Gene therapy

Abstract

A product comprises: (a) a vector for specific expression of liver and / or spleen phagocytes, wherein the vector comprises a transgene operably linked to one or more expression control sequences; and (b) an immune checkpoint inhibitor or a Tr1 cell inhibitor.

Description

TECHNICAL FIELD

[0001] The present invention relates to vectors for phagocyte-specific expression, in particular liver and / or spleen phagocyte-specific expression, and combinations of these vectors with immune checkpoint inhibitors and / or Tr1 cell inhibitors. The invention further relates to cells and pharmaceutical compositions comprising said vectors, and uses in therapy, including the treatment or prevention of cancer, for example liver metastases. BACKGROUND

[0002] The liver is involved in several biological functions, including detoxification, clearance of proteins and cells, and metabolic functions, among others. In order to protect the liver from immunological reactions that can damage it, the liver is characterized by an immunosuppressive environment that limits immunity. Due to its immunosuppressive environment, several tumor types tend to spread to the liver, giving rise to liver metastases.

[0003] The liver is one of the most common sites of cancer metastasis, accounting for almost 25% of all cases. A variety of primary tumors can be the source of metastasis, however, considering the total number of patients affected, colorectal adenocarcinoma is the most common. Liver metastases are associated with a poor prognosis and usually constitute the cause of death in cancer patients.

[0004] Surgical resection remains the gold standard for anatomically resectable liver metastases. Strategies to improve resection chances include neoadjuvant chemotherapy, portal vein embolization to increase future liver remnant, or two-stage resection with one-stage resection of the combination of primary tumor and liver lesions. However, it is reported that patients with colorectal metastases have a five-year survival rate of only 25% to 58% after curative resection of liver lesions, with a median survival time of 74 months. Therefore, there is a great need for improved treatments for cancers such as liver metastases. SUMMARY

[0005] The present inventors have developed a lentiviral vector (LV) platform that, for example, enables engineering of liver-resident macrophages (Kupffer cells) to specifically deliver a transgene, such as interferon-alpha (IFNa), to liver metastases. The present inventors observed that gene-based IFNa delivery to different mouse models of colorectal and pancreatic ductal adenocarcinoma liver metastases significantly delayed tumor growth. While not wishing to be bound by theory, the present inventors observed that the response to IFNa was associated with tumor-associated macrophage (TAM) immune activation, enhanced MHCII-restricted antigen presentation by tumor-infiltrating dendritic cells, and a reduction in CD8 T cell hyperchromasia. In contrast, increased IL10 signaling, enhanced CTLA4 expression, and expansion of Eomes CD4 T cells, a cell type that exhibits features of Type 1 regulatory T (Tr1) cells, were associated with resistance to IFNa gene therapy. The present inventors then observed that targeting regulatory T cell function by immune checkpoint blockade and IFNa LV delivery resulted in a strong synergy, resulting in complete responses in most mice.

[0006] In one aspect, the present application provides a product comprising: (a) a vector for liver and / or spleen phagocyte-specific expression; and (b) an immune checkpoint inhibitor or a Tr1 cell inhibitor. Suitably, the phagocyte targeted in the present application is selected from one or more of: macrophages, such as M2-like macrophages and / or MRC1+ macrophages; dendritic cells; and endothelial cells, such as liver sinusoidal endothelial cells. Suitably, the phagocyte targeted in the present application is selected from one or more of: resident macrophages (e.g. Kupffer cells); liver sinusoidal endothelial cells; spleen macrophages; tumor-associated macrophages; and / or monocyte-derived macrophages. In some embodiments, the phagocyte targeted in the present application is a Kupffer cell.

[0007] The vector can comprise a transgene operably linked to one or more expression control sequences.

[0008] In one aspect, the present application provides a product comprising: (a) a vector for liver and / or spleen phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequences; and (b) an immune checkpoint inhibitor or a Tr1 cell inhibitor.

[0009] In preferred embodiments, the product of the present application comprises an immune checkpoint inhibitor. In some embodiments, the product of the present application comprises a Tr1 cell inhibitor.

[0010] In one aspect, the application provides a product comprising: (a) a vector for liver and / or spleen macrophage-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequences; and (b) an immune checkpoint inhibitor. In one aspect, the application provides a product comprising: (a) a vector for liver and / or spleen macrophage-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequences; and (b) a Tr1 cell inhibitor.

[0011] In some embodiments, the product is in the form of a composition (e.g., a pharmaceutical composition) or a kit.

[0012] In one aspect, the application provides a vector for use in therapy, wherein the vector is for liver and / or spleen macrophage-specific expression, and wherein the vector is used in conjunction with an immune checkpoint inhibitor or a Tr1 cell inhibitor.

[0013] In preferred embodiments, the use of the application comprises combination with an immune checkpoint inhibitor. In some embodiments, the use of the application comprises combination with a Tr1 cell inhibitor.

[0014] In one aspect, the application provides a vector for use in therapy, wherein the vector is for liver and / or spleen macrophage-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequences, and wherein the vector is used in conjunction with an immune checkpoint inhibitor or a Tr1 cell inhibitor.

[0015] In one aspect, the application provides a vector for use in therapy, wherein the vector is for liver and / or spleen macrophage-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequences, and wherein the vector is used in conjunction with an immune checkpoint inhibitor. In one aspect, the application provides a vector for use in therapy, wherein the vector is for liver and / or spleen macrophage-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequences, and wherein the vector is used in conjunction with a Tr1 cell inhibitor.

[0016] In one aspect, the application 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 conjunction with a vector for liver and / or spleen macrophage-specific expression.

[0017] In one aspect, the application provides an immune checkpoint inhibitor or a Trl cell inhibitor for use in therapy, wherein the immune checkpoint inhibitor or Trl cell inhibitor is used in conjunction with a vector for liver and / or spleen phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequences.

[0018] In one aspect, the application provides an immune checkpoint inhibitor for use in therapy, wherein the immune checkpoint inhibitor is used in conjunction with a vector for liver and / or spleen phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequences. In one aspect, the application provides a Trl cell inhibitor for use in therapy, wherein the Trl cell inhibitor is used in conjunction with a vector for liver and / or spleen phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequences.

[0019] In some embodiments, the vector is for Kupffer cell-specific expression.

[0020] In some embodiments, the one or more expression control sequences comprises: (a) a phagocyte-specific promoter and / or enhancer; and / or (b) one or more miRNA target sequences.

[0021] In some embodiments, the one or more expression control sequences comprises a phagocyte-specific promoter and / or enhancer; and / or (b) one or more miRNA target sequences.

[0022] In some embodiments, the one or more expression control sequences comprises: (a) a phagocyte-specific promoter and / or enhancer. In some embodiments, the one or more expression control sequences comprises one or more miRNA target sequences. In some embodiments, the one or more expression control sequences comprises: (a) a phagocyte-specific promoter and / or enhancer; and (b) one or more miRNA target sequences.

[0023] In some embodiments, the phagocyte-specific promoter and / or enhancer is a liver and / or spleen phagocyte-specific promoter and / or enhancer.

[0024] In some embodiments, the one or more miRNA target sequences suppresses expression in cells other than liver phagocytes.

[0025] In some embodiments, the phagocyte is a liver and / or spleen phagocyte.

[0026] In some embodiments, the phagocyte is a macrophage. In some embodiments, the phagocyte is an M2-like macrophage and / or an MRC1+ macrophage; a dendritic cell; or a liver sinusoidal endothelial cell.

[0027] 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.

[0028] In some embodiments, the phagocyte is a liver sinusoidal endothelial cell.

[0029] In some embodiments, the vector comprises, from 5’ to 3’: a phagocyte-specific promoter and / or enhancer— a transgene— one or more miRNA target sequences.

[0030] In some embodiments, the phagocyte-specific promoter and / or enhancer is selected from the group consisting of: an 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; an 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; fragments thereof, or combinations thereof.

[0031] In some embodiments, the phagocyte-specific promoter and / or enhancer is an MRC1 promoter and / or enhancer or a fragment thereof.

[0032] In some embodiments, the MRC1 promoter and / or enhancer or a 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.

[0033] In some embodiments, the MRC1 promoter and / or enhancer or a fragment thereof comprises or consists of the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof.

[0034] In some embodiments, the one or more miRNA target sequences inhibit expression in non-phagocytic cells (e.g., non-liver and / or non-spleen phagocytic cells). In preferred embodiments, the one or more miRNA target sequences inhibit expression in non-liver phagocytic cells (i.e., cells other than liver phagocytic cells).

[0035] In some embodiments, the one or more miRNA target sequences inhibit transgene expression in liver cells and / or liver sinusoidal endothelial cells and / or spleen phagocytic cells.

[0036] The one or more miRNA target sequences can inhibit expression in some population of liver and / or spleen cells.

[0037] In some embodiments, the one or more miRNA target sequences inhibit transgene expression in liver cells. In some embodiments, the one or more miRNA target sequences inhibit transgene expression in liver sinusoidal endothelial cells (LSECs). In some embodiments, the one or more miRNA target sequences inhibit transgene expression in spleen phagocytic cells. In some embodiments, the one or more miRNA target sequences inhibit transgene expression in spleen macrophages. In some embodiments, the one or more miRNA target sequences inhibit transgene expression in liver cells, liver sinusoidal endothelial cells (LSECs), and / or spleen phagocytic cells.

[0038] In some embodiments, the one or more miRNA target sequences comprise: (a) one or more miR-126 target sequences; and / or (b) one or more miR-122 target sequences.

[0039] In some embodiments, the one or more miRNA target sequences comprise one or more miR-126 target sequences. In some embodiments, the one or more miRNA target sequences comprise one or more miR-122 target sequences. In some embodiments, the one or more miRNA target sequences comprise: (a) one or more miR-126 target sequences; and (b) one or more miR-122 target sequences.

[0040] In some embodiments, the one or more miRNA target sequences comprise four miR-126 target sequences and / or four miR-122 target sequences. In some embodiments, the one or more miRNA target sequences comprise four miR-126 target sequences and four miR-122 target sequences.

[0041] 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.

[0042] In preferred embodiments, the vector comprises a transgene operably linked to: (a) an MRC1 promoter and / or enhancer or fragments thereof; and (b) one or more miR-126 target sequences and / or one or more miR-122 target sequences.

[0043] In some embodiments, the transgene encodes a therapeutic polypeptide and / or an antigenic polypeptide.

[0044] In some embodiments, the transgene encodes a therapeutic polypeptide. In some embodiments, the transgene encodes an antigenic polypeptide.

[0045] In some embodiments, the transgene encodes a cytokine. In some embodiments, the cytokine is interferon-a, interferon-b, interferon-g, IL2, IL12, TNF-a, CXCL9, IL1-b, IL15, IL18, IL10, GMCSF, FLT3, IL7, or IL21.

[0046] In some embodiments, the transgene encodes interferon-a. In some embodiments, the transgene encodes IL12. In some embodiments, the transgene encodes IL10.

[0047] In some embodiments, the interferon-a comprises or consists of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.

[0048] In some embodiments, the interferon-a comprises or consists of the amino acid sequence of SEQ ID NO: 8.

[0049] In some embodiments, the transgene encodes a tumor antigen. In some embodiments, the tumor 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.

[0050] In some embodiments, the antigen is an MHC-I restricted antigen. In some embodiments, the antigen is an MHC-II restricted antigen.

[0051] In some embodiments, the tumor antigen is TRP2.

[0052] 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 sequences; and optionally a third vector comprising a third transgene operably linked to one or more expression control sequences. 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 sequences; and a third vector comprising a third transgene operably linked to one or more expression control sequences. The one or more expression control sequences can be as disclosed herein.

[0053] In some embodiments, the transgene encodes a cytokine, and the vector further comprises a second transgene operably linked to one or more expression control sequences; and optionally a third transgene operably linked to one or more expression control sequences. In some embodiments, the transgene encodes a cytokine, and the vector further comprises a second transgene operably linked to one or more expression control sequences; and a third transgene operably linked to one or more expression control sequences. The one or more expression control sequences can be as disclosed herein. The third transgene can be comprised in a second vector.

[0054] Preferably, the transgene and the second transgene are different. Preferably, the transgene, the second transgene, and the third transgene are different. The second and / or third vectors and the second and / or third transgenes can comprise additional features or be operably linked to additional features in the same manner as disclosed herein for the vectors and / or transgenes of the application.

[0055] 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.

[0056] In some embodiments, the second transgene encodes a second cytokine, wherein the cytokine is different from 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.

[0057] In some embodiments, the cytokine is interferon-alpha, and the second cytokine is IL12.

[0058] In some embodiments, the second transgene encodes a tumor antigen. In some embodiments, the tumor 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.

[0059] In some embodiments, the cytokine is interferon-a, and the second cytokine is a tumor antigen.

[0060] In some embodiments, the cytokine is IL12, and the second cytokine is a tumor antigen.

[0061] In some embodiments, the third transgene encodes a tumor antigen. In some embodiments, the tumor 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.

[0062] In some embodiments, the tumor antigen is TRP2.

[0063] In some embodiments, the second transgene encodes a second cytokine, wherein the cytokine is different from the second cytokine, and the third transgene encodes a tumor antigen.

[0064] In some embodiments, the cytokine is interferon-a, the second cytokine is IL12, and the third transgene encodes a tumor antigen.

[0065] In some embodiments, the cytokine is interferon-a, the second cytokine is IL12, and the tumor antigen is TRP2.

[0066] In some embodiments, the transgene is further operably linked to one or more regulatory elements.

[0067] In some embodiments, the transgene is further operably linked to a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0068] In some embodiments, the transgene is further operably linked to a destabilization domain. In some embodiments, the destabilization domain is a dihydrofolate reductase destabilization domain.

[0069] 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.

[0070] 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.

[0071] In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an integrating defective lentiviral vector (IDLV).

[0072] In some embodiments, the viral vector is a viral vector particle.

[0073] 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.

[0074] In some embodiments, the viral vector particle is produced in a viral particle producer or packaging cell that has been genetically engineered to reduce expression of CD47 and / or HLA on the surface of the cell. In some embodiments, the viral vector particle is substantially free of surface-exposed CD47 and / or HLA.

[0075] The vector can express a transgene specifically 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 not substantially expressed in cells other than phagocytes when transduced by the vector; and / or (iii) the transgene is not substantially expressed in lung cells, bone marrow cells, and / or blood cells when transduced by the vector; and / or (iv) the transgene is substantially expressed only in some liver cells and / or some spleen cells; and / or (v) expression of the transgene in Kupffer cells is at least ten times greater than expression in liver cells when transduced by the vector; and / or (vi) the transgene is not substantially expressed in liver cells when transduced by the vector.

[0076] 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 not substantially expressed in cells other than phagocytes when transduced by the vector. In some embodiments, the transgene is not substantially 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 liver cells when transduced by the vector. In some embodiments, the transgene is not substantially expressed in liver cells when transduced by the vector.

[0077] In some embodiments, the transgene is expressed substantially only in liver cells and / or spleen cells, and optionally not substantially in liver cells, when transduced by the vector.

[0078] In some embodiments, the transgene is expressed substantially only in liver cells and / or spleen cells.

[0079] 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), TGFB (transforming growth factor beta) pathway-related proteins, 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 two or more immune checkpoint inhibitors can be used.

[0080] In some embodiments, the immune checkpoint inhibitor inhibits PD-1.

[0081] In some embodiments, the cytokine is interferon-a, the second cytokine is IL12, the tumor antigen is TRP2, and the immune checkpoint inhibitor inhibits PD-1.

[0082] In some embodiments, the TGFB pathway-related 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 av, integrin b5, integrin b6, integrin b8, and LRRC32 (Leucine Rich Repeat Containing 32).

[0083] 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.

[0084] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA4 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody.

[0085] In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody.

[0086] In some embodiments, the transgene encodes interferon-a and the immune checkpoint inhibitor is an anti-PD1 antibody. In some embodiments, the transgene encodes interferon-a and the immune checkpoint inhibitor is an anti-CTLA4 antibody.

[0087] In some embodiments, the cytokine is interferon-a, the second cytokine is IL12, and the tumor antigen is TRP2, and the immune checkpoint inhibitor is an anti-PD1 antibody.

[0088] 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.

[0089] In one aspect, the present application provides a cell comprising the product of the present application.

[0090] In one aspect, the present application provides a cancer vaccine comprising the product of the present application. In one aspect, the present application provides the product of the present application for use in therapy.

[0091] In preferred embodiments, the use in therapy is the treatment or prevention of cancer.

[0092] In one aspect, the application provides a method of treating or preventing cancer, the method comprising administering to a subject in need thereof: (a) a vector for liver and / or spleen phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequences; and (b) an immune checkpoint inhibitor or a Tr1 cell inhibitor.

[0093] The components of the combination can be administered, for example, simultaneously, sequentially or separately.

[0094] In some embodiments, the cancer is a liver metastasis. The metastasis can, for example, be derived from a colorectal cancer or a pancreatic ductal adenocarcinoma (PDAC).

[0095] In some embodiments, the cancer is a primary liver tumor.

[0096] In some embodiments, the product, combination or components thereof are administered systemically. In some embodiments, the product, combination or components thereof are administered by intravenous injection, intra-portal injection or intra-hepatic arterial injection.

[0097] In one aspect, the application provides a product comprising: (a) a vector for liver and / or spleen phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequences; (b) a second vector for liver and / or spleen phagocyte-specific expression, wherein the second vector comprises a second transgene operably linked to one or more expression control sequences, wherein the transgene is different from the second transgene.

[0098] In one aspect, the application provides a vector for use in therapy, wherein the vector is for liver and / or spleen phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequences, and wherein the vector is for use in conjunction with a second vector for liver and / or spleen phagocyte-specific expression, wherein the second vector comprises a second transgene operably linked to one or more expression control sequences, wherein the transgene is different from the second transgene.

[0099] In one aspect, the application provides a method of treating or preventing cancer, the method comprising administering to a subject in need thereof: (a) a vector for liver and / or spleen phagocyte-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequences; and (b) a second vector for liver and / or spleen phagocyte-specific expression, wherein the second vector comprises a second transgene operably linked to one or more expression control sequences, wherein the transgene is different from the second transgene.

[0100] In one aspect, the application provides a product comprising a vector for liver and / or spleen macrophage-specific expression, wherein the vector comprises (a) a transgene operably linked to one or more expression control sequences; and (b) a second transgene operably linked to one or more expression control sequences, wherein the transgene is different from the second transgene.

[0101] In one aspect, the application provides a vector for use in therapy, wherein the vector is for liver and / or spleen macrophage-specific expression, wherein the vector comprises (a) a transgene operably linked to one or more expression control sequences; and (b) a second transgene operably linked to one or more expression control sequences, wherein the transgene is different from the second transgene.

[0102] In one aspect, the application provides a method of treating or preventing cancer, the method comprising administering to a subject in need thereof a vector for liver and / or spleen macrophage-specific expression, wherein the vector comprises (a) a transgene operably linked to one or more expression control sequences; and (b) a second transgene operably linked to one or more expression control sequences, wherein the transgene is different from the second transgene.

[0103] In some embodiments, the transgene encodes a cytokine. In some embodiments, the cytokine is IL12, interferon-a, interferon-β, interferon-γ, IL2, TNF-a, CXCL9, IL1-β, IL15, IL18, IL10, GMCSF, FLT3, IL7, or IL21. In some embodiments, the cytokine is IL12.

[0104] In some embodiments, the second transgene encodes a second cytokine, wherein the cytokine is different from the second cytokine. In some embodiments, the second cytokine is interferon-a, interferon-β, interferon-γ, IL2, IL12, TNF-a, CXCL9, IL1-β, IL15, IL18, IL10, GMCSF, FLT3, IL7, or IL21. In some embodiments, the second cytokine is interferon-a.

[0105] In some embodiments, (a) the transgene encodes a cytokine (e.g., IL12, interferon-a, interferon-b, interferon-g, IL2, TNF-a, CXCL9, IL1-b, IL15, IL18, IL10, GMCSF, FLT3, IL7, or IL21); and (b) the second transgene encodes a second cytokine (e.g., interferon-a, interferon-b, interferon-g, IL2, IL12, TNF-a, CXCL9, IL1-b, IL15, IL18, IL10, GMCSF, FLT3, IL7, or IL21), wherein the cytokine is different from the second cytokine.

[0106] In some embodiments, the transgene encodes IL12, and the second transgene encodes interferon-a.

[0107] In some embodiments, the second transgene encodes a tumor antigen. In some embodiments, the tumor 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.

[0108] In some embodiments, (a) the transgene encodes a cytokine (e.g., IL12, interferon-a, interferon-b, interferon-g, IL2, TNF-a, CXCL9, IL1-b, IL15, IL18, IL10, GMCSF, FLT3, IL7, or IL21); and (b) the second transgene encodes a tumor 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).

[0109] In some embodiments, the transgene encodes IL12, and the second transgene encodes a tumor antigen.

[0110] In some embodiments, the product or combination further comprises a third vector for liver and / or spleen phagocyte-specific expression, wherein the third vector comprises a third transgene operably linked to one or more expression control sequences, wherein the third transgene is different from the transgene and the second transgene.

[0111] In some embodiments, the vector further comprises a third transgene operably linked to one or more expression control sequences, wherein the third transgene is different from the transgene and the second transgene.

[0112] In some embodiments, the product or combination further comprises a second vector for liver and / or spleen macrophage-specific expression, wherein the second vector comprises a third transgene operably linked to one or more expression control sequences, wherein the third transgene is different from the transgene and the second transgene.

[0113] The transgene, the second transgene, and the third transgene can each independently be selected from a cytokine or a tumor antigen, e.g., a cytokine or a tumor antigen as disclosed herein.

[0114] In one aspect, the present application provides a method of treating or preventing a cancer, the method comprising administering to a subject in need thereof: (a) a vector for liver and / or spleen macrophage-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequences; (b) a second vector for liver and / or spleen macrophage-specific expression, wherein the second vector comprises a second transgene operably linked to one or more expression control sequences, wherein the transgene is different from the second transgene; and (c) a third vector for liver and / or spleen macrophage-specific expression, wherein the third vector comprises a third transgene operably linked to one or more expression control sequences, wherein the third transgene is different from the transgene and the second transgene.

[0115] In one aspect, the present application provides a method of treating or preventing a cancer, the method comprising administering to a subject in need thereof a vector for liver and / or spleen macrophage-specific expression, wherein the vector comprises (a) a transgene operably linked to one or more expression control sequences; (b) a second transgene operably linked to one or more expression control sequences; and (c) a third transgene operably linked to one or more expression control sequences.

[0116] 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 from the second cytokine; and (c) the third transgene encodes a tumor 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 tumor antigens can be as disclosed herein.

[0117] In some embodiments, the transgene encodes IL12, the second transgene encodes interferon-alpha, and the third transgene encodes a tumor antigen.

[0118] In one aspect, the application provides a method of treating or preventing cancer, the method comprising administering to a subject in need thereof (i) a vector for liver- and / or spleen-phagocyte specific expression, wherein the vector comprises (a) a transgene operably linked to one or more expression control sequences; (b) a second transgene operably linked to one or more expression control sequences; and (c) a third transgene operably linked to one or more expression control sequences; and (ii) an immune checkpoint inhibitor.

[0119] 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. BRIEF DESCRIPTION OF DRAWINGS

[0120] Figure 1

[0121] Generation of an LV platform capable of in vivo liver macrophage engineering .

[0122] (A) Schematic of Mrc1.GFP and Mrc1.GFP.miRT LVs. (B) Schematic of the experiments shown in panels C-F. (C) LV copy number per cell of the indicated organs analyzed by digital droplet PCR (ddPCR). (D and E) GFP expression in the indicated cell types in the indicated organs analyzed by flow cytometry (FC). (n=5 mice / group, statistical analysis by Mann-Whitney test comparing Mrc1.GFP LV vs. Mrc1.GFP.miRT LV and p values adjusted for multiple testing with Bonferroni correction). In E, Mrc1.GFP LV and Mrc1.GFP.miRT LV were used at 3 10 10 TU / kg. (F) Representative immunofluorescence (IF) images and relative GFP quantification of livers bearing metastases from MC38 cells (mCherry (red), GFP (green), F4 / 80 (gray), and nuclei (blue, left panel)) in the left panel or AKT PF cells (GFP (green), F4 / 80 (gray), and nuclei (blue, right panel)) in the right panel; metastasis (Met), peri-metastatic area (dashed line), and whole liver are indicated. In the left panel, MC38 cells were injected 10 days after LV delivery (LV at 3 10 10 TU / kg); in the right panel, Mrc1.GFP.miRT LV was used in NSG mice at 5 10 9 TU / kg (n=5 mice / group; statistical analysis by bootstrap t-test).

[0123] Figure 2

[0124] In vivo LV-engineered KCs enable rapid, sustained and well-tolerated IFNa production .

[0125] (A) Schematic of KCs engineered with IFNa LV (top) or control LV (bottom). (B) Plasma IFNa levels analyzed by ELISA at the indicated time points after LV injection (n = 10 mice / group for control LV, IFNa LV, or untransduced UT, respectively, n = 5 mice for UT). (C) LV copy number per cell obtained by ddPCR (n = 8 mice / group for control LV, IFNa LV, or UT, respectively; statistical analysis by Kruskal-Wallis with Dunn's test, adjusted p values by Bonferroni correction). (D) Blood cell counts of B cells (left panel), eosinophils (middle panel), and neutrophils (right panel) at the indicated time points after LV injection (as n values in B; statistical analysis by Mann- Whitney test). (E) Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) at 126 days after LV injection (n = 9 mice / group for control LV, n = 10 mice / group for IFNa LV, n = 5 mice for UT; statistical analysis by Kruskal-Wallis test). (F) Histopathological analysis of the indicated organs at 366 days after LV injection (as n values in C).

[0126] Figure 3

[0127] Gene-based forced IFNa expression by KCs unleashes T cell activation and impairs liver metastasis growth .

