGE1, NY- ESO-1, HER2, EGFR, MUC-1 or GAST.

10. The product of, or vector, immune checkpoint inhibitor or Tr1 cell inhibitor for use according to any preceding claim, wherein the vector is a viral vector, optionally wherein the vector is a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a herpes simplex viral vector.

11. The product of, or vector, immune checkpoint inhibitor or Tr1 cell inhibitor for use according to any preceding claim, wherein the immune checkpoint inhibitor inhibits an inhibitory checkpoint molecule selected from the group consisting of CTLA-4 (Cytotoxic T- Lymphocyte-Associated protein 4; CD152), A2AR (Adenosine A2A receptor), B7-H3 (CD276), B7-H4 (VTCN1), BTLA (B and T Lymphocyte Attenuator; CD272), HVEM (Herpesvirus Entry Mediator), IDO (Indoleamine 2,3-dioxygenase), TDO (tryptophan 2,3-dioxygenase), KIR (Killer-cell Immunoglobulin-like Receptor), LAG3 (Lymphocyte Activation Gene-3), PD-1 (Programmed Death 1 receptor), PD-L1 (PD-1 ligand 1), PD-L2 (PD-1 ligand 2), TIM-3 (T- cell Immunoglobulin domain and Mucin domain 3), VISTA (V-domain Ig Suppressor of T cell Activation), B7-1 (CD80), B7-2 (CD86), a TGFB (Transforming growth factor beta) pathway- associated protein, Il13 (interleukin-13), IL4 (interleukin-4), FGL (Fibrinogen Like 1), TIGIT (T cell immunoreceptor with Ig and ITIM domains), CD96 (TACT protein), Ceacam-1 (Carcinoembryonic antigen related cell adhesion molecule 1), CD155 (PVR protein), CD112 (PVR-related protein 2 (PVRL2)), LGALS3 (Galectin 3) and CD47 (integrin associated protein).

12. The product of, or vector, immune checkpoint inhibitor or Tr1 cell inhibitor for use according to any one of claims 1-10, wherein the Tr1 cell inhibitor inhibits a molecule selected from the group consisting of Cd4, Eomes, Gzmk, Lag3, Pdcd1, Ahr, Maf, Prdm1, Ctla4 and Il10ra.

13. The vector, immune checkpoint inhibitor or Tr1 cell inhibitor for use according to any one of claims 2-12, wherein the use is treatment or prevention of cancer.

14. The vector, immune checkpoint inhibitor or Tr1 cell inhibitor for use according to claim 13, wherein the cancer is a liver metastasis or a primary liver tumour.

15. A product comprising: (a) a vector for liver and/or splenic phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequence; (b) a second vector for liver and/or splenic phagocyte-specific expression, wherein the second vector comprises a second transgene operably linked to one or more expression control sequence, wherein the transgene is different to the second transgene.