[0128] (A) Schematic of the experiment in panels B-N. (B and H) Plasma IFNa levels obtained by ELISA at the indicated time points after tumor challenge. Lower or higher doses, respectively, 1.5 10 9 TU / kg or 1.5 10 10TU / kg (in B, left panel, n = 10 mice / group, 10 mice / group, 5 mice / group; in B, right panel, n = 10 mice / group, 10 mice / group, 5 mice / group; in H, n = 11 mice / group, 10 mice / group, 3 mice / group in control LV, IFNa LV, or UT, respectively). (C, E, I, and N) Tumor growth by magnetic resonance imaging (MRI) (C, left panel, n = 9 mice / group, 8 mice / group, 9 mice / group, 8 mice / group; C, right panel, n = 10 mice / group, 10 mice / group, 10 mice / group, 10 mice / group; in E, n = 10 mice / group, 10 mice / group, 10 mice / group, 9 mice / group; in I, n = 9 mice / group, 10 mice / group, 9 mice / group, 10 mice / group, and in N, from left to right, n = 7 mice / group, 8 mice / group; statistical analysis by Mann-Whitney). (D and J) Representative MRI of control LV (left panel) and IFNa LV-treated (right panel) mice with MC38 liver metastases in D 20 days after tumor transplantation or AKTPF liver metastases in J 28 days after tumor transplantation, complete responders (CR), healthy liver (white), and metastases (Met, yellow) are indicated by dotted lines in D or arrows in J. (F, G, K, and L) Percentage of indicated cell types infiltrating MC38.OVA (in F and G) or AKTPF (K and L) liver metastases by FC analysis (n = 10 mice / group, 7 mice / group in control LV or IFNa LV, respectively; statistical analysis by Mann-Whitney test). (M) Representative IF images and relative CD8 T cell quantification of AKTPF liver metastases by CM (n in L; statistical analysis by Mann-Whitney test).

[0129] Figure 4

[0130] Engineering KCs with IFNa LVs enables preferential IFNa signaling around metastatic foci .

[0131] (A) Side-by-side comparison of representative liver sections containing metastatic lesions (Met) from the indicated treatment cohorts by spatial transcriptomic analysis showing H&E staining (left) or by using spatial transcriptomic analysis (right). Spatial spots are indicated in color associated with spatial compartments. (B) Heatmap of normalized enrichment scores (NES) by gene set enrichment analysis (GSEA) of selected gene ontology (GO) terms across different spatial compartments by spatial transcriptomics (Visiium). Gene sets were grouped into cytokine-related effects (red), immune activation status (blue), tumor-related (black), and liver function (tan; n = 3 mice / group, 3 mice / group, 2 mice / group in partial responders, resistant, or controls, respectively). (C) Fold change in average gene expression levels of the indicated genes belonging to the indicated gene categories in spatial compartments and treatment cohorts (as n values in B).

[0132] Figure 5

[0133] IFNa promotes APC immune activation and enhanced MHCII-restricted antigen presentation in responder mice .

[0134] (A) GSEA analysis of scRNA sequencing data showing NES of selected GO terms computed based on genes differentially expressed in antigen presenting cells (APCs) in the indicated comparisons (as n in A; statistical analysis by adaptive multiscale-splitting Monte-Carlo procedure; : padj<0.05; : padj<0.01; : padj<0.001; : p<0.0001). (B) UMAP representation of scRNA sequencing data of APCs from the indicated groups (n=3 mice / group, 3 mice / group, 2 mice / group in partial responders, resistant, or control, respectively). (C) Expression of selected genes showing mean expression (color scale) and percentage (size of shapes) of cells expressing the indicated gene categories in IFNa TAMs and TAMs. (D) GSEA analysis showing NES of selected GO terms on genes differentially expressed in IFNa 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 mice / group, 3 mice / group, 2 mice / group in partial responders, resistant, or control, respectively). (F) Expression profile of genes belonging to the indicated categories showing mean expression (color scale) and percentage (size of shapes) of cells expressing the indicated genes. (G) Combined gene expression scores of genes belonging to the indicated categories in different cell populations from the indicated groups.

[0135] Figure 6

[0136] Therapeutic response to IFNa is associated with T cell activation and counteracted by Eomes CD4 T cell infiltration .

[0137] (A) GSEA analysis showing NES of selected GO terms on genes differentially expressed in the indicated comparisons (n=3 mice / group, 3 mice / group, 2 mice / group in partial responders, resistant, or control, respectively; statistical analysis as in A). (B) UMAP representation of cells from AKT PF liver metastases annotated as T and NK cells (n values as in A). (C) Expression of selected genes showing mean expression (color scale) and percentage (size of shapes) of cells expressing the indicated gene categories in pooled Eomes CD4 T cells and all other T and NK cells. (D) Percentage of the indicated cell populations from the indicated groups (n as in A; statistical analysis as in D). (E) Gene expression profile showing mean expression (color scale) and percentage (size of shapes) of cells expressing the indicated genes in pooled all CD8 T cell subtypes together with genes belonging to the exhaustion-associated gene signature and effector / memory-like associated genes highlighted in yellow and green, respectively. Figure 5 Figure 5

[0138] Figure 7

[0139] Combination of IFNa from engineered KCs with functional suppression of regulatory T cells eradicates liver metastasis .

[0140] ​​(A) Patients were stratified into IFNa signaling low and high cohorts (n=21) based on their IFNa signature score by bulk RNA sequencing of tumors. (B) Tr1 cell signature score detected in bulk RNA sequencing data from human patient CRC-derived liver metastases stratified by their intrinsic IFNa 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, IFNa high signaling; pt. #16, IFNa low signaling) showing CD4 (green), LAG3 (red) and nuclei (blue). (D) Percentage of EOMES CD4+ T cells infiltrating AKT PF liver metastases by FC analysis (n=7 mice / group, 8 mice / group, 5 mice / group, 10 mice / group from left to right as indicated treatment; statistical analysis by Mann-Whitney test, p values adjusted by Bonferroni correction). (E and H) Tumor growth by MRI analysis (n=9, 9, 9, 10 in E; n=7 mice / group, 8 mice / group, 9 mice / group, 9 mice / group from left to right in H; statistical analysis by ANCOVA in E, Mann-Whitney test in H, p values adjusted by Bonferroni correction). (F) Schematic of the experiments shown in G and H. (G) Tumor growth assessed by tumor weight (n=13 mice / group, 8 mice / group, 10 mice / group, 9 mice / group, controls consisted of 10 control LV mice and 3 UT mice; statistical analysis by Mann-Whitney test, p values adjusted by Bonferroni correction).

[0141] Figure 8

[0142] Generation of an LV platform capable of in vivo liver macrophage engineering .

[0143] (A) Schematic (generated with USCS Genome Browser) showing the murine putative Mrc1 promoter. (B) Representative FACS plot showing GFP expression in bone marrow-derived macrophages (BMDM) transduced as indicated. (C) Percentage of GFP+ BMDM analyzed by flow cytometry (FC) (n=3 cell cultures / group). (D) Mean fluorescence intensity of PDL1 (left panel) and MRC1 (right panel) in untreated (NT) BMDM polarized as indicated analyzed by FC (n=3 cell cultures / group). (E) LV copy number per cell calculated by ddPCR analysis (n=3 cell cultures / group). (F) Schematic of the bidirectional LV design, without (top) and with (bottom) the miRT sites delivered systemically (i.v.) to mice. (G) LV copy number per cell calculated by ddPCR analysis (n=8 mice / group, 8 mice / group, 8 mice / group, 3 mice / group from left to right, statistical analysis by Kruskal-Wallis test). (H) Mean 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 areas showing GFP and nuclei (top) and F4 / 80 and nuclei (bottom). (J and K) Percentage (in J) and MFI (in K) of GFP-positive and dlNGFR-positive cells in liver KCs and LSECs analyzed by FC (n=as in A; statistical analysis by Kruskal-Wallis with Dunn test, adjusted p values by Bonferroni correction). (L) GFP expression in different organs after PBS (UT) or Mrc1.GFP.miRT LV delivery to mice challenged with liver metastases from MC38 cells (top) or AKT PF cells (bottom). Single channel of the images shown in F. Liver with metastases from MC38 cells (top) or AKT PF 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 (gray), nuclei (blue).

[0144] Figure 9

[0145] In vivo LV-engineered KCs enable rapid, sustained and well-tolerated IFNa production .

[0146] (A) Blood cell counts of inflammatory monocytes, resident monocytes, CD8 T cells, CD4 T cells, eosinophils, platelets and red blood cells and amount of hemoglobin at the indicated time points after LV injection (n = 10 mice / group, 10 mice / group, 5 mice / group in control LV, IFNa LV or UT mice, respectively, statistical analysis by Mann-Whitney test). (B) Heatmap showing fold change in row mean of auto-reactive antibodies detected in mouse serum against the indicated targets at day 52 (n = 3 mice / group in IFNa LV or control LV, respectively) and day 366 (n = 8 mice / group in IFNa LV, control LV or UT, respectively) after LV injection. Positive control: plasma from lupus mice (18-week-old female NZB / NZW mice, statistical analysis by Mann-Whitney test, adjusted p- values by Bonferroni correction). (C) Histopathological analysis of the indicated organs at day 366 after LV injection, scored as absent (none), minimal, mild, moderate, marked and severe (n = 8 mice / group, 8 mice / group, 5 mice / group in control LV, IFNa LV or UT mice, respectively).

[0147] Figure 10

[0148] Gene-based forced IFNa expression by KCs unleashes T cell activation and impairs liver metastasis growth .

[0149] (A) LV copy number per cell in the liver by ddPCR analysis (left, n = 9 mice / group, 10 mice / group, 3 mice / group; right, 8 mice / group, 7 mice / group, 5 mice / group in control LV, IFNa LV or UT, respectively), (B) B cell counts in blood (left, n = 10 mice / group, 9 mice / group, 5 mice / group; right, n = 9 mice / group, 9 mice / group, 5 mice / group in control LV, IFNa LV or UT, respectively; statistical analysis by Mann-Whitney test), (C) Tumor volume by caliper measurements at the indicated time points (left, n = 1 mice / group, 4 mice / group in IFNa LV complete responders or UT cohorts, right, n = 1 mice / group, 5 mice / group), (D) Plasma IFNa levels by ELISA (day 5, n = 9 mice / group, 6 mice / group; day 11, n = 9 mice / group, 7 mice / group, left) and LV copy number per cell by ddPCR analysis in the liver (n = 10 mice / group, 10 mice / group, right), (E) Representative images of H&E staining of human liver sections containing CRC metastases (left) and murine liver sections containing AKTPF metastases (right); note tumor glands (NG), tumor endothelium (NE) and dirty central necrosis (CDN) highlighted with black arrows, (F) Representative CM images of murine livers containing AKTPF metastases, stained for CD4 (green), CD8 (red), CD11c (green), F4 / 80 (red), CD31 (green), a-SMA (red), E-cadherin (gray) and nuclei (blue) as indicated in the figure, (G) Tumor growth by MRI analysis (n = 10 mice / group, 9 mice / group in control LV or IFNa LV mice, respectively; statistical by Mann-Whitney test), (H) Plasma IFNa levels by ELISA (n = 10 mice / group, 9 mice / group, 5 mice / group in control LV, IFNa LV or UT, respectively, left) and LV copy number per cell by ddPCR analysis in the liver (n = 10 mice / group, 10 mice / group, 5 mice / group in control LV, IFNa LV or UT, respectively, right), (I) LV copy number per cell by ddPCR analysis in the liver (n = 11 mice / group, 6 mice / group in control LV or IFNa LV, respectively), (J) Plasma IFNa levels by ELISA (n = 7 in control LV; n = 8 mice / group in IFNa LV).

[0150] Figure 11

[0151] Engineering KCs with IFNa LVs enables preferential IFNa signaling around metastatic foci .

[0152] (A) Gene expression analysis of tumors by using ddPCR analysis (n=10 mice / group, 7 mice / group in control LV or IFNa LV, respectively). (B) Schematic representation of tumor volumes at day 28 for 3 different cohorts: control (red), partial responders (blue) or resistant (green). (C) UMAP representation of spatial transcriptomic spots based on AKTPF liver metastases (left) and representative H&E images overlaid with transcriptomic spots highlighted with colors according to UMAP clustering (right). (D) Gene expression of selected genes associated with CRC (cancer cell gene signature) or liver function (liver gene signature). Mean gene expression is displayed by color scale and percentage of cells expressing the indicated gene is represented by size of shapes (n=8). (E) Bottom right: Schematic representation showing spatial compartments A to H. Approximate distance to tumor-liver border is indicated in mm and color associated with individual spatial compartments. Top right and left: Slices by using spatial transcriptomic analysis, not including Figure 4 slices shown in A.

[0153] Figure 12

[0154] IFNa promotes APC immune activation and enhanced MHCII-restricted antigen presentation in responder mice .

[0155] (A) UMAP representation, as Figure 4 shown in B, displaying cells from AKTPF liver metastases from different treated cohorts (as in A, n). (B) Fraction of cells belonging to the identified cell populations for each sample (n=3 mice / group, 3 mice / group, 2 mice / group in control, partial responders or resistant, respectively). (C) UMAP representation displaying all cells from all groups (as in A, n), showing expression levels of the indicated genes on a scale from gray (low expression) to blue (high expression). (D) Heatmap showing log fold change of expression of the top 20 upregulated genes in each cluster (n=8). (E) Gene expression of selected genes associated with different clusters of the APC compartment, showing mean expression (color scale) and percentage (size of shapes) of cells expressing the indicated genes (n=8).

[0156] Figure 13

[0157] Therapeutic response to IFNa is associated with T cell activation and counteracted by Eomes CD4 T cell infiltration .

[0158] (A) Heatmap showing the log fold change of expression of the top 20 upregulated genes in each cluster identified in the T cell and NK cell compartments (n=8). (B) Gene expression of selected genes associated with the indicated features in different clusters (as Figure 12 shown in E; n=8). (C) GSEA analysis showing the NES of selected GO terms on genes differentially expressed in CD8 T cells in the indicated comparisons (as Figure 4 in A; statistical analysis as Figure 5 in A).

[0159] Figure 14

[0160] Combination of IFNa from engineered KCs with functional suppression of regulatory T cells eradicates liver metastasis .

[0161] (A) Correlation of IFNa signature score and Trl signature score (n=42; statistical analysis by Spearman correlation coefficient). (B) IF images of sections of human livers containing metastases, stained for CD4 (green), LAG3 (red) and nuclei (blue). Indicated are the liver, metastasis (Met), neoplastic gland (NG), neoplastic endothelium (NE) and central necrosis (CDN). CD4+LAG3+ cells are indicated with arrows. (C) Plasma IFNa levels by using ELISA (left panel, from left to right n=10 mice / group, 9 mice / group, 9 mice / group, 9 mice / group) and LV copy number per cell in the liver by using ddPCR (right panel, from left to right n=9 mice / group, 8 mice / group, 9 mice / group, 10 mice / group). (D) MFI of PD1 expression on CD8 (left panel) or CD4 (right panel) T cells in the blood circulation by using FC analysis (from left to right n=10 mice / group, 9 mice / group, 10 mice / group, 10 mice / group; statistical analysis by Mann-Whitney test, adjusted p values by Bonferroni correction). (E and F) Plasma IFNa levels by using ELISA (in E, left panel, n=10, 8, 10, 9, 3; in F, left panel, from left to right n=10 mice / group, 9 mice / group, 9 mice / group, 9 mice / group), and LV copy number per cell in the liver by using ddPCR analysis (in E, right panel, n=10 mice / group, 8 mice / group, 10 mice / group, 9 mice / group, 3 mice / group; in F, right panel, from left to right n=6 mice / group, 7 mice / group, 9 mice / group, 9 mice / group).

[0162] Figure 15

[0163] Combination of IL12 and IFNa promotes CD8 T cell activation and liver metastasis clearance .

[0164] (a) Schematic of the experiment. (b) Tumor weights at day 22 after 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 the total number of circulating CD8 T cells. (e and f) Percentage of CD8 T cells infiltrating MC38.OVA liver metastases out of total CD45 cells, in e; and PEX tetramer CD8 T cells out of all tetramer CD8 T cells, in f. PEX was identified as indicated in the Methods section. (g-i) Flow cytometry analysis of the liver showing terminal 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 marker in tetramer CD8 T cells, in i. TEX was identified as indicated in the Methods section.

[0165] Figure 16

[0166] Combination of IL12, IFNa and anti-PD 1 monoclonal antibody promotes CD8 T cell activation and liver metastasis clearance .

[0167] (a) Schematic of the experiment. (b) Tumor weights at day 22 after 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 the treatment groups, demonstrating either no tumor (complete responders) or OVA expression (clearance of all OVA-expressing cells from the tumor). (e) Blood analysis by flow cytometry at day 14 showing the percentage of tetramer (anti-OVA) CD8 T cells out of the total number of circulating CD8 T cells. (f and g) Percentage of CD8 T cells infiltrating MC38.OVA liver metastases out of total CD45 cells, in f; and PEX tetramer CD8 T cells out of all tetramer CD8 T cells, in g. (h-j) Flow cytometry analysis of the liver showing terminal 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 marker in tetramer CD8 T cells, in j.

[0168] Figure 17

[0169] Combination of IL12 and IFNa with melanoma-associated antigens from expression by liver macrophages promotes melanoma liver metastasis clearance Figure 18 .

[0170] (a) Schematic of the experiment. (b and c) Magnetic resonance imaging (MRI) analysis at day 13 and day 19 after melanoma inoculation in the indicated groups. (d) Representative pictures of livers from mice in the indicated treatment groups. (e and f) Percentage of CD4 out of total CD45 cells infiltrating B16 liver metastases, in e, and CD8 out of total CD45 cells, in f. (f) Flow cytometry analysis of livers showing percentage of CD4 T cells expressing PD1 out of total CD4 T cells.

[0171] Figure 19

[0172] a, b, UMAP projection of single-cell RNA sequencing (scRNA-seq) of the entire dataset of the indicated tissues. c, d, UMAP projection of scRNA-seq of APC subsets clusters of the indicated tissues. e, f, Expression of selected genes belonging to the indicated classes in APCs and tissues (n = 2 mice / group, 3 mice / group, 3 mice / group, 3 mice / group for liOVA, OVA.Ifna, OVA.112, and OVA.Combo, respectively; statistical analysis by Wilcoxon test with Bonferroni correction, comparison with liOVA; : padj < 0.05; : padj < 0.005; : padj < 0.0005). g, Combined gene expression score of genes belonging to the indicated classes in different cell populations from the indicated tissues and groups.

[0173] Figure 20

[0174] a, b, UMAP projection of scRNA-seq of the entire dataset of the indicated groups and tissues. c, d, GSEA of scRNA-seq data showing NES of selected GO terms calculated based on differentially expressed genes in KCs, macrophages, and monocytes in the indicated comparisons (n = 2 mice / group, 3 mice / group, 3 mice / group, 3 mice / group for liOVA, OVA.Ifna, OVA.112, and OVA.Combo, respectively; statistical analysis by adaptive multiscale-splitting Monte-Carlo procedure; : padj < 0.05; : padj < 0.005; : padj < 0.0005).

[0175] Figure 21

[0176] a, UMAP projection of scRNA-seq of liver T cell and NK cell subsets. b, Gene set enrichment analysis (GSEA) of scRNA-seq data showing the normalized enrichment score (NES) of selected gene ontology (GO) terms computed based on genes differentially expressed in T cells and NK cells in the indicated comparisons (n = 2 mice / group, 3 mice / group, 3 mice / group, 3 mice / group for liOVA, OVA.Ifna, OVA.112, and OVA.Combo, respectively; statistical analysis by adaptive multi-level splitting Monte-Carlo procedure; padj < 0.05; padj < 0.005; padj < 0.0005). c, Combined gene expression scores of genes belonging to the indicated categories in different cell populations from the indicated tissues and groups. d, Combined gene expression scores of selected genes belonging to the indicated categories in liver CD8 + expression of selected genes in the indicated categories in liver CD8 padj < 0.05; padj < 0.005; padj < 0.0005). e, UMAP projection of liver scRNA-seq indicating cells with OVA-specific TCRs. f, GSEA of scRNA-seq data showing the NES of selected GO terms computed based on genes differentially expressed in OVA-specific CD8 + expression of selected genes in the indicated categories in liver CD8 + Clonotype sharing between liver and tumor CD4 + T cell clonotypes. j, CD4 + T cell populations. k, CD4+ T cell numbers divided by TCR clonotype frequency.

[0177] Figure 22

[0178] a, OVA-specific and bystander CD8 + Clonotypic sharing between liver and tumor T cells, grouped by TCR clonotype.b, Liver OVA-specific CD8 + Expression of selected genes in indicated classes of T cells (n = 2 mice / group, 3 mice / group, 3 mice / group, 3 mice / group for liOVA, OVA.Ifna, OVA.Il12, and OVA.Combo; statistical analysis by Wilcoxon test with Bonferroni correction, compared to liOVA. : padj < 0.05; : padj < 0.005; : padj < 0.0005). c, Liver shared and non-shared CD4 + Expression of selected genes in indicated classes of T cells (number of mice and statistical analysis).

[0179] Figure 23

[0180] a, Schematic of the antigen prediction pipeline used to identify neoantigens in the AKT PF LM model.b, Schematic of TA33 LV.c, Schematic of the experiments shown in d-h.d-h, Mice bearing established AKT PF LM were treated with TA33 or TA33.Combo 7 days after tumor challenge (TA33 1 10 7 TU / mouse, TA33.Combo total dose 1.2 10 8 TU / mouse). In d, LM volume was quantified by MRI at day 27 after tumor injection (n = 6 mice / group, 6 mice / group, 9 mice / group for control untreated mice, TA33-treated mice, or TA33.Combo-treated mice; horizontal lines indicate median values, statistical analysis by Kruskal-Wallis with Dunn’s test). In e, f, FC analysis of liver (n = 6 mice / group, 6 mice / group, 8 mice / group for control untreated mice, TA33-treated mice, or TA33.Combo-treated mice; statistics as in i). In g and h, IFNg ELISPOT assay on CD8 + T cells isolated from the spleen of indicated mice (n = 3 mice / group, 3 mice / group).

[0181] Figure 24

[0182] a, Plasma levels of IFNa and IL-12 measured by ELISA 7 days after treatment (n = 6 mice / group, 6 mice / group, 9 mice / group for control untreated mice, TA33 treated mice or TA33.Combo treated mice; horizontal lines represent median values). b, FC analysis of blood performed on day 14 after tumor injection (as in a, number of mice, horizontal lines represent median values, statistical analysis by Kruskal-Wallis with Dunn's test). 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).

[0183] Figure 25

[0184] a-g, Mice bearing established B16-F10 LMs were treated with Trp2.Combo on day 5 after tumor challenge (control mice remained untreated, OVA.Combo total dose was 1.2 10 8 TU / mouse). 3 and 10 days after LV injection, mice were injected with 0.2 mg of a-PD1 or remained untreated. In b, quantification of LM volume by MRI at the indicated time points (n = 5 mice / group, 8 mice / group, 6 mice / group, 7 mice / group for control untreated mice, control + a-PD1 treated mice, Trp2.Combo treated mice or Trp2.Combo + a-PD1 treated mice; horizontal lines represent median values, statistical analysis by Kruskal-Wallis with Dunn's test). In c, representative MRI images of livers from control untreated mice or Trp2.Combo + a-PD1 treated mice. In d-g, FC analysis of the indicated tissues (n = 3 mice / group, 5 mice / group, 6 mice / group, 7 mice / group for control untreated mice, control + a-PD1 treated mice, Trp2.Combo treated mice or Trp2.Combo + a-PD1 treated mice; horizontal lines represent median values, statistical analysis by Kruskal-Wallis with Dunn's test).

[0185] phagocytes

[0186] a, FC analysis of blood (n = 5 mice / group, 8 mice / group, 6 mice / group, 7 mice / group for control untreated mice, control + a-PD1 treated mice, Trp2. Combo treated mice or Trp2. Combo + a-PD1 treated mice, horizontal lines represent median values, statistical analysis by Kruskal-Wallis with Dunn's test). b, Circulating Ly6c+ CD44+ CD8 + Correlation between T cells and tumor volume (n of mice in c, statistical analysis by Spearman correlation). DETAILED DESCRIPTION

[0187] As used herein, the terms "comprising" and "consisting of" are synonymous with "including" or "containing," and are inclusive or open-ended, and do not exclude additional, unrecited members, elements, or steps. The terms "comprising" and "consisting of" also include the term "consisting essentially of."

[0188] macrophages

[0189] The present application relates to phagocyte-specific transgene expression, in particular liver and / or spleen phagocyte-specific transgene expression.

[0190] As used herein, a "phagocyte" is a specialized cell capable of phagocytosis. Phagocytosis can include the recognition of particles larger than 0.5 pm and their uptake into a plasma membrane-derived vesicle, called a phagosome. Phagocytes can take up microbial pathogens and apoptotic cells. Thus, phagocytosis is not only essential for microbial elimination, but also for tissue homeostasis (Rosales, C, and Uribe-Querol, E., 2017. BioMed research international, 2017).

[0191] Suitably, the phagocytes targeted in the present application are liver and / or spleen phagocytes.

[0192] As used herein, a "liver phagocyte" can be a phagocyte that is predominantly present in liver tissue, and a "spleen phagocyte" can be a phagocyte that is predominantly present in spleen tissue.

[0193] Suitably, the phagocytes can be monocytes, macrophages, neutrophils, dendritic cells, eosinophils, fibroblasts, epithelial cells, and / or endothelial cells.

[0194] Suitably, the phagocyte can be a macrophage, a dendritic cell and / or a liver sinusoidal endothelial cell. For example, the phagocyte can be a liver and / or spleen macrophage, a liver and / or spleen dendritic cell and / or a liver sinusoidal endothelial cell.

[0195] Suitably, the phagocyte can be a professional phagocyte (e.g. a liver and / or spleen professional phagocyte), such as a monocyte, a macrophage, a neutrophil, a dendritic cell and an eosinophil. In some embodiments, the phagocyte is a macrophage and / or a dendritic cell.

[0196] Suitably, the phagocyte can be an unprofessional phagocyte, such as a fibroblast, an epithelial cell and / or an endothelial cell. In some embodiments, the phagocyte is an endothelial cell.

[0197] “Professional phagocytes” include monocytes, macrophages, neutrophils, dendritic cells, osteoclasts and eosinophils. These cells are responsible for eliminating microorganisms and presenting them to cells of the adaptive immune system. In addition, fibroblasts, epithelial cells and endothelial cells can also perform phagocytosis. These “unprofessional” phagocytes are not capable of taking up microorganisms, but are important in eliminating apoptotic bodies (Rosales, C and Uribe-Querol, E., 2017. BioMed research international, 2017).

[0198] dendritic cells

[0199] In some embodiments, the phagocyte is a macrophage (e.g. a liver and / or spleen macrophage).

[0200] Macrophages are innate immune cells that clear tissues of pathogens or other biological material. In adult mammals, macrophages are present in all tissues, where they exhibit great anatomical and functional diversity. In tissues, they organize in defined patterns, with each cell occupying its own territory. Macrophages have a role in almost every aspect of organismal biology, from development, homeostasis to repair, until the immune response to pathogens. Specifically, tumours are heavily populated with 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).

[0201] Liver macrophages can include liver-resident macrophages, infiltrating macrophages (e.g., bone marrow (BM)-derived macrophages), avascular peritoneal macrophages, and spleen-derived monocytes. Spleen macrophages can include marginal zone macrophages (MZMΦ), marginal metallophilic macrophages (MMMΦ), and red pulp macrophages (RpMΦ).

[0202] In some embodiments, the phagocyte is an M2-like macrophage and / or an MRC1+ macrophage (e.g., liver and / or spleen M2-like and / or MRC1+ macrophage).

[0203] Depending on the activation state and function of macrophages, they can be classified into Ml-like (classically activated macrophages) and M2-like (alternatively activated macrophages). Ml activation is induced by intracellular pathogens, bacterial cell wall components, lipoproteins, and cytokines such as interferon gamma and tumor necrosis factor alpha. Ml-like macrophages are characterized by inflammatory cytokine secretion and production of nitric oxide (NO), creating an effective pathogen-killing mechanism.

[0204] M2 activation is induced by fungal cells, parasites, immune complexes, complement, apoptotic cells, macrophage colony-stimulating factor, IL-4, IL-13, IL-10, tumor growth factor beta. M2-like macrophages have high phagocytic capacity, produce extracellular matrix (ECM) components, angiogenic factors, and chemokines, and IL-10. In addition to pathogen defense, M2-like macrophages clear apoptotic cells, can dampen inflammatory responses, and promote wound healing. M2-like macrophages are often referred to as anti-inflammatory, pro-resolution, wound-healing, tissue-repair, and regulatory or M2 macrophages (M2MΦ). Szer, T., 2015. Mediators of inflammation, 2015.

[0205] M2-like macrophages can be identified based on gene transcription or protein expression of a set of M2 markers, as described in Szer, T., 2015. Mediators of inflammation, 2015. These markers include transmembrane glycoproteins, scavenger receptors, enzymes, growth factors, hormones, cytokines, and cytokine receptors. Suitably, the M2-like macrophage expresses 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 phagocyte is an MRC1+ macrophage.

[0206]

[0207] ​The mannose receptor C-type 1 (MRC1) is also known as CD206, CLEC13D, and CLEC13DL. MRC1 is a C-type lectin that is found primarily on the surface of macrophages, immature dendritic cells, and liver sinusoidal endothelial cells, and mediates 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.

[0208] In mice and humans, M2-like polarized macrophages (including tumor-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).

[0209] In some embodiments, the phagocyte is a resident macrophage (e.g., a liver resident macrophage or a spleen resident macrophage).

[0210] Most tissues in vivo contain a population of tissue-resident macrophages. Tissue-resident macrophages are known for their role as immune sentinels in the front line of tissue defense, where they are discretely positioned and transcriptionally programmed to respond to pathogens or environmental challenges (Davies, L.C, et al., 2013. Nature immunology, 14(10), p. 986).

[0211] Liver-resident macrophages (also known as “liver macrophages”) include Kupffer cells and motile liver macrophages. Kupffer cells are maintained in adults independently of the bone marrow and serve to clear microorganisms and cellular debris from the blood and to clear senescent red blood cells. Phenotypic markers of Kupffer cells can include F4 / 80 hi , CD11b lo , CD169 + , CD68 + , galectin-3 + , and . Motile liver macrophages have an immune surveillance function and phenotypic markers can include F4 / 80 + , CD11b + , and CD80 hi (Davies, L.C, et al., 2013. Nature immunology, 14(10), p. 986).

[0212] Spleen-resident macrophages include marginal zone macrophages (MZMΦ), marginal metallophilic macrophages (MMMΦ), and red pulp macrophages (RpMΦ). Microanatomically, the spleen is divided into white pulp and red pulp (Rp), which are separated by the marginal zone (MZ). RpMΦ form a vast network inside the Rp and are characterized in mice by expression of F4 / 80 高 and strong autofluorescence. Inside the MZ, two populations of macrophages can be distinguished. MZMΦ typically express C-type lectin SIGN related 1 (SIGNR1) and a type I scavenger receptor called macrophage receptor with collagenous structure (MARCO) on their surface. MMMΦ are defined by expression of sialic acid binding Ig-like lectin-1 (Siglec-1, sialoadhesin, CD169) and MOMA-1, among other molecules.

[0213] In some embodiments, the phagocyte is an infiltrating macrophage (e.g., a liver infiltrating macrophage or a spleen infiltrating macrophage), e.g., a bone marrow (BM)-derived macrophage.

[0214] In some embodiments, the phagocyte is a perivascular peritoneal macrophage (PM).

[0215] PMs are present in the peritoneal cavity, have self-renewal capacity, and exist as two distinct subsets of PMs, namely large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs). LPMs are derived from embryonic precursors and represent the most abundant subset of phenotypes that display F4 / 80 高 CD11b 高 MHCII 低 under steady conditions. Whereas SPMs are a minor subset of phenotypes with F4 / 80 低 CD11b 低 MHCII 高 and are derived from BM-derived myeloid precursors and mainly appear during infection.

[0216] In some embodiments, the phagocyte is a monocyte-derived macrophage (e.g., a liver and / or spleen monocyte-derived macrophage).

[0217] Monocytes circulate in the blood and are recruited to mucosal tissues or sites of inflammation, 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 that contain many phagocytic vesicles. In contrast, dendritic cells are smaller and display dendrites on their surface (Segura, E and Coillard, A., 2019. Frontiers in immunology, 10, p. 1907).

[0218] In some embodiments, the phagocyte is a tumor-associated macrophage (e.g., a liver and / or spleen tumor-associated macrophage).

[0219] Tumor-associated macrophages (TAMs) are a class of macrophages that are present in higher numbers in the microenvironment of solid tumors. Tumor-associated macrophages (TAMs) contribute to tumor progression at different levels by promoting genetic instability, nurturing cancer stem cells, supporting metastasis, and taming protective adaptive immunity. TAMs can have dual supportive and inhibitory effects 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).

[0220] In some embodiments, the phagocyte is an MRC1+ liver macrophage (e.g., Kupffer cell) and / or an MRC1+ spleen macrophage.

[0221] In some embodiments, the phagocyte is a Kupffer cell.

[0222] endothelial cells

[0223] In some embodiments, the phagocyte is a dendritic cell (e.g., a liver and / or spleen dendritic cell).

[0224] Dendritic cells (DCs) are antigen-presenting cells of the mammalian immune system. Their main function is to process antigenic material and present it on the cell surface to T cells of the immune system.

[0225] In normal liver, DCs usually reside only around the portal triad and, like DCs in other peripheral sites, are efficient at capturing, processing and transporting antigen to local lymphoid tissue. In contrast to LSECs and KCs, freshly isolated liver DCs are predominantly immature cells, express surface MHC, but very little of the costimulatory molecules required for T cell activation (Lau, A.H. and Thomson, A.W., 2003. Gut, 52(2), pp.307-314).

[0226] Conventional / myeloid DCs (cDCs) and plasmacytoid DCs (pDCs) 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).

[0227] vectors

[0228] In some embodiments, the phagocyte is an endothelial cell (e.g. a liver and / or spleen endothelial cell). For example, the phagocyte can be a liver sinusoidal endothelial cell (LSEC).

[0229] LSECs have one of the highest endocytic capacities in the human body and can clear soluble macromolecules and small particles via endocytic receptors. Characteristics used to identify LSECs include: (a) their high and rapid endocytic capacity, (b) lack of septa and organization of fenestrae in the sieve plates, and (c) surface markers such as VEGFR3 + VEGFR2 + Ve-cadherin + Factor VIII + or CD31 + , LYVE-1 + , L-SIGN + , Stabilin-1 + , , PROX-1 − (Poisson, J. et al., 2017. Journal of hepatology, 66(1), pp.212-227).

[0230] In some embodiments, the phagocyte is a LSEC.

[0231] In another aspect, the present application provides a vector for LSEC-specific expression, wherein the vector comprises a transgene operably linked to one or more expression control sequences

[0232] phagocyte-specific expression

[0233] In one aspect, the present application provides a product comprising a vector for phagocyte-specific expression, in particular liver and / or spleen phagocyte-specific expression.

[0234] viral vectors

[0235] The vector can be a phagocyte-specific expression vector, in particular a liver and / or spleen phagocyte-specific expression vector. As used herein, the terms "phagocyte-specific expression", "liver phagocyte-specific expression" and "spleen phagocyte-specific expression" can refer to preferential or predominant expression of a transgene (e.g. as a polypeptide or RNA) in phagocytes compared to other cells (e.g. blood, lung and bone marrow cells). In some embodiments, at least 50% of transgene expression occurs in phagocytes. In some embodiments, at least 60%, 70%, 80%, 90% or 95% of transgene expression occurs in phagocytes. In some embodiments, a transgene is expressed substantially only in phagocytes.

[0236] For example:

[0237] (i) expression of a transgene in a phagocyte transduced by a vector can be greater than expression of the transgene in other cells transduced by the vector; and / or

[0238] (ii) a transgene can be expressed substantially not in cells other than phagocytes when transduced by a vector; and / or

[0239] (iii) a transgene can be expressed substantially not in lung cells, bone marrow cells and / or blood cells when transduced by a vector; and / or

[0240] (iv) a transgene can be expressed substantially only in some liver cells and / or some spleen cells; and / or

[0241] (v) expression of a transgene in Kupffer cells can be at least ten times greater than expression in liver cells when transduced by a vector; and / or

[0242] (vi) a transgene can be expressed substantially not in liver cells when transduced by a vector.

[0243] Expression of the transgene can 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 can be used to determine the level of expression in different cell types. Alternatively, if the transgene is a reporter gene (e.g. GFP), immunofluorescence analysis (e.g. by confocal imaging analysis) can be used to determine the level of expression in different cell types.

[0244] Suitably, expression of the transgene in phagocytic cells transduced by the vector can be greater than expression of the transgene in other cells transduced by the vector. For example, expression of the transgene in phagocytic cells transduced by the vector can be at least 10-fold, at least 20-fold, or at least 50-fold, or at least 100-fold greater than expression of the transgene in other cells transduced by the vector.

[0245] Suitably, the transgene is not substantially expressed in cells other than phagocytic cells when transduced by the vector. For example, the percentage of cells other than phagocytic cells expressing the transgene can be 5% or less, 2% or less, 1% or less, or 0%. For example, expression of the transgene in cells other than phagocytic cells can be undetectable.

[0246] Suitably, the transgene is not substantially 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 expressing the transgene can 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 can be undetectable.

[0247] Suitably, the transgene is substantially only expressed in hepatocytes and / or splenocytes. For example, the percentage of cell types other than hepatocytes and / or splenocytes expressing the transgene can be 5% or less, 2% or less, 1% or less, or 0%. For example, expression of the transgene in cell types other than hepatocytes and / or splenocytes can be undetectable.

[0248] Suitably, expression of the transgene in Kupffer cells when transduced by the vector can be at least ten-fold greater than expression in hepatocytes. For example, expression of the transgene in Kupffer cells can be at least ten-fold, at least twenty-fold or at least fifty-fold greater than expression in hepatocytes.

[0249] Suitably, the transgene can not be substantially expressed in hepatocytes when transduced by the vector. For example, the percentage of hepatocytes expressing the transgene can be 5% or less, 2% or less, 1% or less, or 0%. For example, expression of the transgene in hepatocytes can be undetectable.

[0250] Suitably, expression of the transgene in Kupffer cells can be at least ten times greater than expression in LSECs when transduced by the vector. For example, expression of the transgene in Kupffer cells can be at least ten times, at least twenty times, or at least fifty times greater than expression in LSECs.

[0251] Suitably, the transgene can be substantially not expressed in LSECs when transduced by the vector. For example, the percentage of LSECs expressing the transgene can be 5% or less, 2% or less, 1% or less, or 0%. For example, expression of the transgene in LSECs can be undetectable.

[0252] If the vector is an integrating vector (e.g. integrase proficient), copies of the vector can be specifically integrated into phagocytes, in particular liver and / or spleen phagocytes, for example. For example:

[0253] (i) integration of the vector in the liver and spleen can be greater than integration of the vector in other organs (e.g. lymph nodes, brain, small intestine, blood, bone marrow); and / or

[0254] (ii) integration of the vector can substantially occur in the liver, spleen, optionally blood, and optionally bone marrow; and / or

[0255] (iii) integration of the vector can substantially not occur in lymph nodes, brain, small intestine.

[0256] Integration of the vector can be determined by any suitable method known to the skilled person. For example, viral copy number analysis, for example by quantitative digital droplet PCR of different organs.

[0257] Suitably, integration of the vector in the liver and spleen is greater than integration of the vector in other organs such as lymph nodes, brain, small intestine, blood, bone marrow. For example, viral copy number in the liver and spleen can be at least 10 times, at least 20 times, or at least 50 times, or at least 100 times greater than in other organs.

[0258] Suitably, integration of the vector substantially occurs in the 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 can be at least detectable.

[0259] Suitably, integration of the vector substantially does not occur in lymph nodes, brain, small intestine. For example, integration of the vector in lymph nodes, brain, small intestine can be undetectable. All of these biological compartments harbour resident macrophage populations which can potentially express the transgene upon systemic delivery of the vector.

[0260] retroviral and lentiviral vectors

[0261] Suitably, the vector of the application is a viral vector. The vector of the application can be a lentiviral vector, but it is also contemplated that other viral vectors can be used.

[0262] Other suitable viral vectors include those described in Lundstrom, K., 2018. Diseases, 6(2), p. 42. For example, other suitable viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, alphavirus vectors, flavivirus vectors, rhabdovirus vectors, measles virus vectors, Newcastle disease virus vectors, poxvirus vectors, and picornavirus vectors.

[0263] The vector of the application can be in the form of a viral vector particle. Suitably, the viral vector of the application is in the form of a lentiviral vector particle.

[0264] The vector can be an integrating viral vector or a non-integrating viral vector. An “integrating viral vector” is capable of integrating into the host cell genome after transduction into a host cell. A “non-integrating viral vector” is not capable of integrating into the host cell genome after transduction into a host cell, or exhibits very weak integration capacity.

[0265] Methods of making 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), p. 475-489; Ayuso, E., et al., 2010. Current gene therapy, 10(6), p. 423-436; and Goins, W.F., et al., 2008. Methods Mol Biol. 433, p. 97-113.

[0266] adenoviral vectors

[0267] The vector of the application can be a retroviral vector or a lentiviral vector. The vector of the application can be a retroviral vector particle or a lentiviral vector particle.

[0268] Retroviral vectors can be derived from, or can be derivable from, any suitable retrovirus. A large number of different retroviruses have been identified. Examples include murine leukemia virus (MLV), human T cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29), and avian erythroblastosis virus (AEV).

[0269] Retroviruses can be broadly classified into two categories: "simple" and "complex". Retroviruses can even be further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are lentiviruses and spumaviruses.

[0270] The basic structure of retroviral and lentiviral genomes share many common features, such as 5' LTR and 3' LTR. Positioned between or within these features are packaging signals that enable the genome to be packaged, primer binding sites, integration sites that allow integration into the host cell genome, and gag, pol, and env genes that encode packaging components that are required polypeptides for viral particle assembly. Lentiviruses have additional features, such as the rev and RRE sequence in HIV, which enable efficient export of RNA transcripts of the integrated provirus from the nucleus of the infected target cell to the cytoplasm.

[0271] In the provirus, these genes are flanked at both ends by regions called long terminal repeat sequences (LTRs). These LTRs are responsible for provirus integration and transcription. The LTRs also act as enhancer-promoter sequences and can control expression of the viral genes.

[0272] These LTRs are identical sequences themselves, which can be divided into three elements: U3, R, and U5. U3 is derived from a 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 a sequence unique to the 5' end of the RNA. The size of these three elements can vary greatly among different retroviruses.

[0273] In a defective retroviral vector genome, gag, pol, and env can not be present or functional.

[0274] In a typical retroviral vector, at least a portion of one or more protein coding regions necessary for replication can be removed from the virus. This renders the viral vector replication defective. Portions of the viral genome can also be replaced with a library of candidate regulatory moieties operably linked to a regulatory control region and a reporter moiety in the vector genome, thereby generating a vector comprising a candidate regulatory moiety that is capable of transducing a target host cell and / or integrating its genome into the host genome.

[0275] Lentiviral vectors are part of a larger group of retroviral vectors. Briefly, lentiviruses can be divided into a primate group and a non-primate group. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of human acquired immune deficiency syndrome (AIDS); and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototypic "slow virus" visna / maedi virus (VMV), as well as the related caprine arthritis- encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), and the recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[0276] The lentivirus family differs from retroviruses in that lentiviruses have the ability 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 cells that make up, for example, muscle, brain, lung, and liver tissue.

[0277] As used herein, a "lentiviral vector" is a vector comprising at least one component derivable from a lentivirus. Suitably, the component is involved in the biological mechanism by which the vector infects a cell, expresses a gene, or is replicated.

[0278] A lentiviral vector can be a "primate" vector. A lentiviral vector can be a "non-primate" vector (i.e., derived from a virus that does not naturally infect primates, especially humans). An example of a non-primate lentivirus can be any member of the Lentiviridae family that does not naturally infect primates.

[0279] As an example of a lentivirus-based vector, HIV-1 and HIV-2 based vectors are described below.

[0280] HIV-1 vectors contain cis-acting elements that are also found in simple retroviruses. It has been shown that sequences extending into the gag open reading frame are important for packaging of HIV-1. Therefore, HIV-1 vectors usually contain a relevant portion of gag in which the translation 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 the RRE, allowing the transport of full-length or singly spliced mRNA from the nucleus to the cytoplasm. In the absence of Rev and / or RRE, full-length HIV-1 RNA accumulates in the nucleus. Alternatively, constitutive transport elements from certain simple retroviruses, such as Mason-Pfizer monkey virus, can be used to obviate the need for Rev and RRE. Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.

[0281] Most HIV-2-based vectors are very similar in structure to HIV-1 vectors. Like HIV-1 -based vectors, HIV-2 vectors also require the RRE for efficient transport of full-length or singly spliced viral RNA.

[0282] Optionally, the viral vectors used in the present application have a minimal viral genome.

[0283] By "minimal viral genome" it is understood that the viral vector has been manipulated to remove non-essential elements and retain essential elements, thereby endowing it with the required functions of infecting, transducing and delivering a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in WO 1998 / 017815.

[0284] Optionally, the plasmid vector used to produce the viral genome within the host cell / packaging cell will have sufficient lentiviral genetic information to allow packaging of the RNA genome into a viral particle capable of infecting a target cell but not capable of independent replication in the presence of the packaging components, thereby producing an infectious viral particle within the final target cell. Optionally, the vector lacks functional gag-pol and / or env genes and / or other genes essential for replication.

[0285] However, the plasmid vector used to produce the viral genome within the host cell / packaging cell will also comprise transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in the host cell / packaging cell. These regulatory sequences can be the natural sequences associated with the transcribed viral sequences (i.e. the 5' U3 region), or they can be heterologous promoters, such as another viral promoter (e.g. the CMV promoter).

[0286] The vector can be a self-inactivating (SIN) vector in which the viral enhancer and promoter sequences have been deleted. SIN vectors can be generated in vivo and transduce non-dividing cells in vivo with efficiencies similar to those of wild-type vectors. Transcriptional inactivation of the long terminal repeat (LTR) in SIN proviruses should prevent mobilization by replication-competent viruses. This should also enable regulatable expression of genes under the control of internal promoters by eliminating any cis-acting effects of the LTR.

[0287] The vector can be integrase-defective (i.e. integrase-deficient). Integrase-defective lentiviral vectors can be generated, for example, by packaging the vector with catalytically inactive integrase, such as HIV integrase carrying a D64V mutation in the catalytic site; or by modifying or deleting the essential att sequences in the vector LTR; or a combination of the above.

[0288] In some embodiments, the vector is an integrase-defective lentiviral vector. In some embodiments, the vector is an integrase-competent lentiviral vector.

[0289] Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors (LVs) can be efficient when delivered systemically and specifically target the liver and are preferentially internalized by the liver and spleen 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 an excellent tool to deliver genes of interest to the hepatocyte population.

[0290] Suitably, the vector is VSV-G pseudotyped. In some embodiments, the vector is a VSV-G pseudotyped lentiviral vector particle.

[0291] Gene transfer into professional phagocytes and antigen-presenting cells (APCs) is constrained by the presence of CD47 molecules on LV particles. CD47-null LVs show retained infectivity and significantly increased susceptibility to phagocytosis. CD47-null LVs transduce professional phagocytes more efficiently ex vivo and in vivo compared to CD47-carrying LVs and induce significantly higher elevations in cytokine responses after systemic administration to mice. CD47-null LVs allow increased gene transfer efficiency into human primary monocytes and have increased susceptibility to phagocytosis by primary human macrophages ex vivo and in vivo when administered systemically to mice compared to previously available LVs. For example, VSV-G pseudotyped LVs lacking CD47 molecules on the surface are even more efficiently taken up by professional phagocytes of the liver and spleen than VSV-G pseudotyped LVs carrying CD47.

[0292] Allogeneic human leukocyte antigens (HLA), such as MHC-I, can also be recognized by the immune system. For example, antibodies can directly bind to HLA epitopes. Thus, cells and enveloped viruses comprising HLA proteins derived from an allogeneic source can be targeted and neutralized by the immune system. A reduction or lack of surface-exposed HLA molecules is advantageous in viruses used as vaccines, as the viruses will be less likely to be neutralized by antibodies that bind to HLA.

[0293] Suitable methods of producing CD47-null and / or HLA-null vectors are described in WO 2019 / 219836.

[0294] In some embodiments, the vector is substantially free of surface-exposed CD47 and / or HLA molecules. In some embodiments, the vector is a VSV-G pseudotyped lentiviral vector particle that is substantially free of surface-exposed CD47 and / or HLA molecules.

[0295] As used herein, the term“substantially free of’ means a significant reduction in the number of molecules expressed on the surface of the vector as compared to the number of molecules expressed on the surface of a vector produced in a cell that has not been genetically engineered to reduce expression of the molecules, but otherwise is substantially identical, such that the vector exhibits a therapeutically useful increase in the ability to transduce macrophages, phagocytes, antigen presenting cells, and / or monocytes and / or induce a cytokine response following systemic administration.

[0296] 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 that does not comprise any surface-exposed CD47 molecules and / or HLA molecules.

[0297] adeno-associated viral vectors

[0298] The vector of the application can be an adenoviral vector. The vector of the application can be an adenoviral vector particle.

[0299] Adenoviruses are double-stranded linear DNA viruses that do not go through an RNA intermediate. There are over 50 different human adenovirus serotypes, which are grouped into six subgroups based on genetic sequence homology. The natural target of adenoviruses are respiratory and gastrointestinal epithelial cells, often causing only mild symptoms. Serotypes 2 and 5, which have 95% sequence homology, are most commonly used in adenoviral vector systems and are typically associated with upper respiratory tract infections in adolescents.

[0300] Adenoviruses have been used as vectors for gene therapy and heterologous gene expression. This large (36 kb) genome can accommodate up to 8 kb of foreign insert DNA and is able to replicate efficiently in complementing cell lines, producing high titers of virus (up to 10 12of the adenovirus. Thus, adenoviruses are one of the best systems to study expression of genes in primary non-replicating cells.

[0301] Replicating cells are not required for expression of viral genes or foreign genes from the adenovirus genome. Adenoviral vectors enter cells via receptor-mediated endocytosis. Once inside the cell, adenoviral vectors rarely integrate into the host chromosome. Rather, they function episomally (independently of the host genome) in the host cell nucleus. Thus, use of recombinant adenoviruses mitigates problems associated with random integration into the host genome.

[0302] herpes simplex viral vectors

[0303] The vector of the application can be an adeno-associated virus (AAV) vector. The vector of the application can be in the form of an AAV vector particle.

[0304] An AAV vector or AAV vector particle can comprise an AAV genome or a fragment or derivative thereof. An AAV genome is a polynucleotide sequence that can encode functions required for production of AAV particles. These functions include those involved 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-defective and rely on the provision of helper functions in trans to complete the replication and packaging cycle. Thus, AAV genomes are typically replication-defective.

[0305] An AAV genome can be in single-stranded form (sense or antisense), or alternatively, in double-stranded form. Use of the double-stranded form allows bypassing of the DNA replication step in the target cell, and thus can accelerate transgene expression.

[0306] AAVs that occur in nature can be classified according to various biological systems. An AAV genome can be from any naturally derived serotype, isolate or clade of AAV.

[0307] AAVs can be referred to according to their serotype. A serotype corresponds to a variant subspecies of AAV that has a unique reactivity due to its expression profile of capsid surface antigens, whereby it can be used to distinguish it from other variant subspecies. Typically, an AAV vector particle of a particular AAV serotype does not efficiently cross-react with neutralizing antibodies specific for any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11.

[0308] AAVs can also be referred to according to clade or clone. This refers to the phylogenetic relationship of naturally derived AAVs and generally refers to phylogenetic groups of AAVs that can be traced to a common ancestor and includes all descendants thereof. In addition, AAVs can be referred to according to a particular isolate, i.e., a genetically isolated strain of a particular AAV found in nature. The term genetically isolated strain describes a population of AAVs that undergoes limited genetic mixing with other naturally occurring AAVs, thereby defining a recognizable different population at the genetic level.

[0309] Generally, the AAV genome of a naturally derived serotype, isolate, or clade of AAVs comprises at least one inverted terminal repeat sequence (ITR). The ITR sequence provides a functional origin of replication in cis and allows for integration and excision of the vector from the cellular genome. The ITR can be the only sequence required for the transgene to be linked in cis. Suitably, one or more ITR sequences flank the transgene.

[0310] The AAV genome can also comprise packaging genes, such as rep and / or cap genes that encode the packaging functions of the AAV particle. A promoter can be operably linked to each of the packaging genes. Particular examples of such promoters include the p5, p19, and p40 promoters. For example, the p5 and p19 promoters are typically used to express the rep genes, while the p40 promoter is typically used to express the cap genes. The rep genes encode one or more of the proteins Rep78, Rep68, Rep52, and Rep40 or variants thereof.

[0311] The cap genes encode one or more capsid proteins, such as VP1, VP2, and VP3 or variants thereof.

[0312] The AAV genome can be the full genome of a naturally occurring AAV. For example, a vector comprising the full genome of an AAV can be used to make an AAV vector or vector particle.

[0313] Suitably, the AAV genome is derivatized for the purposes of administration to a patient. Such derivatization is standard in the art and the present application encompasses the use of any known derivative of an AAV genome, as well as derivatives that can be generated by the application of techniques known in the art. The AAV genome can 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.

[0314] Derivatives of the AAV genome include any truncated or modified form of the AAV genome that allows expression of a transgene from the AAV vector of the application in vivo. Typically, the AAV genome can be significantly truncated to comprise minimal viral sequences, yet retain the functions described above. This can reduce the risk of recombination of the vector with wild-type virus, and avoid triggering a cellular immune response due to the presence of viral gene proteins in the target cell.

[0315] Typically, the derivative will comprise at least one inverted terminal repeat sequence (ITR), optionally more than one ITR, such as two ITRs or more. One or more of these ITRs can be derived from an AAV genome of a different serotype, or can be a chimeric or mutant ITR. Suitable mutant ITRs are ITRs in which the trs (terminal resolution site) has been deleted. This deletion allows the genome to continue replication to generate single-stranded genomes containing both the coding sequence and the complementary sequence, i.e. self-complementary AAV genomes. This allows bypassing of DNA replication in the target cell, and thus enables accelerated transgene expression.

[0316] The AAV genome can comprise one or more ITR sequences from any naturally derived serotype, isolate or clade of AAV or variants thereof. The AAV genome can comprise at least one (such as two) AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11 ITR, or variants thereof.

[0317] One or more ITRs can flank the transgene at either end. Inclusion of one or more ITRs can aid the formation of concatemers of the AAV vector in the nucleus of the host cell, for example after conversion of single-stranded vector DNA to double-stranded DNA under the action of the host cell DNA polymerase. Formation of such episomal concatemers protects the AAV vector during the life cycle of the host cell, allowing long-term expression of the transgene in vivo.

[0318] Suitably, the ITR element will be the only sequence retained in the derivative from the native AAV genome. Suitably, the derivative can not comprise the rep and / or cap genes of the native genome, and any other sequences of the native genome. This can reduce the likelihood of integration of the vector into the host cell genome. Additionally, reducing the size of the AAV genome allows increased flexibility to incorporate other sequence elements (such as regulatory elements) within the vector in addition to the transgene.

[0319] Thus, in derivatives of the application the following can be removed: one inverted terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes. However, derivatives can additionally comprise one or more rep and / or cap genes or other viral sequences of the AAV genome. Naturally occurring AAV integrates at a specific site on human chromosome 19 with high frequency and shows negligible random integration frequency, such that retention of integration ability in AAV vectors can be tolerated in a therapeutic setting.

[0320] The application additionally encompasses providing sequences of the AAV genome in a different order and configuration to the native AAV genome. The application also encompasses replacing one or more AAV sequences or genes with a chimeric gene composed of sequences from another virus or with sequences from more than one virus. Such chimeric genes can be composed of sequences from two or more related viral proteins from different viral species.

[0321] AAV vector particles can be encapsidated by capsid proteins. Suitably, AAV vector particles can be in the form of transcapsidated, in which an AAV genome or derivative with the ITR of one serotype is packaged in a capsid of a different serotype. AAV vector particles also include mosaic forms, in which a mixture of unmodified capsid proteins from two or more different serotypes make up the viral capsid. AAV vector particles also include chemically modified forms carrying ligands adsorbed to the surface of the capsid. For example, such ligands can include antibodies for targeting specific cell surface receptors.

[0322] When derivatives comprise capsid proteins (i.e. VP1, VP2 and / or VP3), the derivatives can be chimeric, shuffled or capsid-modified derivatives of one or more naturally occurring AAV. In particular, the application encompasses providing capsid protein sequences from different serotypes, clades, clones or isolates of AAV within the same vector (i.e. pseudotyped vectors). The AAV vector can be in the form of a pseudotyped AAV vector particle.

[0323] Chimeric, shuffled or capsid-modified derivatives will generally be selected to provide one or more desired functions of the AAV vector. Thus, these derivatives can exhibit improved gene delivery efficiency and / or reduced immunogenicity (humoral or cellular) compared to AAV vectors comprising naturally occurring AAV genomes. Improved gene delivery efficiency can be achieved, for example, by enhanced binding of receptors or co-receptors at the cell surface, enhanced internalization, enhanced intracellular and nuclear transport, enhanced uncoating of the viral particle and enhanced conversion of the single-stranded genome to the double-stranded form.

[0324] Chimeric capsid proteins include those generated by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This can be performed, for example, by a marker rescue method 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 with the desired properties. The capsid sequences of the different serotypes can be altered by homologous recombination within the cell to generate new chimeric capsid proteins.

[0325] Chimeric capsid proteins also include those generated by engineering 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.

[0326] Shuffled or chimeric capsid proteins can also be generated by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be produced by random fragmentation of the sequences of related AAV genes, for example, those encoding capsid proteins of multiple different serotypes, followed by reassembly of the fragments in a self-priming polymerase reaction, which can also result in crossover exchanges in regions of sequence homology. Libraries of hybrid AAV genes produced in this way by shuffling capsid genes of several serotypes can be screened to identify viral clones with the desired functions. Similarly, error-prone PCR can be used to randomly mutate AAV capsid genes to produce a diverse library of variants, which can then be selected for desired properties.

[0327] Genetic modifications can also be made to the sequence of the capsid gene to introduce specific deletions, substitutions, or insertions relative to the native wild-type sequence. In particular, the capsid gene can be modified by inserting the sequence of an unrelated protein or peptide within the open reading frame of the capsid coding sequence or at the N- and / or C-terminus of the capsid coding sequence. The unrelated protein or peptide can advantageously be one that acts as a ligand for a particular cell type, conferring enhanced binding to the target cell or increasing the specificity of the vector for targeting a particular cell population. The unrelated protein can also be one that aids in the 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 chosen so as not to interfere with other functions of the viral particle, for example, internalization, trafficking of the viral particle.

[0328] The capsid protein can be an artificial or mutant capsid protein. As used herein, the term "artificial capsid" means that the capsid particle comprises an amino acid sequence that does not occur in nature or comprises an amino acid sequence that 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 variation in the amino acid sequence as compared to the sequence of the parent capsid from which the artificial capsid protein is derived, where the artificial capsid amino acid sequence and the parent capsid amino acid sequence are aligned.

[0329] other viral vectors

[0330] The vector of the application can be a herpes simplex virus vector. The vector of the application can be a herpes simplex virus vector particle.

[0331] Herpes simplex virus (HSV) is a neurotropic DNA virus with advantageous properties as a delivery vector. HSV is highly infectious, and thus HSV vectors are efficient vehicles for the delivery of foreign genetic material to cells. Viral replication is easily disrupted by null mutations in immediate early genes, which can be complemented in trans in vitro, enabling direct production of high-titer, pure preparations of non-pathogenic vectors. The genome is large (152 Kb), and many viral genes are dispensable for in vitro replication, allowing them to be replaced by large or multiple transgenes. The potential for latent infection of wild-type virus results in persistence of episomal virus in sensory nerve nuclei for the duration of the host life cycle. The vectors are non-pathogenic, cannot reactivate, and persist long-term. Latent active promoter complexes can be exploited in vector design to achieve long-term stable transgene expression in the nervous system.

[0332] Due to the broad expression pattern of cellular receptors recognized by the virus, HSV vectors transduce a broad range of tissues. Increased understanding of the processes involved in cellular entry has allowed targeting of the tropism of HSV vectors.

[0333] expression control sequences

[0334] Other suitable viral vectors include those described in Lundstrom, K., 2018. Diseases, 6(2), p. 42.

[0335] The vector of the application can be an alphavirus vector. The vector of the application can be an alphavirus vector particle. The vector of the application can be a flavivirus vector. The vector of the application can be a flavivirus vector particle.

[0336] Self-amplifying ssRNA viruses include 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) that have positive-sense genomes. Alphaviruses have been applied primarily to preclinical gene therapy research for cancer treatment. Alphavirus vectors can be delivered as naked RNA, as a tiered plasmid DNA vector, and as recombinant replication-defective or replication-competent particles.

[0337] The vector of the present application can be a rhabdovirus vector. The vector of the present application can be a rhabdovirus vector particle. The vector of the present application can be a measles virus vector. The vector of the present application can be a measles virus vector particle.

[0338] Rhabdoviruses (e.g., rabies and vesicular stomatitis virus) and measles viruses carry negative-strand genomes. In rhabdoviruses, recombinant vesicular stomatitis virus (VSV) has been applied to preclinical gene therapy research. Measles viruses (e.g., MV-Edm) have found multiple gene therapy applications.

[0339] The vector of the present application can be a Newcastle disease virus vector. The vector of the present application can be a Newcastle disease virus vector particle.

[0340] The ssRNA paramyxovirus Newcastle disease virus (NDV) replicates specifically in tumor cells and has therefore been frequently applied to cancer gene therapy.

[0341] The vector of the present application can be a poxvirus vector. The vector of the present application can be a poxvirus vector particle.

[0342] Poxviruses are characterized by their dsDNA genomes, which can accommodate foreign DNA of more than 30 kb in size. Poxviruses have found several applications as gene therapy vectors. For example, vaccinia virus vectors have been shown to have potential for the treatment of cancer. Vaccinia virus is a large, enveloped poxvirus with a linear, double-stranded DNA genome of about 190 kb. Vaccinia virus can accommodate foreign DNA of up to about 25 kb in size, which also makes it useful for the delivery of large genes. A number of attenuated vaccinia virus strains suitable for gene therapy applications are known in the art, such as the MVA and NYVAC strains.

[0343] The vector of the present application can be a picornavirus vector. The vector of the present application can be a picornavirus vector particle.

[0344] Picornaviridae. Coxsackievirus, which belongs to the Picornaviridae family of small RNA viruses, has been used as an oncolytic vector.

[0345] MRC1 -derived expression control sequences

[0346] The vector of the application can comprise one or more expression control sequences. Suitably, the transgene is operably linked to one or more expression control sequences.

[0347] As used herein, an "expression control sequence" is any nucleotide sequence that controls the expression of a transgene, for example to promote and / or increase expression in some cell types and / or to reduce expression in other cell types.

[0348] The expression control sequences and the transgene can be in any suitable arrangement in the vector, provided that the expression control sequence(s) is operably linked to the transgene. As used herein, the term "operably linked" means that the parts (e.g. the transgene and the expression control sequence(s)) are connected in such a way as to enable them to perform their functions substantially unimpeded.

[0349] The expression control sequence can be a phagocyte-specific expression control sequence, in particular a liver and / or spleen phagocyte-specific expression control sequence (e.g. such that the vector specifically expresses the transgene in phagocytes, in particular liver and / or spleen phagocytes). The expression control sequence includes promoters, enhancers, and 5' and 3' untranslated regions (e.g. miRNA target sequences).

[0350] The one or more expression control sequences can comprise: (a) a phagocyte-specific promoter and / or enhancer; and / or (b) one or more miRNA target sequences.

[0351] In some embodiments, the one or more expression control sequences comprise a phagocyte-specific promoter and / or enhancer, and optionally one or more miRNA target sequences.

[0352] The vector can comprise, for example from 5' to 3': a phagocyte-specific promoter and / or enhancer - transgene - one or more miRNA target sequences.

[0353] phagocyte-specific promoters

[0354] Suitably, the vector of the application can comprise one or more MRC1 -derived expression control sequences.

[0355] As used herein, “MRC1 -derived expression control sequence” is an expression control sequence that includes any 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.

[0356] Regulatory features present in the MRC1 gene can 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-gene and promoter-gene associations. Regulatory features present in the MRC1 gene include the MRC1 promoter, MRC1 enhancers, and MRC1 5’ and 3’ UTRs. The mannose receptor regulatory sequence is at least partially immediately upstream of the transcription start site (Eichbaum, Q, et al., Blood, 1997. 90(10): p. 4135-43).

[0357] MRC1 promoters

[0358] The vector of the application can comprise a phagocyte-specific promoter, in particular a liver and / or spleen phagocyte-specific promoter. Suitably, the transgene is operably linked to a phagocyte-specific promoter, in particular a liver and / or spleen phagocyte-specific promoter.

[0359] A “promoter” is a region of DNA that directs the initiation of transcription of a gene. The promoter is located near the transcription initiation site of a gene, upstream (towards the 5’ region of the sense strand) of the DNA.

[0360] As used herein, a “phagocyte-specific promoter” can be a promoter that enables phagocyte-specific expression of a transgene operably linked to the promoter

[0361] Exemplary phagocyte-specific promoters include the MRC1 promoter; the ITGAM promoter; the CD86 promoter; the CD274 promoter; the CD163 promoter; the LYVE1 promoter; the STAB1 promoter; the ITGAX promoter; the SIRPA promoter; the TIE2 promoter; the CHIL3 promoter; the CD68 promoter; the CSF1R promoter; the VCAM1 promoter; the PTGS1 promoter; and the C1QA promoter.

[0362] Engineered promoter variants derived from any of these promoters can be used, provided that the variant retains the ability to drive phagocyte-specific expression of a transgene operably linked to the promoter. The skilled person will obtain such variants using methods known in the art. The variants can be at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any of the promoters.

[0363] Fragments of any of these promoters (or variants thereof) can be used, provided that the fragment retains the ability to drive phagocyte-specific expression of a transgene operably linked to the promoter. The skilled person will be able to obtain such fragments using methods known in the art. The fragments can 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.

[0364] In some embodiments, the phagocyte-specific promoter is selected from the group consisting of: the MRC1 promoter; the ITGAM promoter; the CD86 promoter; the CD274 promoter; the CD163 promoter; the LYVE1 promoter; the STAB1 promoter; the ITGAX promoter; the SIRPA promoter; the TIE2 promoter; the CHIL3 promoter; the CD68 promoter; the CSF1R promoter; the VCAM1 promoter; the PTGS1 promoter; and the C1QA promoter; or a variant and / or fragment thereof.

[0365] In preferred embodiments, the phagocyte-specific promoter is the MRC1 promoter or a variant and / or fragment thereof.

[0366] exemplary human MRC1 promoters

[0367] In one aspect, the application provides a vector comprising the MRC1 promoter. Suitably, a transgene is operably linked to the MRC1 promoter.

[0368] Any suitable method can be used to identify the MRC1 promoter, for example by using a promoter prediction tool or by using the sequence immediately upstream of the MRC1 open reading frame. Suitably, the MRC1 promoter can be the sequence immediately upstream of the MRC1 open reading frame for about 0.2kb to 5kb, 0.5kb to 5kb, 1kb to 2kb, or about 1.8kb.

[0369] In some embodiments, the MRC1 promoter comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 1. Suitably, the MRC1 promoter comprises or consists of a nucleotide sequence that 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.

[0370] In some embodiments of the application, the MRC1 promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof.

[0371] exemplary mouse MRC1 promoters :

[0372]

[0373] (SEQ ID NO: 1)

[0374] In some embodiments of the application, 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.

[0375] In some embodiments of the application, the MRC1 promoter comprises or consists of the nucleotide sequence SEQ ID NO: 2 or a fragment thereof.

[0376] exemplary XhoI-human.MRC1.promoter :

[0377]

[0378] (SEQ ID NO: 2)

[0379] In some embodiments of the application, 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.

[0380] 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.

[0381] In some embodiments of the application, the MRC1 promoter comprises or consists of the nucleotide sequence SEQ ID NO: 31, or a fragment thereof.

[0382] inducible promoters

[0383]

[0384] (SEQ ID NO: 31)

[0385] phagocyte-specific enhancers

[0386] Suitably, the phagocyte-specific promoter can be an inducible promoter

[0387] As used herein, an “inducible promoter” is a promoter that is active only under specific conditions. For example, expression of a transgene can be induced by a small molecule or drug (e.g., that binds to a promoter, regulatory sequence, or to a transcriptional repressor or activator molecule) or by the use of an environmental trigger. Types of inducible promoters include chemically inducible promoters (e.g., Tet-on systems); temperature inducible promoters (e.g., Hsp70 or Hsp90 derived promoters); and light inducible promoters. Suitably, the promoter is chemically inducible.

[0388] Any suitable method can be used to engineer an inducible phagocyte-specific promoter.

[0389] Alternatively, the phagocyte-specific promoter can be a constitutive promoter. As used herein, a “constitutive promoter” is a promoter that is active at all times.

[0390] mouse Mrc1 enhancer 1

[0391] The vectors of the application can comprise a phagocyte-specific enhancer. Suitably, the transgene is operably linked to a phagocyte-specific enhancer.

[0392] An “enhancer” is a region of DNA that can be bound by a protein (activator) 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) from a gene, upstream or downstream of the start site.

[0393] As used herein, a “phagocyte-specific enhancer” can be an enhancer that enables phagocyte-specific expression of a transgene operably linked to the enhancer.

[0394] Exemplary phagocyte-specific enhancers include an MRC1 enhancer; an ITGAM enhancer; a CD86 enhancer; a CD274 enhancer; a CD163 enhancer; a LYVE1 enhancer; a STAB1 enhancer; an 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.

[0395] Engineered enhancer variants derived from any of these enhancers can be used, provided that the variant retains the ability to drive phagocyte-specific expression of a transgene operably linked to the enhancer. The skilled person will obtain such variants using methods known in the art. The variant can be at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to any enhancer.

[0396] A fragment of any of these enhancers (or variants thereof) can be used, provided that the fragment retains the ability to drive phagocyte-specific expression of a transgene operably linked to the enhancer. The skilled person will be able to obtain such fragments using methods known in the art. The fragment can be at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, or at least 1000 nucleotides in length.

[0397] In some embodiments, the phagocyte-specific enhancer is selected from the group consisting of: an MRC1 enhancer; an ITGAM enhancer; a CD86 enhancer; a CD274 enhancer; a CD163 enhancer; a LYVE1 enhancer; a STAB1 enhancer; an 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 variants and / or fragments thereof.

[0398] In preferred embodiments, the phagocyte-specific enhancer is an MRC1 enhancer or a variant and / or fragment thereof.

[0399] The vectors of the application can 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 the phagocyte-specific promoter and / or enhancer.

[0400] In some embodiments, the phagocyte-specific promoter and / or enhancer is selected from the group consisting of: an 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; an 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 variants and / or fragments thereof.

[0401] The phagocyte-specific promoter and the phagocyte-specific enhancer can be a combination of any of the above, for example the MRC1 promoter and the ITGAM enhancer.

[0402] In preferred embodiments, the phagocyte-specific promoter and / or enhancer is the MRC1 promoter and / or enhancer or a variant and / or fragment thereof.

[0403] Exemplary MRC1 enhancers can include:

[0404] human MRC1 enhancer 1

[0405] ACAGAACCAGCAGTATAGGGAAGGCCGTGGTGTTGTGGGACTCACATGATATTATTTATGATATCTTGGAAATTAGAGCAAAGACAGGTTAGGCATTGTGGTCAGAGGAGCTGGGTTATGACACCGAGGAAACAAGCTGACCCTTGAATTAAAACATATTGACGCCATAGCAATAAGAGGATGGAACCACATTGCCCTCTGCTGTTGGGGAATCATGGCCGCTGCCCCCATTCTGCAGTTAAGAGACCCGGTACTGCCCTCTGCTGGCTGGATGCACATGTTTCCACATTCTGGATTAGTATCCTTTTGAATTTAAATTTAAAAACAGTCTCCTGCTGCCTGCCAGTGACTCACTGTGGCCTCTTTATGTTGTTAGTAGCTTTGTTTTACTCTGGCAGATAGAAAATATGTTACAGGTCGCCATCTTGGTTCCGGGACTCAGCA

[0406] (SEQ ID NO: 17)

[0407] mouse Mrc1 enhancer 2

[0408] AGCCCCACCATGTTATTGATGGCCAAACAATACGCATGCTGACAGCCATTATCTGTGGCCTCTGATGCTATTAGCCAAACCATGTTATTGATGGTCAAACAATACGCATGCTGACAGCCATTATCTGGGACTCAGAAAGTTCTGCATATTCAAGTCAGGCCAGAGGATCCGAGTTCTAATGTTAAGAGAAACCAACACACCAACAAGCAAATAAACAAACCTACCCTTGAACCAAAATATACATCAATACCTCCGTTGCAAATGGATAAATGGAACTGCATTGCCCTCTGCTGTTGGGGAATCTTGGCAACCATTTCAACTCTATGGCTGGAGATGACTTACTGCTCTGTTTATTTTCCATCCTCCTGCTTAGATTATTGCTTTCAAAGTTTCCAGAATAGAAGAAGTCAGTGGTGGCCAGTTGTCCTTTAATGGTCTCTTATCTACCAATGGCTAGTATCCTTTTTGCATTATCGTAGCTCTACTCTTGTAGATGTTAAATT

[0409] (SEQ ID NO: 18)

[0410] human MRC1 enhancer 2

[0411] ACATGGGAGGCAAGGCGGAAGGAGCATGAGGCTGACCTAGCAGGCAGGAAGCACAGAAATCACATTTTGAGCTACATAGAAGAAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAATCAGGAAGTGAGACTAGTCTATAAAACTGCAAAGCCTACTCCCACTGACATACTTCCTTTAGCAATGCACAGCTGCCACAACCCTCCCAAATCCTGCCACCAACTGGAGACCAAGGGTTACAATAAGTAGACCTAAGGGAGGGGTACTTTTCTTTTCAACCACTGCAGTGGAGCACACCTCTATGTCCAACATGAAGGAAATAGAGGCTGGAAGACCAGAAATTCAAGGTCACCCACCAGCTCATCGCCAGTTGCAGATCAGTTTGAGCTACAGGCTATCTGCCTCAAATATAAAACTAAACAGAAAGTCAATAAAAAGGCCACACTTGGGGAAGTGGATAATAGGGTCAAATATTAGTAAACACCTCTTCTTCCCCATTGTTAAAGCCTGCTCCCTCCAGTTCCTCTGACTTTACTGTTACATAACAGATCTTGGACCTGTGACTGCTGTGTTTACAACATACTCAGTGACCCCTAACTTCTAATCATGAAACACATTTACCCGGTTCCAGGATGCCATCTCTCCACCTACAGCTCACCATGGAAGCATTTTGCCTCTTAGCAAAGGTCTTTGGTTTCTCGTGGGTGGCA

[0412] (SEQ ID NO: 19)

[0413] mouse Mrc1 enhancer 3

[0414]

[0415] (SEQ ID NO: 20)

[0416] human MRC1 enhancer 3

[0417]

[0418] (SEQ ID NO: 21)

[0419] mouse Mrc1 enhancer 4

[0420]

[0421] (SEQ ID NO: 22)

[0422] human MRC1 enhancer 4

[0423] AATAAACGTCTAGGAACATTTACCCTAAAGTACTGCCCTCTCTATGTGAACAAACTTAAGCCTGTGTTCTTTCCTTTTTGTGAACAGACGCGAGGCAATTTTTAATCTATAATGAAGATCACAAGCGCTGCGTGGACGCTCTAAGTGCCATCTCAGTTCAGACGGCAACTTGCAACCCGGAAGCTGAATCCCAGAAATTCCGCTGGGTGTCAGATTCTCAGATCATGAGTGTTGCTTTCAAATTATGTTTGGGAGTGCCATCAAAAACTGACTGGGCTTCCGTCACCCTGTATGCCTGTGATTCGAAAAGTGAATATCAGA

[0424] (SEQ ID NO: 23)

[0425] mouse Mrc1 enhancer 5

[0426] TGGAAGAGTTGGAAACTTTTGACCTAAAAGATCGTCCTTGTTACATGAATCCACTTAGCCATGCTTGCTTTCTTCTTCTTTTCCTGCTTCTTTCTTTTTAAACAGACACCAGGCAATTTTTAATCTATAATGAAGATCACAAGCGCTGCGTGGATGCAGTGAGTCCCAGTGCCGTCCAAACCGCAGCTTGCAACCAGGATGCCGAATCACAGAAATTCCGATGGGTGTCCGAATCTCAGATTATGAGTGTTGCATTTAAATTATGCCTGGGAGTGCCATCAAAAACGGACTGGGTTGCTATCACTCTCTAT

[0427] (SEQ ID NO: 24)

[0428] human MRC1 enhancer 5

[0429] TGTCAGGTTCTCTGGAGCACCCTCTCACCTGTTCAGACTAATTTCCTAAGTTCGGCGGGTCCCGGACCAAGATGGCGACCCGCTACATTTCATTCTTACATGCAGGGGATGAGCGCACTGTTTCACCACTTTGATTGCCTTTTTTGAGCATGGTAGATATTCAGTAAGCAACCCATGGATTGAATTCTACTTTATGTTTAATGCAGGACGAAAGGCGGGATGTGTTGCCATGAAAACCGGAGTGGCAGGTGGCTTATGGGATGTTTTGAGTTGTGAAGAAAAGGCAAAATTTGTGTGCAAACATTGGGCAGAAGGAGTGACTCGCCCACCAGAGCCCACAACAACTCCTGAACCCAAATGTCCAGAAAACTGGGGTACCACCAGTAAAACCAGCATGTGTTTCAAAGTAAGGATCACTCGCCAAAT

[0430] (SEQ ID NO: 25)

[0431] mouse Mrc1 enhancer 6

[0432] CATCCTCATTTTATTTTATGTACTTCTTTGTTCGTTAAAGCTGGCATTCCTTACAGTTCTATGAGGCAGGTCTTGGTATTTGCATTTGGAGAGGAGAAAGCAAGTTCAGAGCGTTTGAGTAACTTACCTAAAATCTCTAGTTGAGACGTGTCTCATTTTGAAATCTGTGAAAAACTTTGGTCCTGGAAAACCTACGTAGACCTTGGGAAGAAGGAAGGAAAAAGGGAAGGAAGGAGGGAGGGAGAGAGAAGCAGTAAACTATTTTTGCCATTATGGTGAATTTGATAATATAAAATATTTTATCATTAAATGCCTGTGTAGGGGGCACTTTGCCAAATGTTAGAAATATAAAGTGTTACAAACCCCCCTGCATCTGAGATCATAATTGGGCATCAGAACCCTGATGCTCGGTTCTGAGTGCCTTCTGTGAGCACGGCAGGCCTTCAGCAGGCACCTGTCAAGTGAATTCTACTTCATATATTTAATGCAGGGCGAAAGCCAGGGTGTGTTGCCATGAGAACCGGGATTGCAGGGGGCTTATGGGATGTTTTGAAATGTGATGAAAAGGCAAAATTTGTGTGCAAGCACTG

[0433] (SEQ ID NO: 26)

[0434] human MRC1 enhancer 6

[0435] GAGTGATTGTGCATGAACTTGTGGAGACCTCAATTGTTCTTGCAACTTGTCTCTTCTATTACTATTGCAAAAGGAATGGCTAAGTCTTTCTTGAAAGAATTCATATAGTTCTCTTTCAGAGACCTGCAGCAGTTACCACTTTGGGGAACTAGAGAAAAGTTATTTTTAAGTTTCTCTGGAATGAAAGGCACAATTCTATAATTTGGCCTTATTGCTTAATCCACCAGTTTTAAGTTCCTTGTTTGTAAAATATGAATGTTAGTAACTCTTCTTCTTTAAAATCTCGTTATATCATCAAGCTTG

[0436] (SEQ ID NO: 27)

[0437] mouse Mrc1 enhancer 7

[0438]

[0439] (SEQ ID NO: 28)

[0440] human MRC1 enhancer 7

[0441]

[0442] (SEQ ID NO: 29)

[0443] exemplary XhoI-human.MRC1.enhancer

[0444]

[0445] (SEQ ID NO: 30)

[0446] In some embodiments, the MRC1 enhancer comprises or consists of at least 70% of the same nucleotide sequence as any of SEQ ID NO: 17-30, or a fragment thereof. Suitably, the MRC1 enhancer comprises or consists of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the same nucleotide sequence as any of SEQ ID NO: 17-30, or a fragment thereof.

[0447] In some embodiments of the present invention, the MRC1 enhancer comprises or consists of a nucleotide sequence of any of SEQ ID NO: 17-30 or a fragment thereof.

[0448] In some embodiments of the invention, the MRC1 enhancer comprises or consists of a nucleotide sequence identical to at least 70% of SEQ ID NO: 32, or a fragment thereof. Suitably, the MRC1 enhancer comprises or consists of a nucleotide sequence identical to at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of SEQ ID NO: 32, or a fragment thereof.

[0449] In some embodiments of the present invention, the MRC1 enhancer comprises or consists of a nucleotide sequence SEQ ID NO: 32 or a fragment thereof.

[0450] miRNA target sequences :

[0451] CTCGAGAGCCCCACCATGTTATTGATGGCCAAACAATACGCATGCTGACAGCCATTATCTGTGGCCTCTGATGCTATTAGCCAAACCATGTTATTGATGGTCAAACAATACGCATGCTGACAGCCATTATCTGGGACTCAGAAAGTTCTGCATATTCAAGTCAGGCCAGAGGATCCGAGTTCTAATGTTAAGAGAAAACCAACACACCAACAAGCAAATAAACAAACCTACCCTTGAACCAAAATATACATCAAT ACCTCCGTTGCAAATGGATAAATGGAACTGCATTGCCCTCTGCTGTTGGGGAATCTTGGCAACCATTTCAACTCTATGGCTGGAGATGACTTACTGCTCTGTTTTATTTTCCATCCTCCTGCTTAGAT TATTGCTTTCAAAGTTTCCAGAATAGAAGAAGTCAGTGGTGGCCAGTTGTCCTTTAATGGTCTCTTATCTACCAATGGCTAGTATCCTTTTTGCATTATCGTAGCTCTACTCTTGTAGATGTTAAATT

[0452] (SEQ ID NO: 32)

[0453] exemplary miRT-126

[0454] The vector of the present invention may contain one or more miRNA target sequences. Suitably, the transgene is operatively linked to one or more miRNA target sequences.

[0455] MicroRNA (miRNA) genes are distributed across all human chromosomes except the Y chromosome. They can be located in non-coding regions of the genome or within introns of protein-coding genes. Approximately 50% of miRNAs occur in clusters, which are transcribed into polycistronic primary transcripts. Similar to protein-coding genes, miRNAs are typically transcribed from polymerase II promoters to produce so-called primary miRNA transcripts (pri-miRNA). This pri-miRNA is then processed through a series of endonuclease cleavage steps performed by two enzymes, Drosha and Dicer, belonging to the type III RNase family. A specific nuclear complex composed of Drosha and the DiGeorge syndrome crisis region gene (DGCR8) cleaves a stem-loop of approximately 60 nucleotides from the pri-miRNA, called the pre-miRNA. This specific nuclear complex trims the two strands near the base of the primary stem-loop, retaining a 5' phosphate group and a 2-bp 3' overhang. The pre-miRNA is then actively transported from the nucleus to the cytoplasm by RAN-GTP and Exportin. Subsequently, Dicer performs double-strand cleavage at the stem-loop end not defined by Drosha cleavage, generating a 19bp to 24bp double-stranded structure, which consists of a mature miRNA and the opposing strand of the double-stranded structure (called miRNA). Consistent with the rules of thermodynamic asymmetry, only one strand of the duplex is selectively loaded into the RNA-induced silencing complex (RISC) and accumulates into mature microRNA. This strand is typically the one whose 5' end is less tightly paired with its complementary sequence, as demonstrated by single nucleotide mismatches introduced at the 5' end of each strand of the siRNA duplex. However, some miRNAs support the accumulation of both strands of the duplex to a similar degree.

[0456] MicroRNAs trigger RNAi, much like small interfering RNAs (siRNAs) are widely used for experimental gene knockdown. The main difference between miRNAs and siRNAs is their biogenesis. Once the guide strand of a small RNA molecule is loaded into the RISC, it preferentially interacts with the mRNA target sequence located in the 3' untranslated region (3'UTR) of a protein-coding gene. Nucleotides 2 through 8, counted from the 5' end of the miRNA (the so-called seed sequence), have been shown to be essential for triggering RNAi. If the entire guide strand sequence is perfectly complementary to the mRNA target (as is often the case with siRNAs and plant miRNAs), the mRNA undergoes endonuclease cleavage at the intervention of the Argonaute (Ago) protein (also known as the "slicer" of the small RNA duplex) and integrates into the RNA-induced silencing complex (RISC). DGRC (Digeminal syndrome crisis region gene 8) and TRBP (TAR (HIV) RNA-binding protein 2) are double-stranded RNA-binding proteins that promote the biogenesis of mature miRNAs via Drosha and Dicer RNase III, respectively. The guide strand of the miRNA duplex is incorporated into the effector complex RISC, which recognizes specific targets and induces posttranscriptional gene silencing through incomplete base pairing. Several mechanisms have been proposed for this regulatory modality: miRNAs can induce repression of translation initiation, deadenylate target mRNAs for degradation, or isolate targets into cytoplasmic P-bodies.

[0457] On the other hand, if only the seed sequence is perfectly complementary to the target mRNA, while the remaining bases show incomplete pairing, RNAi works through multiple mechanisms, leading to translational repression. Eukaryotic mRNA degradation mainly occurs through the following process: shortening of the polyA tail at the 3' end of the mRNA and decapping at the 5' end, followed by 5'-3' exonuclease digestion and accumulation of miRNA in discrete cytoplasmic regions (so-called P bodies, which are rich in components of the mRNA decay pathway).

[0458] Transgenic expression can 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 can prevent or reduce transgenic expression in that cell by binding to their corresponding miRNA target sequences located in a vector or polynucleotide (Brown, BD et al. (2007) Nat Biotechnol 25: 1457-1467).

[0459] The appropriate miRNA target sequence for inhibiting transgene expression in specific cells will be known to those skilled in the art. Any suitable method may be used to identify the appropriate miRNA target sequence, for example by performing microarrays containing known miRNAs (e.g., from miRbase).

[0460] Containing more than one copy of the miRNA target sequence in the vector can improve the system's effectiveness. It is also envisioned that different miRNA target sequences may be included. For example, the transgene can be operatively linked to more than one miRNA target sequence, which may or may not be different. The miRNA target sequences may be tandem, but other arrangements are envisioned. The vector may, for example, contain 1, 2, 3, 4, 5, 6, 7, or 8 copies of the same or different miRNA target sequences. Suitablely, the vector contains four copies of each miRNA target sequence.

[0461] The target sequence can be fully or partially complementary to the miRNA. As used herein, the term “fully complementary” can mean that the target sequence has a nucleic acid sequence that is 100% complementary to the sequence of the miRNA that recognizes it.

[0462] As used herein, the term "partially complementary" can mean that the target sequence is only partially complementary to the sequence of the miRNA that recognizes it, wherein the partially complementary sequence is still recognized by the miRNA. In other words, in the context of this invention, a partially complementary target sequence can effectively recognize the corresponding miRNA and achieve the prevention or reduction of transgene expression in cells expressing that miRNA.

[0463] A copy of the miRNA target sequence can be separated by a spacer sequence. The spacer sequence can contain, for example, at least one, at least two, at least three, at least four, or at least five nucleotide bases.

[0464] Vectors driving transgene expression from M2-like macrophage-specific promoters (e.g., the MRC1 promoter) can be used to drive selective transgene expression in Kupffer cells (KC) and, to a lesser extent, selective transgene expression in MRC1+ splenic macrophages and hepatic sinusoidal endothelial cells (LSECs). miRNA target sequences can be used to further increase the specificity of the vector. One or more miRNA target sequences can inhibit transgene expression in some hepatocyte populations and / or some splenic cell populations. One or more miRNA target sequences can inhibit transgene expression in some hepatic macrophages and / or some splenic macrophages. For example, expression can target LSECs.

[0465] As used herein, the term "inhibited expression" can refer to reduced expression of a transgene operatively linked to one or more miRNA target sequences in a relevant cell type, compared to transgene expression under otherwise substantially identical conditions but in the absence of one or more miRNA target sequences. In some embodiments, transgene expression is inhibited by at least 50%. In some embodiments, transgene expression is inhibited by at least 60%, 70%, 80%, 90%, or 95%. In some embodiments, transgene expression is substantially blocked.

[0466] Appropriately, one or more miRNA target sequences inhibit transgene expression in hepatic sinusoidal endothelial cells (LSECs) and / or hepatocytes.

[0467] In some implementations, one or more miRNA target sequences inhibit transgene expression in hepatocytes and / or LSECs. For example, the vector may contain one or more copies of (i) a miRNA target sequence that inhibits transgene expression in LSECs; and / or (ii) one or more copies of a miRNA target sequence that inhibits transgene expression in hepatocytes.

[0468] Suitablely, one or more miRNA target sequences include: (i) one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8) miR-126 target sequences; and / or (ii) one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8) miR-122 target sequences.

[0469] The miR-126 target sequence is an exemplary miRNA target sequence for inhibiting transgene expression in LSECs. miR-126 is a microRNA expressed in endothelial cells (such as LSECs) and reduces the expression of target genes when it binds to its target sequence.

[0470] In some embodiments of the invention, the miR-126 target sequence comprises or consists of a nucleotide sequence identical to at least 70% of SEQ ID NO: 3, or a fragment thereof. Suitably, the miR-126 target sequence comprises or consists of a nucleotide sequence identical to at least 80%, at least 90%, or at least 95% of 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.

[0471] exemplary miRT-122

[0472] CGCATTATTACTCACGGTACGA

[0473] (SEQ ID NO: 3)

[0474] The miR-122 target sequence is an exemplary miRNA target sequence for inhibiting 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.

[0475] In some embodiments of the invention, the miR-122 target sequence comprises or consists of a nucleotide sequence identical to at least 70% of SEQ ID NO: 4, or a fragment thereof. Suitably, the miR-122 target sequence comprises or consists of a nucleotide sequence identical to at least 80%, at least 90%, or at least 95% of 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.

[0476] ​

[0477] ACAAACACCATTGTCACACTCCA

[0478] (SEQ ID NO: 4)

[0479] Additional miRNA target sequences that inhibit transgene expression in LSEC and / or hepatocytes can be identified by any suitable method, such as miRNA expression analysis as described in Oda, S. et al., 2018. The American journal of pathology, 188(4), pp. 916-928.

[0480] In some embodiments, one or more miRNA target sequences include: (i) two or more miR-126 target sequences; and / or (ii) two or more miR-122 target sequences. In some embodiments, one or more miRNA target sequences include: (i) four miR-126 target sequences; and / or (ii) four miR-122 target sequences. Suitably, the target sequences are separated by spacer sequences.

[0481] In some embodiments of the invention, one or more miRNA target sequences comprise at least 70% of the same nucleotide sequence or fragment thereof as one or more of SEQ ID NO: 5-7. Suitably, one or more miRNA target sequences comprise at least 80%, at least 90%, or at least 95% of the same nucleotide sequence or fragment thereof as one or more of SEQ ID NO: 5-7.

[0482] In some embodiments of the present invention, one or more miRNA target sequences comprise or consist of nucleotide sequences of one or more of SEQ ID NO: 5-7 or fragments thereof.

[0483] Exemplary miRT-1224 x miRT

[0484] TCTAGATAAACAAACACCATTGTCACACTCCATTCGAAACAAACACCATTGTCACACTCCAACGCGTACAAACACCATTGTCACACTCCAATGCATACAACACCATTGTCACACTCCACCCGGGTCGAGCTCGGT ACC

[0485] (SEQ ID NO: 5)

[0486] Exemplary miRT-1264 x miRT

[0487] GGTACCAGCAAACGCATTATTACTCACGGTACGACCATCGCATTATTACTCACGGTACGAACTTCGCATTATTACTCACGGTACGACGAACGCATTATTACTCACGGTACGACACGTGTCGGTACC

[0488] (SEQ ID NO: 6)

[0489] Exemplary miRT-122 and miR1264 x miRT

[0490] GGTACCAGCGCTACAAACACCATTGTCACACTCCAACATACAAACACCATTGTCACACTCCAGATTACAAACACCATTGTCACACTCCACAGAACAAACACCATTGTCACACTCCAGTTTTAAACGCATTATTACTCACGGTACGACCATCGCATTATTACTCACGGTACGAACTTCGCATTATTACTCACGGTACGACGAACGCATTATTACTCACGGTACGACACGTGTCGGTACC

[0491] (SEQ ID NO: 7)

[0492] In some embodiments of the invention, one or more miRNA target sequences comprise or consist of a nucleotide sequence identical to at least 70% of SEQ ID NO: 36, or a fragment thereof. Suitably, one or more miRNA target sequences comprise or consist of a nucleotide sequence identical to at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of SEQ ID NO: 36, or a fragment thereof.

[0493] In some embodiments of the present invention, one or more miRNA target sequences comprise or consist of the nucleotide sequence SEQ IDNO: 36 or a fragment thereof.

[0494] Exemplary AfeI-4 x miRT122-4 x miRT126-PmlI

[0495] AGCGCTACAAACACCATTGTCACACTCCAACATACAAACACCATTGTCACACTCCAGATTACAAACACCATTGTCACACTCCACAGAACAAACACCATTGTCACACTCCAGTTTTAAACGCATTATTACTCACGGTACGACCATCGCATTATTACTCACGGTACGAACTTCGCATTATTACTCACGGTACGACGAACGCATTATTACTCACGGTACGACACGTGTC

[0496] (SEQ ID NO: 36)

[0497] In some implementations, one or more miRNA target sequences inhibit transgene expression in some liver and / or some spleen macrophages. For example, one or more miRNA target sequences may inhibit transgene expression in M2-like macrophages. For example, one or more miRNA target sequences may inhibit transgene expression in Kupffer cells and / or MRC1+ spleen macrophages.

[0498] In some implementations, one or more miRNA target sequences inhibit transgene expression in splenic phagocytes (e.g., splenic macrophages).

[0499] miRNA target sequences that inhibit transgene expression in some liver and / or some spleen macrophages can be identified by any suitable method, such as miRNA expression analysis as described in Zhang, Y. et al., 2013. International journal of molecular medicine, 31(4), pp. 797-802.

[0500] Other expression control sequences

[0501] The vector of the present invention may also include one or more regulatory elements that can function pre- or post-transcriptionally. Suitably, the transgene is operatively linked to one or more regulatory elements that can function pre- or post-transcriptionally. The one or more regulatory elements can promote transgene expression in phagocytes.

[0502] "Regulatory elements" are any nucleotide sequences that promote peptide expression, such as those used to increase transcript expression or enhance mRNA stability.

[0503] Suitable regulatory elements include, for example, promoters, enhancer elements, posttranscriptional regulatory elements, and polyadenylation sites.

[0504] Post-transcriptional regulatory elements

[0505] The vector of the present invention may contain one or more post-transcriptional regulatory elements. Suitably, the transgene is operatively linked to one or more post-transcriptional regulatory elements. Post-transcriptional regulatory elements can improve gene expression.

[0506] The vector of the present invention may contain a post-transcriptional regulatory element (WPRE) of marmot hepatitis virus. Suitably, the transgene is operatively linked to the WPRE.

[0507] In some embodiments of the invention, the WPRE comprises or consists of a nucleotide sequence identical to at least 70% of SEQ ID NO: 35, or a fragment thereof. Suitably, the WPRE comprises or consists of a nucleotide sequence identical to at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of SEQ ID NO: 35, or a fragment thereof.

[0508] In some embodiments of the present invention, the WPRE comprises or consists of a nucleotide sequence SEQ ID NO: 35 or a fragment thereof.

[0509] Exemplary SalI-WPRE

[0510] GTCGACCCGACAGTTTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTC CTCCTTGTATAAATCCTGGTTGCTGTCCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCC TCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGAC GTCCTTCTGCTACGTCCCTCTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACC

[0511] (SEQ ID NO: 35)

[0512] Destabilizing domain

[0513] The vector of the present invention may contain a nucleotide sequence encoding a destabilization domain. Suitably, the transgene is operatively linked to the destabilization domain, i.e., co-located with the transgene product, such that when the transgene is translated, a fusion protein comprising the destabilization domain fused with the transgene product is produced.

[0514] Destabilizing domains (DDs) represent fusion protein components that are inherently unstable and destabilize other proteins upon incorporation, leading to protein degradation. A well-known example of a DD is the Shield system, which incorporates rapamycin-binding protein (FKBP12) as a built-in destabilizing domain into a protein to induce protein degradation in the cell. In the absence of its specific ligand (Shield-1), the protein is degraded by the proteasome (Banaszynski, LA, et al., 2006. Cell, 126(5), pp. 995-1004).

[0515] Another exemplary destabilizing domain is dihydrofolate reductase (DHFR) or a variant thereof. In mammalian cells, fusion proteins containing DHFR proteins are rapidly ubiquitinated and degraded by the proteasome system. The antibiotic trimethoprim (TMP) or small TMP-based molecules can bind to DHFR proteins and prevent their degradation, allowing fusion proteins to evade degradation (Peng, H., et al., 2019. Molecular Therapy-Methods & Clinical Development, 15, pp. 27-39).

[0516] The vector of the present invention may contain a dihydrofolate reductase coding sequence or a variant or derivative thereof. Suitably, the transgene is operatively linked to the dihydrofolate reductase coding sequence (or a variant or derivative thereof), i.e., co-located with the transgene product, such that when the transgene is translated, a fusion protein comprising the dihydrofolate reductase coding sequence (or a variant or derivative thereof) fused with the transgene product is produced.

[0517] Polyadenylation sequence

[0518] The vector of the present invention may contain a polyadenylated sequence. Suitably, the transgene is operatively linked to the polyadenylated sequence. A polyadenylated sequence may be inserted after the transgene to improve transgene expression.

[0519] Polyadenylation sequences typically include a polyadenylation signal, a polyadenylation site, and a downstream element: the polyadenylation signal contains a sequence motif recognized by the RNA cleavage complex; the polyadenylation site is a cleavage site that adds a poly-A tail to the mRNA; and the downstream element is a GT-rich region, which is usually located downstream of the polyadenylation site and is important for efficient processing.

[0520] Kozak sequence

[0521] The vector of the present invention may contain a Kozak sequence. Suitably, the transgene is operatively linked to the Kozak sequence. The Kozak sequence may be inserted before the start codon to improve the initiation of translation.

[0522] In some embodiments of the invention, the Kozak sequence comprises or consists of a nucleotide sequence identical to at least 70% of SEQ ID NO: 33, or a fragment thereof. Suitably, the Kozak sequence comprises or consists of a nucleotide sequence identical to at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of SEQ ID NO: 33, or a fragment thereof.

[0523] In some embodiments of the present invention, the Kozak sequence comprises or consists of a nucleotide sequence SEQ ID NO: 33 or a fragment thereof.

[0524] Exemplary BamHI-KOZAC

[0525] GGATCCGCCACC

[0526] (SEQ ID NO: 33)

[0527] Transgene

[0528] The vector of the present invention may contain one or more transgenes. Suitably, one or more expression control sequences are operatively linked to the transgenes.

[0529] There are no particular restrictions on genetically modified organisms (GMOs), and any suitable GMO can be used.

[0530] Genetically modified organisms (GMOs) can encode naturally occurring human genes or their variants and / or segments.

[0531] Genetically modified organisms (GMOs) can be therapeutic GMOs.

[0532] Genetically modified organisms (GMOs) can encode therapeutic peptides and / or antigenic peptides.

[0533] In some embodiments, the transgene contains a nucleotide sequence encoding a signal peptide, preferably wherein the signal peptide is operatively linked to the encoded polypeptide (e.g., a therapeutic polypeptide and / or an antigenic polypeptide). The signal peptide may be, for example, a natural signal peptide of the encoded polypeptide. In some embodiments, the transgene does not contain a nucleotide sequence encoding a signal peptide.

[0534] Therapeutic polypeptide

[0535] Appropriately, the genetically modified organism (GMO) encodes therapeutic peptides.

[0536] As used herein, "therapeutic peptide" is any peptide that can be used in a therapy. For example, a therapeutic peptide may contain therapeutic cytokines that can activate an immune response.

[0537] In some implementations, the transgene encodes cytokines, such as cytokines that can activate immune responses, particularly anti-tumor responses.

[0538] Cytokines are molecular messengers that allow cells of the immune system to communicate with each other to produce a coordinated, robust, but self-limiting response to target antigens. Cytokines directly stimulate immune effector cells and stromal cells at the tumor site, enhancing the recognition of tumor cells by cytotoxic effector cells. Cytokines can have broad antitumor activity (Lee, S and Margolin, K., 2011. Cancers, 3(4), pp. 3856-3893).

[0539] For example, any cytokine that can activate an immune response, particularly an anti-tumor response, can be used. Exemplary cytokines include IFNα, IFNβ, and IFN2. IL-2, IL-12, TNFα, CXCL9, and IL-1β. Other exemplary cytokines include IL-10, IL-15, or IL-18. Other exemplary cytokines include GMCSF, FLT3, IL-7, or IL-21.

[0540] Variants of any of these cytokines may be used, provided that the variant retains the ability to activate an immune response, particularly an antitumor response. Those skilled in the art will be able to obtain 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 with any cytokine.

[0541] Fragments of any of these cytokines (or their variants) may be used, provided that the fragment retains the ability to activate an immune response, particularly an antitumor response. Those skilled in the art will be able to obtain such fragments using methods known in the art. For example, the fragment may retain residues or domains required to activate an immune response.

[0542] In some embodiments, the transgene encodes a cytokine selected from IFNα, IFNβ, IFNγ, IL-2, IL-12, TNFα, CXCL9, and IL-1β, or variants and / or fragments thereof. In some embodiments, the transgene encodes a cytokine selected from IL10, IL15, or IL18, or variants and / or fragments thereof. In some embodiments, the transgene encodes a cytokine selected from GMCSF, FLT3, IL7, or IL21.

[0543] Interferon

[0544] There are three main types of interferons (IFNs). Human type I IFN genes encode a family of 17 different proteins (including 13 subtypes of IFNα, plus IFNβ, IFNε, IFNκ, and IFNω). Only a single type II IFN, IFNγ, exists. Type III IFNs consist of IFNλ1, IFNλ2, IFNλ3, and IFNλ4.

[0545] All IFNs have the potential to act on tumor cells to exert a direct anti-tumor effect, or act on immune cells to exert an indirect anti-tumor effect (Parker, BS, et al., 2016. Nature Reviews Cancer, 16(3), p. 131).

[0546] In some embodiments, the transgene encodes interferon, such as type I interferon (e.g., IFNα, IFNβ), type II interferon (e.g., IFNγ), or type III interferon (e.g., IFNλ, IFNλ2, IFNλ3, IFNλ4). In some embodiments, the transgene encodes type I interferon (e.g., IFNα, IFNβ).

[0547] IFN alpha

[0548] Interferon-alpha (IFNα) (a type 1 interferon) is a pleiotropic cytokine that plays a crucial role in protecting organisms from viral infections. IFNα's antitumor functions are well-established, including direct tumor cell killing, activation of adaptive and innate immune functions, and angiogenesis suppression. IFNα has been approved for clinical use in several types of tumors, including melanoma, renal cell carcinoma, and Kaposi's sarcoma. However, recombinant IFNα alone is not well tolerated when administered systemically, therefore alternative therapeutic options for IFNα are currently preferred.

[0549] The vector of the present invention can reduce the systemic toxicity associated with IFNα delivery by selectively delivering therapeutic IFNα to tumors. The route of administration and the intended target cells, namely phagocytes, facilitate the natural loss of the vector through their physiological metabolic turnover.

[0550] In some embodiments, the transgene encodes IFNα. An exemplary human interferon-α (IFNα) used in this invention is UniProtKB P01562.

[0551] In some embodiments of the invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 70% of SEQ ID NO: 8, or a fragment thereof. Suitably, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 80%, at least 90%, or at least 95% of SEQ ID NO: 8, or a fragment thereof.

[0552] In some embodiments of the present invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence SEQ IDNO: 8 or a fragment thereof.

[0553] Exemplary human interferon-alpha :

[0554] MASPFALLMVLVVLSCKSSCSLGCDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSSLSTNLQERLRRKE

[0555] (SEQ ID NO: 8)

[0556] In some embodiments of the invention, the transgenic material comprises or consists of a nucleotide sequence identical to at least 70% of SEQ ID NO: 34, or a fragment thereof. Suitably, the transgenic material comprises or consists of a nucleotide sequence identical to at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of SEQ ID NO: 34, or a fragment thereof.

[0557] In some embodiments of the present invention, the transgene comprises or consists of a nucleotide sequence SEQ ID NO: 34 or a fragment thereof.

[0558] Exemplary human IFNA transgene :

[0559] ATGGCCTCGCCCTTTGCTTTACTGATGGTCCTGGTGGTGCTCAGCTGCAAGTCAAGCTGCTCTCTGGGCTGTGATCTCCCTGAGACCCACAGCCTGGATAACAGGAGGACCTTGATGCTCCTGGCACAAATGAGCAGAATCT CTCCTTCCTCCTGTCTGATGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCCAGCCATCTCTGTCCTCCATGAGCTGATCCAGCAGATCTTCAACCTCTTTACCACAAAAGAT TCATCTGCTGCTTGGGATGAGGACCTCCTAGACAAATTCTGCACCGAACTCTACCAGCAGCTGAATGACTTGGAAGCCTGTGTGATGCAGGAGGAGAGGGTGGGAGAAACTCCCCTGATGAATGCGGACTCCATCTTGGCTG TGAAGAAATACTTCCGAAGAATCACTCTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCCCTCTCTTTATCAACAAACTTGCAAGAAAGATTAAGGAGGAAGGAATAA

[0560] (SEQ ID NO: 34)

[0561] IFN beta

[0562] In some embodiments, the transgene encodes IFNβ. An exemplary human IFNβ used in this invention is UniProtKB P01574.

[0563] In some embodiments of the invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to or fragments thereof of SEQ ID NO: 9 by at least 70%. Suitably, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to or fragments thereof of SEQ ID NO: 9 by at least 80%, at least 90%, or at least 95%.

[0564] In some embodiments of the present invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence SEQ ID NO: 9 or a fragment thereof.

[0565] Exemplary human interferon-beta :

[0566] MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN

[0567] (SEQ ID NO: 9)

[0568] IFN

[0569] In some implementation schemes, the transgenic code encodes IFN. Exemplary human IFN used in this invention It is UniProtKB P01579.

[0570] In some embodiments of the invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 70% of SEQ ID NO: 10, or a fragment thereof. Suitably, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 80%, at least 90%, or at least 95% of SEQ ID NO: 10, or a fragment thereof.

[0571] In some embodiments of the present invention, the transgenic encoding polypeptide comprises or consists of a polypeptide sequence SEQ ID NO:10 or a fragment thereof.

[0572] Exemplary human interferon-gamma :

[0573] MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ

[0574] (SEQ ID NO: 10)

[0575] Other cytokines

[0576] IL-2

[0577] Interleukin-2 (IL-2) and other members of the IL-2-related family of T-cell growth factors (such as IL-4, IL-7, IL-9, IL-15, and IL-21) utilize a common receptor signaling system that leads to the activation and expansion of CD4+ and CD8+ T cells (Lee, S and Margolin, K., 2011. Cancers, 3(4), pp. 3856-3893).

[0578] In some embodiments, the transgene encodes IL-2 or IL-2-related cytokines (e.g., IL-7, IL-15, IL-21). An exemplary human IL-2 used in this invention is UniProtKB P60568.

[0579] In some embodiments of the invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 70% of SEQ ID NO: 11, or a fragment thereof. Suitably, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 80%, at least 90%, or at least 95% of SEQ ID NO: 11, or a fragment thereof.

[0580] In some embodiments of the present invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence SEQ IDNO: 11 or a fragment thereof.

[0581] Exemplary human interleukin-2 :

[0582] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0583] (SEQ ID NO: 11)

[0584] IL-12

[0585] In some embodiments, the transgene encodes IL-12. Exemplary human IL-12 α and β subunits used in this invention are UniProtKB P29459 and P29460.

[0586] In some embodiments of the invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 70% of SEQ ID NO: 12, or a fragment thereof. Suitably, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 80%, at least 90%, or at least 95% of SEQ ID NO: 12, or a fragment thereof.

[0587] In some embodiments of the present invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence SEQ ID NO: 12 or a fragment thereof.

[0588] Exemplary human interleukin-12 subunit alpha :

[0589] MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRI RAVTIDRVMSYLNAS

[0590] (SEQ ID NO: 12)

[0591] In some embodiments of the invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 70% of SEQ ID NO: 13, or a fragment thereof. Suitably, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 80%, at least 90%, or at least 95% of SEQ ID NO: 13, or a fragment thereof.

[0592] In some embodiments of the present invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence SEQ ID NO: 13 or a fragment thereof.

[0593] Exemplary human interleukin-12 subunit beta :

[0594] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSD PQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0595] (SEQ ID NO: 13)

[0596] In some embodiments, the transgene encodes a single-stranded IL12. The single-stranded IL12 may comprise an IL12 subunit β (e.g., the amino acid sequence SEQ ID NO: 13 or a fragment thereof, or a sequence or fragment thereof that is at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 13) and an IL12 subunit α (the amino acid sequence SEQ ID NO: 12 or a fragment thereof, or a sequence or fragment thereof that is at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 12). The single-stranded IL12 may be a fusion protein comprising IL12 subunit β and IL12 subunit α. IL12 subunit β and IL12 subunit α may be conjugated via a linker sequence. The linker sequence may comprise an amino acid sequence SEQ ID NO: 42 or a fragment thereof, or a sequence or fragment thereof that is at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 42, or a combination thereof.

[0597] RRAGGGGSGGGGSGGGGSRT

[0598] (SEQ ID NO: 42)

[0599] In some embodiments, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 70% of SEQ ID NO: 37 or 46, or a fragment thereof. Suitably, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 80%, at least 90%, or at least 95% of SEQ ID NO: 37 or 46, or a fragment thereof.

[0600] In some embodiments of the present invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence SEQ ID NO: 37 or 46 or a fragment thereof.

[0601] Exemplary single chain human interleukin-12 sequence :

[0602] MCPQKLTISWFAIVLLVSPLMAIAGQLMWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRC EAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHS YFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSRAGGGGSGGGGSGGGGSRTRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDK TSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMS YLNAS

[0603] (SEQ ID NO: 37)

[0604] WELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLT FSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKR EKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSRAGGGGSGGGGSGGGGSRTRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPL ELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

[0605] (SEQ ID NO: 46)

[0606] In some embodiments of the invention, the transgenic material comprises or consists of a nucleotide sequence identical to at least 70% of SEQ ID NO: 40, or a fragment thereof. Suitably, the transgenic material comprises or consists of a nucleotide sequence identical to at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of SEQ ID NO: 40, or a fragment thereof.

[0607] In some embodiments of the present invention, the transgene comprises or consists of a nucleotide sequence SEQ ID NO: 40 or a fragment thereof.

[0608]

[0609] (SEQ ID NO: 40)

[0610] IL10

[0611] In some implementations, the transgene encodes IL-10.

[0612] In some embodiments of the invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 70% of SEQ ID NO: 38, or a fragment thereof. Suitably, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 80%, at least 90%, or at least 95% of SEQ ID NO: 38, or a fragment thereof.

[0613] In some embodiments of the present invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence SEQ ID NO: 38 or a fragment thereof.

[0614] Exemplary human interleukin-10 :

[0615] MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0616] (SEQ ID NO: 38)

[0617] In some embodiments of the invention, the transgenic material comprises or consists of a nucleotide sequence identical to at least 70% of SEQ ID NO: 39, or a fragment thereof. Suitably, the transgenic material comprises or consists of a nucleotide sequence identical to at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of SEQ ID NO: 39, or a fragment thereof.

[0618] In some embodiments of the present invention, the transgene comprises or consists of a nucleotide sequence SEQ ID NO: 39 or a fragment thereof.

[0619] ATGCCAGGCTCCGCCCTGCTGTGCTGTCTGCTGCTGCTGACCGGCATGAGGATCAGCAGAGGACAGTACTCCCGGGAGGACAACAATTGCACCCACTTCCCTGTGGGACAGTCCCACATGCTGCTGGAGCTGCG CACAGCTTTTTCTCAGGTGAAGACCTTCTTTCAGACAAAGGACCAGCTGGATAACATCCTGCTGACCGACAGCCTGATGCAGGATTTCAAGGGCTACCTGGGATGTCAGGCCCTGTCCGAGATGATCCAGTTTT ATCTGGTGGAGGTGATGCCTCAGGCTGAGAAGCACGGCCCCGAGATCAAGGAGCACCTGAATTCTCTGGGAGAGAAGCTGAAGACACTGCGGATGCGCCTGAGGAGATGCCACAGGTTCCTGCCTTGTGAGAAC AAGTCTAAGGCCGTGGAGCAGGTGAAGAGCGACTTTAATAAGCTGCAGGATCAGGGCGTGTACAAGGCCATGAACGAGTTCGATATCTTTATCAATTGCATCGAGGCTTATATGATGATCAAGATGAAGAGCTGA

[0620] (SEQ ID NO: 39)

[0621] IL15

[0622] In some implementations, the transgene encodes IL-15.

[0623] In some embodiments of the invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 70% of SEQ ID NO: 44, or a fragment thereof. Suitably, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 80%, at least 90%, or at least 95% of SEQ ID NO: 44, or a fragment thereof.

[0624] In some embodiments of the present invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence SEQ IDNO: 44 or a fragment thereof.

[0625] Exemplary human interleukin-15 :

[0626] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0627] (SEQ ID NO: 44)

[0628] IL18

[0629] In some implementations, the transgene encodes IL-18.

[0630] In some embodiments of the invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to or fragments thereof of SEQ ID NO: 45 or 47 by at least 70%. Suitably, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to or fragments thereof of SEQ ID NO: 45 or 47 by at least 80%, at least 90%, or at least 95%.

[0631] In some embodiments of the present invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence SEQ ID NO: 45 or 47 or a fragment thereof.

[0632] Exemplary human interleukin-18 sequence :

[0633] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0634] (SEQ ID NO: 45)

[0635] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISAYGDSRARGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0636] (SEQ ID NO: 47)

[0637] TNF alpha

[0638] In some embodiments, the transgene encodes tumor necrosis factor α (TNFα). An exemplary human TNFα used in this invention is UniProtKB P01375.

[0639] In some embodiments of the invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 70% of SEQ ID NO: 14, or a fragment thereof. Suitably, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 80%, at least 90%, or at least 95% of SEQ ID NO: 14, or a fragment thereof.

[0640] In some embodiments of the present invention, the transgenic encoding polypeptide comprises or consists of a polypeptide sequence SEQ ID NO:14 or a fragment thereof.

[0641] Exemplary human TNF alpha :

[0642] MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANG VELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0643] (SEQ ID NO: 14)

[0644] CXCL9

[0645] In some implementations, the transgene encodes CXC motif chemokine 9 (CXCL9).

[0646] The exemplary human CXCL9 used in this invention is UniProtKB Q07325.

[0647] In some embodiments of the invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 70% of SEQ ID NO: 15, or a fragment thereof. Suitably, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 80%, at least 90%, or at least 95% of SEQ ID NO: 15, or a fragment thereof.

[0648] In some embodiments of the present invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence SEQ IDNO: 15 or a fragment thereof.

[0649] Exemplary human CXCL9 :

[0650] MKKSGVLFLLGIILLVLIGVQGTPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNPDSADVKELIKKWEKQVSQKKKQKNGKKHQKKKVLKVRKSQRSRQKKTT

[0651] (SEQ ID NO: 15)

[0652] IL-1 beta

[0653] In some embodiments, the transgene encodes interleukin-1β (IL-1β). An exemplary human IL-1β used in this invention is UniProtKB P01584.

[0654] In some embodiments of the invention, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 70% of SEQ ID NO: 16, or a fragment thereof. Suitably, the transgenic encoding polypeptide comprises or consists of an amino acid sequence identical to at least 80%, at least 90%, or at least 95% of SEQ ID NO: 16, or a fragment thereof.

[0655] In some embodiments of the present invention, the transgenic encoding polypeptide comprises or consists of a polypeptide sequence SEQ ID NO:16 or a fragment thereof.

[0656] Exemplary human IL-1 beta :

[0657] MAEVPELASEMMAYYSGNEDDLFFEADGPKQMKCSFQDLDLCPLDGGIQLRISDHHYSKGFRQAASVVVAMDKLRKMLVPCPQTFQENDLSTFFPFIFEEEPIFFDTWDNEAYVHDAPVRSLNCTLRDSQQKSL VMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPAVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSS

[0658] (SEQ ID NO: 16)

[0659] Antigenic polypeptide

[0660] Appropriately, transgenes encode antigenic peptides.

[0661] As used herein, “antigenic peptide” is any peptide that can induce an immune response. Specifically, antigenic peptides can be internalized and presented by antigen-presenting cells (APCs). Antigen presentation allows for the specificity of adaptive immunity and can contribute to immune responses against both intracellular and extracellular pathogens. APCs also naturally play a role in combating tumors by stimulating B cells and cytotoxic T cells to produce antibodies against tumor-associated antigens and kill malignant cells, respectively.

[0662] Antigens can be patient-specific.

[0663] Tumor antigen

[0664] In some implementations, the transgene encodes tumor antigens, such as tumor-specific antigens or tumor-associated antigens.

[0665] As used in this article, "tumor antigen" is an antigenic substance (such as an antigenic polypeptide) produced in tumor cells. "Tumor-specific antigen" is present only on tumor cells and not on any other cells. "Tumor-associated antigen" is present on some tumor cells as well as some normal cells.

[0666] Any suitable tumor antigen can be used. Suitable tumor antigens are well known to those skilled in the art; for example, tumor antigens are recorded in the Cancer Antigenic Peptide Database.

[0667] Tumor antigens are described, for example, in Lu et al. (2021) Hepatology 73: 821-832 and Wu et al. (2022) Medicine in Drug Discovery 16: 100144.

[0668] Some tumors are rich in certain tumor antigens. Therefore, some tumor antigens are used as tumor markers and can also be used as tumor antigen vaccines in cancer therapy.

[0669] Similar to vaccines against pathogens, cancer vaccines involve the delivery of inactivated cancer cells or tumor antigens (TAs) in combination with adjuvants. Cancer vaccines also involve ex vivo attack of dendritic cells (DCs) with TAs. Despite years of experimentation, most cancer vaccines have yielded disappointing results, resulting in only one cancer vaccine being approved for clinical use. Identifying new vaccine delivery systems that bypass the barriers to effective cancer vaccines should make them therapeutically applicable.

[0670] The vector of this invention can represent an effective strategy for designing tumor vaccines.

[0671] In some implementations, the transgene encodes tumor antigens that are abundant on liver metastases.

[0672] In some implementations, the transgene encodes a tumor antigen selected from the following: 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.

[0673] In some implementations, the transgenic code encodes OVA.

[0674] In some embodiments of the invention, the transgenic material comprises or consists of at least 70% of the same nucleotide sequence as SEQ ID NO: 41 or 43, or fragments thereof. Suitably, the transgenic material comprises or consists of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the same nucleotide sequence as SEQ ID NO: 41 or 43, or fragments thereof.

[0675] In some embodiments of the present invention, the transgene comprises or consists of a nucleotide sequence SEQ ID NO: 41 or 43 or a fragment thereof.

[0676] In some implementations, the transgene encodes TRP2.

[0677] In some embodiments of the invention, the transgenic material comprises or consists of at least 70% of the same nucleotide sequence as SEQ ID NO: 48 or 49, or fragments thereof. Suitably, the transgenic material comprises or consists of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the same nucleotide sequence as SEQ ID NO: 48 or 49, or fragments thereof.

[0678] In some embodiments of the present invention, the transgene comprises or consists of a nucleotide sequence SEQ ID NO: 48 or 49 or a fragment thereof.

[0679] This invention considers the combined use of the cytokine gene therapy of this invention and the tumor vaccine of this invention.

[0680] In another aspect, the present invention provides a product (e.g., a composition or kit) comprising a first vector of the present invention and a second vector of the present invention, the first vector comprising a transgene encoding a cytokine (preferably IL12) and the second vector comprising a transgene encoding a tumor antigen.

[0681] In another aspect, the present invention provides a product (e.g., a composition or kit) comprising cells containing a first vector of the present invention, the first vector containing a transgene encoding a cytokine (preferably IL12); and a second vector of the present invention containing a transgene encoding a tumor antigen.

[0682] In another aspect, the present invention provides a product (e.g., a composition or kit) comprising a first vector of the present invention and cells comprising a second vector of the present invention, the first vector comprising a transgene encoding a cytokine (preferably IL12) and the second vector comprising a transgene encoding a tumor antigen.

[0683] In another aspect, the present invention provides a product (e.g., a composition or kit) comprising a first cell containing a first vector of the present invention and a second cell containing a second vector of the present invention, the first vector containing a transgene encoding a cytokine (preferably IL12) and the second vector containing a transgene encoding a tumor antigen.

[0684] The composition may be a pharmaceutical composition as disclosed herein.

[0685] In another aspect, the present invention provides a first vector of the invention comprising a transgene encoding a cytokine (preferably IL12) for use in a therapy, wherein the first vector is administered simultaneously, sequentially, or separately to a subject in combination with a second vector of the invention comprising a transgene encoding a tumor antigen.

[0686] In another aspect, the present invention provides a second vector of the invention comprising a transgene encoding a tumor antigen for use in a therapy, wherein the second vector is administered simultaneously, sequentially, or separately to a subject in combination with a first vector of the invention comprising a transgene encoding a cytokine (preferably IL12).

[0687] In another aspect, the present invention provides the use of a first vector of the invention comprising a transgene encoding a cytokine (preferably IL12) for manufacturing a drug, wherein the first vector is administered simultaneously, sequentially, or separately to a subject in combination with a second vector of the invention comprising a transgene encoding a tumor antigen.

[0688] In another aspect, the present invention provides the use of a second vector of the present invention comprising a transgene encoding a tumor antigen for manufacturing a drug, wherein the second vector is administered simultaneously, sequentially, or separately to a subject in combination with a first vector of the present invention comprising a transgene encoding a cytokine (preferably IL12).

[0689] In a preferred embodiment, the use in the therapy is for treating or preventing cancer.

[0690] In another aspect, the present invention provides a method for treating or preventing cancer, the method comprising administering to a subject in need a first vector of the present invention comprising a transgene encoding a cytokine (preferably IL12) and a second vector of the present invention comprising a transgene encoding a tumor antigen. The first and second vectors may be administered, for example, simultaneously, sequentially, or separately.

[0691] In some implementations, the first and / or second carriers are administered via intravenous injection, portal vein injection, or hepatic artery injection.

[0692] In another aspect, the present invention provides a cell comprising a first vector of the present invention and / or a second vector of the present invention, the first vector comprising a transgene encoding a cytokine (preferably IL12) and the second vector comprising a transgene encoding a tumor antigen.

[0693] Exemplary vector

[0694] In a preferred embodiment, the vector from 5' to 3' comprises: an MRC1 promoter, a transgene, and one or more miRNA target sequences as defined herein. In other preferred embodiments, the vector from 5' to 3' comprises: an MRC1 enhancer, an MRC1 promoter, a transgene, and one or more miRNA target sequences as defined herein.

[0695] In some embodiments, the vector from 5' to 3' comprises: an MRC1 promoter, a transgene encoding IFNα, and one or more miRNA target sequences that inhibit transgene expression in hepatocytes and / or sinusoidal endothelial cells and / or splenic phagocytes.

[0696] In some embodiments, the vector from 5' to 3' comprises: an MRC1 enhancer, an MRC1 promoter, a transgene encoding IFNα, and one or more miRNA target sequences that inhibit transgene expression in hepatocytes and / or sinusoidal endothelial cells and / or splenic phagocytes.

[0697] In some embodiments, the vector from 5' to 3' comprises: an MRC1 enhancer, an MRC1 promoter, a Kozak sequence, a transgene encoding IFNα, a WPRE, and one or more miRNA target sequences that inhibit transgene expression in hepatocytes and / or sinusoidal endothelial cells and / or splenic phagocytes.

[0698] In some embodiments, the vector from 5' to 3' comprises: a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 31; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 34; and a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 36.

[0699] In some embodiments, the vector from 5' to 3' comprises: a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 32; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 31; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 34; and a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 36.

[0700] In some embodiments, the vector from 5' to 3' comprises: a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 32; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 31; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 33; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 34; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 35; and a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 36.

[0701] In some embodiments, the vector from 5' to 3' comprises: an MRC1 promoter, a transgene encoding IL10, and one or more miRNA target sequences that inhibit transgene expression in hepatocytes and / or sinusoidal endothelial cells and / or splenic phagocytes.

[0702] In some embodiments, the vector from 5' to 3' comprises: an MRC1 enhancer, an MRC1 promoter, a transgene encoding IL10, and one or more miRNA target sequences that inhibit transgene expression in hepatocytes and / or sinusoidal endothelial cells and / or splenic phagocytes.

[0703] In some embodiments, the vector from 5' to 3' comprises: an MRC1 enhancer, an MRC1 promoter, a Kozak sequence, a transgene encoding IL10, a WPRE, and one or more miRNA target sequences that inhibit transgene expression in hepatocytes and / or sinusoidal endothelial cells and / or splenic phagocytes.

[0704] In some embodiments, the vector from 5' to 3' comprises: a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 31; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 39; and a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 36.

[0705] In some embodiments, the vector from 5' to 3' comprises: a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 32; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 31; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 39; and a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 36.

[0706] In some embodiments, the vector from 5' to 3' comprises: a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 32; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 31; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 33; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 39; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 35; and a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 36.

[0707] In some embodiments, the vector from 5' to 3' comprises: an MRC1 promoter, a transgene encoding IL12, and one or more miRNA target sequences that inhibit transgene expression in hepatocytes and / or sinusoidal endothelial cells and / or splenic phagocytes.

[0708] In some embodiments, the vector from 5' to 3' comprises: an MRC1 enhancer, an MRC1 promoter, a transgene encoding IL12, and one or more miRNA target sequences that inhibit transgene expression in hepatocytes and / or sinusoidal endothelial cells and / or splenic phagocytes.

[0709] In some embodiments, the vector from 5' to 3' comprises: an MRC1 enhancer, an MRC1 promoter, a Kozak sequence, a transgene encoding IL12, WPRE, and one or more miRNA target sequences that inhibit transgene expression in hepatocytes and / or sinusoidal endothelial cells and / or splenic phagocytes.

[0710] In some embodiments, the vector from 5' to 3' comprises: a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 31; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 40; and a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 36.

[0711] In some embodiments, the vector from 5' to 3' comprises: a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 32; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 31; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 40; and a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 36.

[0712] In some embodiments, the vector from 5' to 3' comprises: a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 32; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 31; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 33; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 40; a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 35; and a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 36.

[0713] Immune checkpoint inhibitor

[0714] 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 inhibitory checkpoint molecules or otherwise antagonizes them. When expressed on the cell surface, inhibitory checkpoint molecules suppress or attenuate T cell-mediated immune responses against said cells. For example, expression of inhibitory checkpoint molecules can prevent cells from being killed by a T cell response. This mechanism is particularly detrimental in cases where cancer cells express inhibitory checkpoint molecules, as this can allow cancer cells to evade the host T cell response. Therefore, when inhibitory checkpoint molecules on tumor cells are inhibited by immune checkpoint inhibitors, an enhanced host T cell response against the tumor cells should occur.

[0715] In some implementations, immune checkpoint inhibitors inhibit inhibitory checkpoint molecules 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 attenuation factor; 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), and PD-L2 (PD-1 ligand 2). TIM-3 (T cell immunoglobulin domain and mucin domain 3), VISTA (Ig inhibitor of the V domain for T cell activation), B7-1 (CD80), B7-2 (CD86), TGFB (transforming growth factor β) pathway-associated protein, Il13 (interleukin-13), IL4 (interleukin-4), FGL (fibrinogen-like 1), TIGIT (T cell immune receptor with Ig and ITIM domains), CD96 (TACT protein), Ceacam-1 (carcinoembryonic antigen-associated cell adhesion molecule 1), CD155 (PVR protein), CD112 (PVR-associated protein 2 (PVRL2)), LGALS3 (galactolectin 3), and CD47 (integrin-associated protein). Combinations of checkpoint inhibitors can also be used.

[0716] In some implementations, the TGFB pathway-related proteins are selected from the group consisting of: TGFB1 (transforming growth factor β-1), TGFB2 (transforming growth factor β-2), TGFB3 (transforming growth factor β-3), LTBP1 (potential transforming growth factor β-binding protein 1), TGFBR1 (transforming growth factor β receptor 1), TGFBR2 (transforming growth factor β receptor 2), integrin αv, integrin β5, integrin β6, integrin β8, and LRRC32 (leucine-rich repeat sequence 32).

[0717] In some implementations, the immune checkpoint inhibitor is an antibody. In some implementations, the immune checkpoint inhibitor antibody is selected from the group consisting of: anti-CTLA4 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-PDL2 antibody, and anti-LAG-3 antibody.

[0718] In some embodiments, the immune checkpoint inhibitor is a CTLA4 inhibitor, preferably an anti-CTLA4 antibody.

[0719] 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.

[0720] In some implementations, the immune checkpoint inhibitor is a PD-L1 inhibitor; preferably, the PD-L1 inhibitor is an anti-PD-L1 antibody.

[0721] In some implementations, the immune checkpoint inhibitor is a PD-L2 inhibitor, and preferably the PD-L2 inhibitor is an anti-PD-L2 antibody.

[0722] In some implementations, the immune checkpoint inhibitor is a LAG-3 inhibitor; preferably, the LAG-3 inhibitor is an anti-LAG-3 antibody.

[0723] As used herein, the term "antibody" should be understood as a polypeptide or fragment thereof that is substantially encoded by one or more immunoglobulin genes and specifically binds to and recognizes an antigen (e.g., a cell surface marker). As used herein, the term "antibody" refers to a whole or complete antibody molecule (e.g., IgM, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA, IgD, or IgE) or any antigen-binding fragment thereof.

[0724] Antibodies can be polyclonal or monoclonal. Monoclonal antibodies are produced by the same immune cells (e.g., hybridomas resulting from the fusion of an antibody-producing B cell line and a cancerous B cell line). A monoclonal antibody targeting a specific antigen will recognize a single specific epitope on said antigen. In contrast, polyclonal antibodies are produced by multiple different cell lines and thus recognize several different epitopes on a specific antigen.

[0725] Antigen-binding fragments of antibodies include, for example, single-chain antibodies, single-chain Fv fragments (scFv), Fd fragments, Fab fragments, Fab' fragments, or F(ab')2 fragments. scFv fragments are single polypeptide chains that include the heavy and light chain variable regions of the antibody from which the scFv originates. In addition, intracellular antibodies, microantibodies, triantibodies, and biantibodies (see, for example, Todorovska et al. (2001) J Immunol Methods 248(1):47-66; Hudson and Ortt (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 antibodies and are compatible with the uses described herein. As used herein, the term antibody also includes antibody fragments produced by modifying the whole antibody or antibody fragments synthesized de novo using recombinant methods.

[0726] Suitable methods for generating antibodies against a specific antigen or antigen-binding fragments thereof are known in the art (see, for example, Greenfield (2014) Antibodies: A Laboratory Manual, 2nd edition 201-221).

[0727] TR1 cell inhibitor

[0728] Type 1 regulatory (Tr1) cells are a class of regulatory T cells involved in peripheral immunity. Tr1 cells are a subset of CD4+ T cells. Tr1 cells can regulate tolerance and can be specific to self- or non-self antigens. An important natural role of Tr1 cells is the suppression of tissue inflammation in autoimmunity and graft-versus-host disease.

[0729] In some implementations, the Tr1 cell inhibitor inhibits molecules selected from the group consisting of: Cd4, Eomes, Gzmk, Lag3, Pdcd1, Ahr, Maf, Prdm1, Ctla4, and Il10ra.

[0730] In some implementations, Tr1 cell inhibitors are antibodies.

[0731] Combination

[0732] As used herein, the terms “combination” or “in combination,” “used in combination with,” or “combination formulation” can refer to the simultaneous, sequential, or separate administration of two or more agents.

[0733] As used in this article, the term "simultaneously" means that the drug is administered at the same time (i.e., at the same time).

[0734] As used in this article, the term "in sequence" means the application of medicines one after another.

[0735] As used herein, the term “separate” means that the agents are administered independently of each other, but within a time interval that allows the agents to exhibit a combined effect (preferably a synergistic effect). Thus, “separate” administration may allow one agent to be administered, for example, 1 minute, 5 minutes, or 10 minutes after another agent.

[0736] Variants, derivatives, analogs, and fragments

[0737] In addition to the specific proteins and nucleotides mentioned herein, this invention also covers their variants, derivatives, and fragments.

[0738] In the context of this invention, a “variant” of any given sequence is a sequence in which a particular sequence of residues (whether amino acid residues or nucleic acid residues) has been modified in a manner such that the polypeptide or polynucleotide in question retains at least one of its intrinsic functions. Variant sequences can be obtained by adding, deleting, substituting, modifying, replacing, and / or altering at least one residue present in a naturally occurring polypeptide or polynucleotide.

[0739] As used herein, the term "derivative" in relation to the proteins or polypeptides of the present invention includes any substitution, alteration, modification, replacement, deletion, and / or addition of one (or more) amino acid residues from or to a sequence, provided that the resulting protein or polypeptide retains at least one of its endogenous functions.

[0740] Typically, amino acid substitutions can be made, for example, from 1, 2, or 3 to 10 or 20 substitutions, provided that the modified sequence retains the desired activity or ability. Amino acid substitutions may include the use of analogs that are not naturally occurring.

[0741] The proteins used in this invention can also have amino acid residue deletions, insertions, or substitutions, which produce silencing changes, thereby yielding functionally equivalent proteins. Intentional amino acid substitutions can be made based on the similarity of residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties, as long as endogenous function is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids without polarized head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0742] Conservative substitutions can be performed, for example, according to the table below. Amino acids in the same block in the second column, and amino acids in the same row in the third column, can substitute for each other:

[0743]

[0744] Typically, variants may share some identity with wild-type amino acid sequences or wild-type nucleotide sequences.

[0745] In the context of this invention, a variant sequence is considered to contain an amino acid sequence that is at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the subject sequence, suitably at least 95%, 96%, 97%, 98%, or 99%. Although variants can also be considered based on similarity (i.e., amino acid residues with similar chemical properties / functions), in the context of this invention, it is preferred to express them based on sequence identity.

[0746] In the context of this invention, a variant sequence is considered to contain a nucleotide sequence that is at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the subject sequence, suitably at least 95%, 96%, 97%, 98%, or 99%. Although variants can also be considered based on similarity, in the context of this invention, variants are preferably expressed based on sequence identity.

[0747] Where appropriate, reference to a sequence having a percentage identity with any of the SEQ ID NOs detailed herein means a sequence having said percentage identity over the entire length of the referenced SEQ ID NO.

[0748] Sequence identity comparisons can be performed visually, or more commonly, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage identity between two or more sequences.

[0749] Percentage identity can be calculated on consecutive sequences, that is, one sequence is aligned with another, 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 "vacancy-free" alignment. Typically, such vacancy-free alignments are only performed on a relatively short number of residues.

[0750] While this is a very simple and consistent approach, it fails to account for the fact that, for example, in otherwise identical sequence pairs, an insertion or deletion in an amino acid or nucleotide sequence can lead to subsequent residue or codon misalignments, potentially resulting in a significantly reduced percentage of identity during global alignment. Therefore, most sequence alignment methods are designed to produce optimal alignments that account for possible insertions and deletions without over-penalizing the overall identity score. This is achieved by inserting “gaps” in the sequence alignment in an attempt to maximize local identity.

[0751] However, these more sophisticated methods assign a "vacancy penalty" to each vacancy that appears in the alignment, so that for the same number of identical amino acids or nucleotides, a sequence alignment with as few vacancies as possible (reflecting a higher correlation between the two compared sequences) will receive a higher score than a sequence alignment with many vacancies. Affine vacancy cost is typically used, which charges a relatively high cost for the presence of a vacancy and imposes a small penalty on each subsequent residue within that vacancy. This is the most commonly used vacancy scoring system. A high vacancy penalty will naturally result in an optimized alignment with fewer vacancies. Most alignment programs allow modification of the vacancy penalty. However, when using such software for sequence alignment, the default value is preferred. For example, when using the GCG Wisconsin Bestfit package, the default vacancy penalty for amino acid sequences is -12 for a vacancy and -4 for each extension.

[0752] Therefore, calculating the maximum percentage identity first requires generating the best alignment, while taking into account gap penalties. A suitable computer program for performing such alignments is the GCG Wisconsin Bestfit software 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 software 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 GNEWORKS comparison tool suite. Both BLAST and FASTA can be used for offline and online searches (see Ausubel et al. (1999) ibid, pp. 7-58 to 7-60). However, for some applications, the GCG Bestfit program is preferred. Another tool, BLAST 2 Sequences, can also be used to compare protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174(2):247-50; FEMS Microbiol. Lett. (1999) 177(1):187-8).

[0753] Although a final percentage of identity can be measured, the alignment process itself is not typically based on all-or-nothing pairwise comparisons. Instead, a scaled similarity score matrix is ​​usually used, which assigns a score 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 in the BLAST program suite). The GCG Wisconsin program typically uses either public defaults or a custom symbolic comparison table (if provided) (see the user manual for more details). For some applications, it is preferred to use the public defaults of the GCG package, or, in the case of other software, a default matrix such as BLOSUM62.

[0754] Once the software produces an optimal alignment, it can calculate percentage sequence identity. The software typically includes this as part of the sequence comparison and generates a numerical result. Percentage sequence identity can be calculated as the percentage of identical residues relative to the total number of residues in the mentioned SEQ ID NO.

[0755] "Fragment" is also a variant, and the term generally refers to selected regions of a polypeptide or polynucleotide that are of interest functionally or, for example, in assays. Therefore, a "fragment" refers to an amino acid or nucleic acid sequence that is part of a full-length polypeptide or polynucleotide.

[0756] Such variants, derivatives, and fragments can be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. In cases where insertion is desired, synthetic DNA encoding the insert fragment and 5' and 3' flanking regions corresponding to the naturally occurring sequence on either side of the insertion site can be prepared. The flanking regions will contain convenient restriction sites corresponding to the sites in the naturally occurring sequence, allowing the sequence to be cleaved with appropriate enzymes, and the synthetic DNA is ligated into the nick. This DNA is then expressed according to the invention to prepare the encoded protein. These methods merely illustrate many standard techniques known in the art for manipulating DNA sequences, and other known techniques may also be used.

[0757] Cell

[0758] In one aspect, the present invention provides a cell comprising the product of the present invention.

[0759] In some embodiments of the product of the present invention, the carrier is contained within a cell.

[0760] In one aspect, the present invention provides a cell comprising the carrier of the present 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.

[0761] The cells may contain the first and / or second vector of the present invention (and / or optionally, a third vector).

[0762] Methods of making cells

[0763] The vectors of the present invention can be introduced into cells using various techniques known in the art, such as transfection, transduction, and transformation. Suitablely, the vectors of the present invention are introduced into cells by transfection or transduction.

[0764] In one aspect, the present invention provides a method for preparing the cells of the present invention. This method may include introducing the vector of the present invention into the cells, for example by transfection or transduction.

[0765] Suitable, the cells may be derived from a sample isolated from the subject (e.g., peripheral blood, bone marrow, or cord blood). The cells can be further separated from the sample by any suitable method.

[0766] The cells of the present invention can be produced by a method comprising the following steps:

[0767] (i) separating or providing a cell-containing sample from the subject; and

[0768] (ii) Transducing or transfecting a cell-containing sample using the vector of the present invention to provide an engineered cell population.

[0769] Cells can be cultured before or after the introduction of the vector of the present invention. These steps can be performed in a closed and sterile cell culture system.

[0770] Hematopoietic stem / progenitor cells and differentiated cells

[0771] Suitablely, the cells may be hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs) (e.g., myeloid / monocyte progenitor cells), or differentiated cells (e.g., macrophages or monocytes). Suitablely, the cells may be autologous and / or allogeneic.

[0772] Hematopoietic stem cells (HSCs) are pluripotent stem cells that can be found in sources such as peripheral blood, bone marrow, and cord blood. HSCs are capable of self-renewal and differentiation into any blood cell lineage. They can recolonize the entire immune system, as well as erythrocyte-like and myeloid cells in all hematopoietic tissues, such as bone marrow, spleen, and thymus. They provide for the lifelong reproduction of all hematopoietic cell lineages.

[0773] Hematopoietic progenitor cells (HPCs) have the ability to differentiate into specific cell types. However, compared to stem cells, they have become even more specific: they are induced to differentiate into their "target" cells. The difference between HSCs and HPCs is that HSCs can replicate indefinitely, while HPCs can only divide a limited number of times.

[0774] Compared to stem cells or progenitor cells, differentiated cells are more specialized. Differentiated cells include differentiated cells of the hematopoietic 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 hematopoietic lineage can be distinguished from HSCs and HPCs by detecting cell surface molecules that are not expressed or are expressed at low levels 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 (erythrocytes), CD2, CD3, CD4, CD8 (T), and CD56 (NK).

[0775] The cells of this invention can 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 cells can be isolated from a subject and can be introduced into the vector of this invention prior to administration to the patient by the methods described herein.

[0776] Adoptive cell transfer can be allogeneic or autologous. "Autologous cell transfer" should be understood as obtaining the starting cell population from the same subject as the subject receiving the transduced cell population. Autologous transfer is advantageous because it avoids the problems associated with immune incompatibility and can be used on the subject regardless of the availability of a genetically matched donor. "Allogeneic cell transfer" should be understood as obtaining the starting cell population from a different subject than the subject receiving the transduced cell population. Optionally, the donor will be genetically matched to the subject receiving the cells to minimize the risk of immune incompatibility. Alternatively, the donor may be unmatched and unrelated to the patient. The appropriate dose of the transduced cell population is effective, such as for treatment and / or prophylaxis. The dose to be administered may depend on the subject to be treated and the condition, and can be readily determined by a technician.

[0777] Producer cell and packaging cell

[0778] Suitablely, the cells may be producer cells. The term "producer cell" includes cells that produce viral particles after transient transfection, stable transfection, or vector transduction, or any cell engineered to stably contain the elements required for viral particle production. Suitable producer cells are well known to those skilled in the art. Suitable producer cell lines include HEK 293 (e.g., HEK 293T), HeLa, and A549 cell lines.

[0779] Suitablely, the cells may be packaging cells. The term "packaging cell" includes cells containing some or all of the elements required for packaging infectious recombinant viruses. Packaging cells may lack the recombinant viral vector genome. Typically, such packaging cells contain one or more vectors capable of expressing viral structural proteins. Cells containing only some of the elements required to produce enveloped viral particles can be used as intermediates for generating virus particle-producing cell lines through subsequent steps such as transient transfection, transduction, or stable integration of each additional required element. These intermediates are covered by the term "packaging cell." Suitable packaging cells are well known to those skilled in the art.

[0780] In some embodiments, the cells are genetically engineered to reduce the expression of CD47 and / or HLA on the cell surface. In some embodiments, the cells contain genetically engineered disruption of the gene encoding CD47 and / or the gene encoding β2-microglobulin and / or one or more genes encoding the MHC-Iα chain. In some embodiments, the cells contain genetically engineered disruption in all copies of the gene encoding CD47. The expression of CD47 and / or HLA on the cell surface may be reduced such that the cells are substantially free of surface-exposed CD47 and / or HLA molecules. In some embodiments, the cells do not contain any surface-exposed CD47 and / or HLA molecules.

[0781] In one aspect, the present invention provides a method for preparing viral vector particles of the present invention. The method may include culturing viral particle producing or packaging cells containing the vector of the present invention under conditions suitable for generating viral particles. The method may include: (a) introducing the vector of the present invention into viral particle producing or packaging cells, for example by transfection or transduction; and (b) culturing the cells under conditions suitable for generating viral particles. Such conditions are well known to those skilled in the art.

[0782] Pharmaceutical composition

[0783] A pharmaceutical composition is a composition comprising a therapeutically effective amount of a pharmaceutically active agent or thereof. Preferably, it comprises a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof).

[0784] In some implementations, the pharmaceutical composition is a cancer vaccine. A "cancer vaccine" is a vaccine that treats existing cancer or prevents the development of cancer.

[0785] "Pharmaceutical acceptable" means that the formulation is sterile and pyrogen-free. The carrier, diluent, and / or excipients must be "acceptable," meaning compatible with the carrier and will not cause harm to the recipient. Typically, the carrier, diluent, and excipients will be sterile and pyrogen-free saline or infusion media; however, other acceptable carriers, diluents, and excipients may be used.

[0786] Acceptable carriers, diluents, and excipients for therapeutic use are well known in the pharmaceutical industry. The selection of a drug carrier, excipient, or diluent is based on the intended route of administration and standard pharmaceutical practice. A pharmaceutical composition may contain, or in addition to, any suitable binder, lubricant, suspending agent, coating agent, or solubilizer as a carrier, excipient, or diluent.

[0787] Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohols, siloxanes, waxes, petroleum gels, vegetable oils, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, fragrance oils, monoglycerides and diglycerides of fatty acids, petroleum ether fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0788] The products, carriers, inhibitors, cells, or pharmaceutical compositions according to the invention can be administered in a manner suitable for treating and / or preventing the diseases described herein. The amount and frequency of administration will be determined by factors such as the subject's condition and the type and severity of the subject's disease, although appropriate dosage can be determined through clinical trials. Pharmaceutical compositions can be formulated accordingly.

[0789] The products, carriers, inhibitors, cells, or pharmaceutical compositions according to the invention can be administered parenterally, for example intravenously or via infusion techniques. The products, carriers, inhibitors, cells, or pharmaceutical compositions can be administered in the form of a sterile aqueous solution, which may contain other substances, such as sufficient salts or glucose, to make the solution isotonic with blood. The aqueous solution can be suitably buffered (preferably buffered to a pH of 3 to 9). Pharmaceutical compositions can be formulated accordingly. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished using standard pharmaceutical techniques well known to those skilled in the art.

[0790] The products, carriers, inhibitors, cells, or pharmaceutical compositions according to the present invention can be administered systemically, for example by intravenous injection.

[0791] The products, carriers, inhibitors, cells, or pharmaceutical compositions according to the invention can be administered topically, for example, by targeted administration to the liver. Suitably, the products, carriers, inhibitors, cells, or pharmaceutical compositions can be administered via intravenous injection in the portal vein or via intrahepatic artery injection.

[0792] The pharmaceutical composition may comprise the product of the present invention, a carrier, an inhibitor, or cells in an infusion medium, such as a sterile isotonic solution. The pharmaceutical composition may be encapsulated in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0793] Products, carriers, inhibitors, cells, or pharmaceutical compositions can be administered in single or multiple doses. In particular, products, carriers, inhibitors, cells, or pharmaceutical compositions can be administered in a single, one-time dose. Pharmaceutical compositions can be formulated accordingly.

[0794] Products, carriers, inhibitors, cells, or pharmaceutical compositions may be administered at different doses (e.g., measured in vector genomes (vg) / kg). In any case, the physician will determine the actual dose most suitable for any individual subject, and the dose will vary depending on the specific subject's age, weight, and response.

[0795] The pharmaceutical composition may also contain one or more other therapeutic agents. The product, carrier, inhibitor, cell, or pharmaceutical composition may be administered in combination with one or more other therapeutic agents.

[0796] This invention also includes the use of kits comprising the products, carriers, inhibitors, cells, and / or pharmaceutical compositions of the present invention. Preferably, the kit is used for the methods and uses described herein, such as the therapeutic methods described herein. Preferably, the kit includes instructions for use of the kit components.

[0797] Methods for treating and / or preventing disease

[0798] In one aspect, the present invention provides products, carriers, inhibitors, cells, or pharmaceutical compositions according to the invention, which are used as pharmaceuticals.

[0799] In related aspects, the present invention provides the use of products, carriers, inhibitors, cells or pharmaceutical compositions according to the invention in the manufacture of pharmaceuticals.

[0800] In a related aspect, the present invention provides a method for administering a product, carrier, inhibitor, cell, or pharmaceutical composition according to the invention to a subject in need. Suitably, the subject is a human subject.

[0801] Cancer

[0802] The products, carriers, inhibitors, cells, or pharmaceutical compositions according to the present invention may be used for the prevention or treatment of cancer in a subject. Suitably, the subject is a human subject.

[0803] In one aspect, the present invention provides products, carriers, inhibitors, cells, or pharmaceutical compositions according to the invention for the prevention or treatment of cancer.

[0804] In related aspects, the present invention provides the use of products, carriers, inhibitors, cells or pharmaceutical compositions according to the present invention for the manufacture of medicaments for the prevention or treatment of cancer.

[0805] In a related aspect, the present invention provides a method for preventing or treating cancer, the method comprising administering to a subject in need a product, carrier, inhibitor, cell, or pharmaceutical composition according to the present invention.

[0806] Participants may have cancer. Alternatively, participants may be at risk of developing cancer.

[0807] Subjects may have previously been identified as being at risk of developing cancer. This increased risk may have been determined through genetic screening and / or by reviewing the subject's family history. Subjects may have been identified as expressing one or more genetic markers that indicate an increased risk of developing cancer.

[0808] Appropriately, those skilled in the art will know of genetic risk factors (e.g., genetic markers) associated with an increased risk of developing cancer. Those skilled in the art can use any suitable method or technique known in the art to determine whether a subject has an increased risk of developing cancer.

[0809] Subjects may have previously received cancer treatment. Subjects may be in cancer remission. Subjects may be resistant to chemotherapy.

[0810] Liver metastasis

[0811] In some implementations, the cancer is liver cancer, such as secondary liver cancer (e.g., liver metastases).

[0812] In some implementations, the subject has secondary liver cancer (e.g., liver metastases) or is at risk of developing secondary liver cancer, and the product, carrier, inhibitor, cell, or pharmaceutical composition is used to prevent or treat secondary liver cancer.

[0813] In some implementations, the subject has a primary cancer (e.g., primary cancer of colorectal cancer, pancreatic cancer, or breast cancer), and the product, carrier, inhibitor, cell, or pharmaceutical composition is used to prevent or treat secondary liver cancer (e.g., liver metastases).

[0814] Metastasis is a secondary malignant growth of cancer cells that develops in places far from the primary site of cancer. Metastases most commonly occur when cancer cells detach from the main tumor and enter the bloodstream or lymphatic system.

[0815] The liver is one of the most common sites of cancer metastasis, accounting for nearly 25% of all cases. The high frequency of liver involvement in metastatic disease can be explained by different hypotheses about metastatic spread. According to the “mechanical or hemodynamic hypothesis,” the dual blood supply to the liver via the portal vein and hepatic artery promotes the retention of circulating cancer cells, which explains the high incidence of liver metastases in patients with gastrointestinal cancer. On the other hand, according to the “seed-and-soil” hypothesis, some primary tumors selectively target the liver as a metastatic site, for example, patients with uveal melanoma with chromosome 3 deletion, and patients with breast cancer with a combination of human growth factor receptor 2 (HER-2) positivity and estrogen (ER) and progesterone receptor (PR) positivity (de Ridder, J. et al., 2016. Oncotarget, 7(34), p. 55368).

[0816] Most liver metastases are cancers, particularly adenocarcinomas. The primary tumor can be any primary tumor, and the primary tumor can be unknown. However, the most common primary tumors in patients with adenocarcinoma originate from the colorectal, pancreatic, or breast tissue (de Ridder, J et al., 2016. Oncotarget, 7(34), p. 55368).

[0817] Subjects can be diagnosed with liver metastases using any suitable method known to those skilled in the art. For example, subjects can be diagnosed using CT imaging with liver protocol, colonoscopy, and EGD.

[0818] The products, carriers, inhibitors, cells, or pharmaceutical compositions of the present invention can be used in combination with any other suitable therapy for the treatment or prevention of liver metastases. For example, in combination with surgical resection and / or chemotherapy for liver metastases.

[0819] Those skilled in the art will understand that they can combine all the features of the invention disclosed herein without departing from the scope of the invention as disclosed herein.

[0820] Preferred features and embodiments of the invention will now be described with the aid of non-limiting examples.

[0821] Unless otherwise stated, the present invention will be practiced using conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology, which are within the capabilities of those skilled in the art. These techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, EF, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Ausubel, FM, et al. (1995 and regular supplements) Current Protocols in Molecular Biology, Chapters 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, JM, and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, DM, and Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. These general texts are incorporated herein by reference.

[0822] Examples

[0823] Example 1

[0824] Results

[0825] Generation of LV platform capable of in vivo liver macrophage engineering

[0826] We first generated LV ( ) 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 KC) and is upregulated by alternative activated macrophages (such as tumor-associated macrophages (TAM)). We then cloned the GFP coding sequence downstream of the Mrc1 promoter sequence (originating from Mrc1.GFP LV) and generated VSV G pseudotyped Mrc1.GFP LV stock solution ( Figure 1 A). Mrc1.GFP LV drove robust transgene expression in IL4-exposed (M2-like) bone marrow-derived macrophages (BMDM), but not in LPS / IFNγ-exposed (M1-like) BMDM (Fig. S1B-E). Intravenous injection (iv) of Mrc1.GFP LV into immunocompromised mice resulted in selective GFP expression in hepatocytes (KC and sinusoidal endothelial cells, LSEC) and some spleen cells (Mrc1-positive macrophages). We did not observe GFP expression or integrated LV copies in hematopoietic cells, bone marrow, or other organs such as the lungs, lymph nodes, small intestine, and brain. Figure 1 B to Figure 1 D). To further fine-tune gene expression in KC, we utilized microRNA (miRNA) regulation. We first used a bidirectional LV containing a selected miRNA target sequence (miRT) downstream of GFP to verify the inhibition of off-target gene expression in cell types. Four tandem copies of mirT-122-5p completely blocked GFP expression in hepatocytes while retaining it in KC ( Figure 8 F to Figure 9 I), while four copies of miRT-126-3p prevent GFP expression in LSECs but not in Kupffer cells ( Figure 8 J and Figure 8 Based on these results, we incorporated four copies each of miRT-122-5p and miRT-126-3p downstream of GFP in Mrc1.GFP LV, producing Mrc1.GFP.miRT LV (K). Figure 1A). To investigate the in vivo expression of the novel Mrc1.GFP.miRT LV in the presence of liver tumors, we generated experimental liver metastases by implanting either MC38 colorectal cancer cells expressing mCherry (red fluorescent protein) or colorectal epithelial cancer cells derived from APCΔ716;KrasG12D;Tgfbr2- / -;Trp53R270H;Fbxw7- / - mice (hereinafter referred to as AKTPF cancer cells). We injected Mrc1.GFP or Mrc1.GFP.miRT LV iv into mice challenged with liver metastases. Consistent with our findings in tumor-free mice, Mrc1.GFP selectively drove GFP expression in KC, LSEC, and spleen Mrc1-positive macrophages, while GFP expression in LSEC was almost completely attenuated in the presence of miRNA regulation (Mrc1.GFP.miRT LV). Figure 1 E). In MC38 and AKTPF-derived metastatic lesions, we observed enhanced GFP expression around liver metastatic areas, indicating enrichment of transduced KCs (including true monocyte-derived tumor-associated macrophages) and / or upregulation of Mrc1 promoter activity in these regions. Figure 1 F and Figure 8 L). We did not observe GFP expression in other organs (such as the brain, small intestine, lungs, and lymph nodes). Figure 8 In summary, the selective biodistribution and expression of the newly developed Mrc1.GFP.miRT LV in KC, along with its enriched expression in the peritumoral region, support the feasibility of in vivo genetic engineering of KC (including liver metastasis-associated macrophages) to deliver therapeutic molecules to liver metastases.

[0827] In vivo LV engineered KCs enable rapid, sustained, and well-tolerated IFN alpha production

[0828] We then used engineered KC to deliver IFNα to liver metastases. For this purpose, we replaced the GFP with the IFNα DNA coding sequence in Mrc1.GFP.miRT LV, derived from what we refer to herein as IFNα LV. To efficiently transduce KC, we generated LVs based on a manufacturing process designed to produce high-titer LV stock solutions with low levels of contaminants such as plasmids and endotoxins, which could lead to bystander innate immune activation or adverse systemic effects. We then engineered KC in vivo by intravenously injecting either IFNα LV or an LV with the same regulatory elements but lacking transgenes (referred to herein as control LV) into immune-active mice at previously reportedly highly effective hepatocyte-targeting doses. Figure 2A). In mice with IFNα-enriched LV-engineered KC, we observed rapid transgene output, reflected by an increase in plasma IFNα concentration, peaking at 700 pg / ml to 1,000 pg / ml after 3 weeks and subsequently stabilizing between 200 pg / ml and 700 pg / ml. These IFNα levels remained stable for up to 240 days compared to the control LV group, eventually decreasing to almost undetectable levels by day 360. Figure 2 B). The number of integrated LV copies in the liver of IFNα-LV-treated mice was lower than that found in control LV-treated mice, indicating long-term anti-selection of IFNα-LV-transduced hepatocytes (including KC). Figure 2 C). Compared with control LV-treated mice, KC IFNα expression decreased over time, resulting in fewer circulating B cells and eosinophils. Compared with control LV-treated mice, CD4 and CD8 T lymphocytes, inflammatory and resident monocytes, neutrophils, platelets, erythrocytes, and hemoglobin levels remained unchanged. Figure 2 D and Figure 9 A). To investigate whether the decrease in B cells is associated with B cell activation and autoantibody production, we measured the presence of autoantibodies in the plasma of mice treated with saline (PBS), control LV, or IFNα LV at days 52 and 366. We found no difference in autoantibody levels among all analysis groups ( Figure 9 B). Furthermore, we did not observe any changes in the levels of liver or tissue damage markers (i.e., alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indicating that there was no hepatotoxicity in mice treated with IFNα LV. Figure 2 E). To further investigate whether exogenous IFNα expression of KC induces inflammation, tissue damage, or other changes, we performed histopathological analysis on most relevant organs at the end of the experiment. No treatment-related abnormalities were observed in any of the analysis compartments. Figure 2 F and Figure 9 C). In summary, these results indicate that KC-driven IFNα expression leads to robust and long-term levels of plasma IFNα that are safe and well-tolerated, at least in mice.

[0829] Gene-based forced IFN alpha expression by KCs releases T cell activation and impairs liver metastasis growth

[0830] We delivered two different doses systemically (1.5). 10 9 TU / kg or 1.5 10 10 IFNα LV at TU / kg was used to engineer KC ( ) in mice previously challenged with mc38-based experimental liver metastases. Figure 3A). Consistent with previous results, we found a sustained dose-dependent level of IFNα in plasma ( Figure 3 B) and the dose-dependent LV copy number integrated in the liver ( Figure 10 A), and in mice treated with IFNα LV, a negative correlation was found between plasma IFNα levels and the number of circulating B cells (A). Figure 10 B). We monitored liver metastasis growth using magnetic resonance imaging and found that both IFNα LV doses delayed tumor progression and achieved complete response (CR) and long-term survival in 3 mice (1 at a lower dose and 2 at a higher dose). Figure 3 C and Figure 3 D). CR mice re-challenged with MC38 tumors exhibited impaired growth, indicating the induction of adaptive immune memory against tumor-associated antigens (D). Figure 10 C). To better investigate the induction of tumor-responsive T cells after KC engineering, we systemically delivered control LV or IFNα LV to syngeneically active mice previously challenged with experimental liver metastases using MC38 cells expressing ovalbumin (OVA, used as a substitute tumor antigen). Consistent with previous results, IFNα expression in KC delayed liver metastasis growth (C). Figure 3 E and Figure 10 D). We found that, compared with control LV-treated mice, tumor-specific T cells, identified by MHCI staining of a pentamer complexed with the OVA immunogenic peptide (SIINFEKL), were enriched in tumors from IFNα LV-treated mice. Figure 3 F). Furthermore, 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 (F). Figure 3 G). These data suggest that IFNα derived from engineered KC can delay tumor progression by biasing TAMs toward an inflammatory phenotype and facilitating T cell activation and the expansion of tumor-reactive T cell clones.

[0831] To further investigate the effects of forced IFNα expression on the tumor microenvironment, we used AKTPF CRC cells to generate experimental liver metastases. AKTPF liver metastases recapitulated some histopathological features of human CRC liver metastases, such as epithelial gland structures formed by CRC cells, visceral necrosis areas, fibrosis, angiogenesis, and immune infiltration. Figure 10 E and Figure 10 F). In two independent experiments, KC expressing IFNα delayed tumor progression and led to complete remission (CR) in 5 out of 10 treated mice. Figure 3 I, Figure 3 J and Figure 10G to Figure 10 I). Based on our previous findings in the MC38 experimental metastasis model, we observed that KC-derived IFNα biased TAM towards an M1-like phenotype and increased the number of tumor-infiltrating CD8 T lymphocytes ( Figure 3 K to Figure 3 M). To further investigate the effect of exogenous IFNα expression from KC on liver metastases from sources different from CRC, we challenged syngeneic mice with KrasG12DTrp53R172H pancreatic ductal adenocarcinoma (PDAC) cells (K8484) via intrahepatic injection and treated them with either control or IFNα LV. We found that, compared with the control group, 6 out of 8 treated mice in the IFNα LV group achieved complete remission (CR), which strongly inhibited tumor growth. Figure 3 N and Figure 10 In summary, these experiments indicate that KC engineering via systemic delivery of IFNα LV results in IFNα expression from KC, which in turn robustly suppresses tumor growth, at least in part, by skewing the TAM phenotype and promoting CD8 T cell recruitment.

[0832] Engineering of KCs by IFN alpha LVs enables preferential IFN alpha signaling around metastatic foci

[0833] To investigate the underlying mechanisms of the observed tumor response, we performed a comprehensive transcriptomic analysis of AKTPF liver metastases in mice treated with either control LV or IFNα LV. We observed increased expression of interferon-stimulated genes, indicating IFNα activity in metastatic lesions of the IFNα LV group ( Figure 11 A). We then employed spatial transcriptomics to investigate the presence of regions preferentially transduced by IFNα in the metastatic liver. To this end, we assigned mice to three distinct groups: (1) control: mice treated with control LV, (2) responders: IFNα-treated mice with a reduced metastatic volume compared to the control, and (3) resistant: IFNα-treated mice with a similar metastatic volume to the control. Figure 11 B). Note that due to the lack of tumors at the time of analysis, we were unable to analyze mice that achieved a full response. We then analyzed 36 mm of livers containing metastatic lesions. 2 Spatial transcriptomics was performed on the slices. Unsupervised clustering analysis was then performed to cluster the spatial spots based on similar transcriptomic profiles. Figure 11 C). Spatial spots belonging to clusters 1 and 6 showed high expression of genes associated with adenocarcinoma and were presumed to be assigned to liver metastatic regions. Conversely, clusters 0, 2–5, and 7 showed high expression of genes associated with normal liver and were presumed to be assigned to liver tissue ( Figure 11D). We then used an unsupervised method that weighted the properties of the surrounding spatial spots (i.e., liver or metastatic lesions) to infer the estimated distance from the metastatic lesion interface to each spatial spot. We grouped the spatial spots into different spatial compartments based on their relative distance from the metastatic / liver parenchymal boundary, including internal metastases (spatial compartment AC), anterior metastases (spatial compartment D), peripheral metastases (spatial compartment EG), and intact liver regions (spatial compartment H). Figure 4 A and Figure 11E). As expected, we found that, across all groups, genes belonging to cancer-related biological processes or pathways (e.g., angiogenesis, the p53 pathway, epithelial-to-mesenchymal transition) were enriched in metastatic regions (internal and anterior regions) compared to regions outside the liver metastases (peripheral metastases and intact liver). Consistent with this observation, epithelial cell-related genes such as epithelial cell adhesion molecule (Epcam), cadherin 1 (Cdh1), and villin 1 (Vil1) were highly expressed in both internal and anterior metastatic regions. Conversely, hepatocyte-related genes (e.g., albumin, Alb; apolipoprotein 2, Apoa2; and cytochrome p450 family 27a1, Cyp27a1) and sets of genes belonging to liver-related pathways (e.g., lipoogenesis or bile acid metabolism) were upregulated in intact liver regions. Consistent with the enhanced transgenic expression of engineered KC in the peri-hepatic metastatic region following systemic Mrc1.GFP.miRT LV delivery, we found that genes associated with responses to type I interferon (e.g., cytokine signaling inhibitor 1, Socs1; signal transducer and transcription activator 1, Stat1; and NLR family CARD domain-containing 5, Nlrc5) were enriched in liver metastatic and peri-metastatic regions of the IFNα LV group (responders and resistant), consistent with the ability of the LV platform to preferentially engineer KC near liver metastases. Furthermore, upregulation of genes associated with type I interferon activity and belonging to the Gene Ontology (GO) category (such as responses to interferon-γ, viral responses, positive regulation of cytokine production, and T cell activation) was associated with regions of type I interferon signaling. Furthermore, in responders compared to resistant or control groups, genes belonging to adaptive immune activation GO categories (such as the regulation of adaptive immune responses (e.g., CD3γ subunits of the TCR complex, Cd3g; CD8, Cd8a; and TCRα subunits, Trac) and immune effector processes) were upregulated in internal, anterior, and peripheral metastatic regions, corresponding to sites of enhanced IFNα activity. Interestingly, genes associated with antigen presentation were also highly expressed in metastatic lesions in both responders and resistant groups compared to controls. Importantly, in the resistant group, but not in controls or responders, we found increased IL10 signaling in anterior and peripheral metastatic regions, suggesting that IL10 may play a role in counteracting IFNα effects in resistant mice. Notably, markers associated with T cell depletion and tolerogenic phenotypes, such as transforming growth factor β1 (Tgfb1), amesericulture proteins (Eomes), and granzyme K (Gzmk), were also upregulated in the resistant group in internal, anterior, and peripheral metastatic regions. Figure 4 B and Figure 4C). In summary, IFNα expression in KC was associated with selective immune activation in liver metastases and surrounding metastatic regions in responder mice. However, in resistant mice, immune activation appeared to be attenuated compared to responders and was associated with enrichment of IL10 signaling in the metastatic / hepatic parenchymal boundary region.

[0834] IFN alpha promotes APC immune activation and enhanced MHCII-restricted antigen presentation in responder mice

[0835] We performed single-cell transcriptomic analysis on live cells from the same metastatic lesions using spatial transcriptomic sequencing. Figure 11 A). We employed an unsupervised clustering approach to identify different cell types, such as: (1) APCs, (2) T and NK cells, (3) B cells, (4) neutrophils, (5) endothelial cells, (6) hepatocytes, and (7) cancer cells, which were manually annotated based on their transcriptomic profiles. Figure 12 A to Figure 12 D). We then focused on cells belonging to the APC cluster. We found that genes belonging to the GO class associated with IFNα, IFNγ, or LPS signaling were relatively enriched in all IFNα-LV treated groups. On the other hand, genes associated with IL10, PGE2, and IL4 signaling were upregulated in the resistant group compared to the responder group, suggesting that these genes may play a role in inducing resistance to gene-based IFNα treatment activity. Genes associated with antigen presentation, namely MHC protein complexes and antigen processing and presentation, were upregulated in some responders compared to the resistant or control groups, and showed the lowest expression in the control group. Figure 5 A). We then performed sub-clustering analysis to better define cell populations and differentially expressed genes within APC clusters. Within APC, we identified overlapping cell clusters in all three experimental groups, except for the TAM cluster, which was remodeled by IFNα treatment, indicating gene expression reprogramming upon exposure to gene-based IFNα delivery. Based on this observation and considering the major effect of IFNα on TAM genetic programming, we termed all TAMs belonging to clusters present in tumors treated with IFNα LV as IFNα-TAMs, and those present in the control LV group as TAMs. All other cell clusters were manually annotated based on their gene expression profiles ( Figure 5 B and Figure 12E). Through differential gene expression analysis among TAM subsets, we found that for all three groups, genes upregulated in IFNα-TAM compared to TAM were enriched in biological processes associated with IFNα / IFNγ responses, 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), and antigen processing-associated transporter 1 (Tap1), consistent with the role of IFNα-TAM in positively regulating immune activation. Notably, two genes associated with immunosuppression, Il10 and Tgfb1, were upregulated in the IFNα LV group, suggesting key roles for these genes in resistant mice. On the other hand, pro-tumor genes typically associated with the pro-tumor activity of TAMs, such as matrix metallopeptidase 8 (Mmp8), transmembrane protein 176B (Tmem176B), trigger receptor 2 (Trem2) expressed on bone marrow cells, and fibroin 1 (Fn1), are upregulated in TAMs relative to IFNα-TAMs. Figure 5 C and Figure 5 D). Notably, compared to the resistant and control groups, the responder group was enriched for specialized APCs, namely classical dendritic cells (cDCs) and monocyte-derived DCs (MoDCs). Figure 5 E). Consistent with this observation, we found that in APCs from responder mice, genes associated with MHCII-restricted antigen presentation, such as those encoding 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. Notably, MHCII-restricted genes, such as those encoding MHCII subunits (H2-T22, H2-T23, H2-D1, and B2m), Tap1, Tap2, Tap-binding protein (Tapbp), and proteasome S20 subunits β8 and 9 (Psmb8 and Psmb9), were upregulated in all IFNα LV-treated (resistant and responder) groups. Consistent with IL10, which plays a presumed role in resistance to KC-derived IFNα expression, IL10-related genes, such as Tgfb, CCAAT enhancer-binding protein β (Cebpb), IL4 receptor (Il4r), Socs3, and CC motif chemokine ligand 24 (Ccl24), were upregulated in resistant APCs compared to responders and controls. Figure 5F). Among all APC populations, DCs, cDCs, and KCs expressing Ccr7, as well as Mo DCs, expressed the highest levels of genes associated with MHCII-restricted antigen presentation. Therefore, the differences in expression levels of genes associated with MHCII-restricted antigen presentation in APCs may at least be partly attributable to the enhanced infiltration of specialized APCs (such as Mo DCs and cDCs) in the responder population. On the other hand, the upregulation of genes associated with MHCII-restricted antigen presentation may be due to the direct effects of IFNα on cells (F). Figure 5 (G). In summary, IFNα released from KC promotes APC remodeling to the immunostimulatory phenotype by enhancing antigen presentation function. However, MHCII-restricted function and DC infiltration appeared to be reduced in resistant mice compared to responder mice. Simultaneously, enhanced IL10 signaling in APCs from resistant mice supports the association between lack of response, IL10 upregulation, and impaired MHCII-restricted antigen presentation.

[0836] Therapeutic response to IFN alpha is associated with T cell activation and counteracted by Eomes CD4 T cell infiltration

[0837] We then performed differential expression analyses in the T and NK cell compartments across the three experimental groups. Similar to TAM, genes involved in IFNα and IFNγ signaling were enriched in all IFNα-LV treated groups. Conversely, genes involved in immune activation (i.e., regulation of T cell-mediated cytotoxicity, natural killer cell activation, or cell killing) were specifically upregulated in a subset of responder groups. Figure 6 A). We then performed unsupervised sub-clustering analysis to identify distinct cell populations within the T and NK cell compartments and manually annotated the resulting clusters. We found overlapping cell clusters in all three experimental groups ( Figure 6 B and Figure S6A). Selectively in resistant mice, we observed a population of CD4 T cells that were transcriptionally similar to the previously described Tr1 cells (here referred to as Eomes CD4 T cells) (Bonnal et al. (2021) Nature Immunology 22: 735-745; Roncarolo et al. (2018) Immunity 49: 1004-1019), expressing CD4 T cell markers such as Ctla4, granzyme K (Gzmk), Lag3, and PD1 (Pdcd1), as well as immunosuppression-associated genes such as the IL10 receptor (Il10ra), Il10, and the transcription factor Eomes, and lacking expression of the transcription factor Foxp3 (…). Figure 6 C and Figure 13 B). On the other hand, selective enrichment within the responder population revealed a population of CD8 T effector 1 cells ( Figure 6D). The latter exhibited transcriptomic features similar to those of tissue-resident effector memory T cells, which had previously been associated with responses to immunotherapy ( Figure 6 E and Figure 13 (B) (Kim et al. (2021) Liver international: official journal of the International Association for the Study of the Liver 41: 764-776). Furthermore, we found that IFNα released by KC increased IFNα and IFNγ signaling across all CD8 T cell populations. Notably, compared to control or resistant mice, genes belonging to T cell desaturation, such as Pdcd1, Lag3, TIM3 (Havcr2), Ctla4, Eomes, thymocyte selection-associated high-mobility cassette protein (Tox), Ccl3, Ccl4, and caspase 3 (Casp3), were downregulated in responders. Conversely, genes associated with adaptive immune responses and T cell-mediated immunity and cytotoxicity, such as transcription factor 7 (Tcf7), T-box transcription factor 21 (Tbx21), Cd69, integrin subunit αe (Itgae), integrin subunit α1 (Itga1), Cd7, Il2, tumor necrosis factor α (Tnf), and Il12a, were upregulated more in responders than in resistant mice. Figure 6 E and Figure 13 (C). In summary, these data indicate that IFNα released by engineered KC promotes adaptive immunity in responder mice by remodeling T cell infiltrates, enriching effector phenotypes associated with responses to immunotherapy, and attenuating T cell desaturation. Conversely, in resistant mice, enhanced desaturation of infiltrating Eomes CD4 T cells and CD8 T cells may block antitumor effects.

[0838] IFN alpha from engineered KCs eradicates liver metastasis in conjunction with functional suppression of regulatory T cells

[0839] We then investigated whether, as in mice, IFNα signaling was positively correlated with the presence of Eomes CD4 T cells in the tumor microenvironment in human CRC liver metastases. To this end, we utilized a large dataset of RNA sequencing data from human CRC liver metastases collected from our center and found that patients with high IFNα signaling scores showed higher levels of Eomes CD4 signatures (…). Figure 7 A, Figure 7 B and Figure 14A). We then performed immunostaining on CRC liver metastasis samples from two patients in this cohort, one with a high IFNα signaling score and the other with a low IFNα signaling score. We found that in the high IFNα group, most CD4 T cells expressed detectable levels of LAG3, while in the low IFNα group, CD4 T cells did not show detectable LAG3 expression. Figure 7 C and Figure 14 (B) Notably, LAG3 has previously been reported as a marker of T cell depletion and human Tr1 cells. This observation suggests that Eomes CD4 T cells are positively correlated with endogenous IFNα signaling and may at least partially counteract immune activation in the tumor microenvironment.

[0840] In mice resistant to IFNα LV, we observed increased IL10 signaling, impaired MHCII-restricted antigen presentation, enhanced Eomes CD4 T cell infiltration, and enhanced CD8 T cell desaturation. This observation is consistent with previous studies indicating that IL10 may play a role in the differentiation, accumulation, and effector function of Eomes CD4 T cells, which have been described as suppressing antigen presentation through perforin-mediated direct killing of dendritic cells (DCs) and inhibiting T cell activity in CRC liver metastases through IL10 secretion. Based on these observations, we inhibited IL10 signaling using a monoclonal antibody that blocks the IL10 receptor (α-IL10R). Mice challenged with AKTPF liver metastases and treated with IFNα or control LV were treated with α-IL10R or unrelated IgG. Accumulation of EOMES CD4 T cells induced by IFNα-blocked IL10R (α-IL10R) was observed. Figure 7 D), indicating that IL10 signaling is essential for IFNα-induced accumulation in these cells of liver metastases. However, the combination of IFNα and α-IL10R achieved lower therapeutic efficacy than IFNα LV or α-IL10R alone. Figure 7 E), indicating that IL10 signaling may also be essential for the therapeutic activity of IFNα. In fact, we found that the combination of α-IL10R and IFNα LV induced the highest increase in PD1 expression on circulating CD4 and CD8 T cells in peripheral blood (E). Figure 14 C and Figure 14 (D), which is consistent with the role of IL10 in restoring T cells and preventing their depletion, which may be especially necessary in the case of IFNα T cell exposure.

[0841] We observed that Ctla4 was expressed in Eomes CD4 T cells, chromosomal CD4 and CD8 T cells, and Foxp3 T-regulated (Treg) cells.Figure 13 B). Furthermore, in resistant mice, CTLA4 was strongly upregulated in CD8 T cells. Notably, CTLA4 in Tr1 cells may play a crucial role in suppressing T cell function and attenuating antigen presentation by isolating the co-stimulatory molecules CD80 / CD86 in APCs. Based on this observation, we combined KC-based IFNα delivery with an anti-CTLA4 blocking monoclonal antibody (α-CTLA4, Figure 7 F) combination. In two different experimental models of CRC liver metastases, MC38 ( Figure 7 G and Figure 14 E) and AKTPF ( Figure 7 H and Figure 14 In F), the combination of IFNα and α-CTLA4 via KC strongly inhibited liver metastasis growth compared to either treatment alone. Notably, in mice with AKTPF liver metastases, we observed that up to 70% of mice exhibited a complete response after the IFNα and CTLA4 combination. This result indicates that enhancing antigen presentation in APCs by inhibiting the regulation of CTLA4 function in Eomes CD4 T cells, desaturated CD4 / CD8 T cells, and CD4 Treg cells strongly enhances the therapeutic activity of IFNα LV, revealing a major contribution of CTLA4 to the development of therapy resistance.

[0842] Overall, these findings demonstrate a strong synergistic effect between our strategy of delivering gene-based IFNα from the tumor bed via KC and checkpoint blockade targeting regulatory T cell function.

[0843] Discussion

[0844] We developed a novel LV platform to engineer KCs close to liver metastases and utilized this strategy to deliver IFNα into CRC and PDAC liver metastasis models. IFNα released from KC led to: i) reprogramming of TAMs and the shift of invasive DCs towards immune activation and antigen presentation; ii) increased recruitment, activation, and reduced desaturation of CD8 T cells; and iii) enrichment of CD8 T cell subsets with tissue-resident effector memory cell characteristics, previously associated with positive responses to immunotherapy. This immune cell remodeling resulted in the suppression of metastases in most mice. In-depth analysis of resistant mice revealed the emergence of an Eomes-expressing CD4 T cell population that is transcriptionally similar to Tr1 cells, which is associated with immunosuppression and tolerogenic functions. Furthermore, APCs from resistant mice showed increased IL10 signaling and reduced MHCII-restricted antigen presentation, while CD8 T cells showed increased desaturation markers. The co-administration of CTLA4 blockade and IFNα LV overcomes these resistance mechanisms, allowing for a near-complete treatment response and providing proof of principle for a novel treatment strategy with the potential to be translated into cancer patients with significant unmet medical needs.

[0845] Efficient KC engineering was achieved through intravenous administration of LV to the liver, preferential biodistribution, and the incorporation of the Mrc1 promoter and miRNA target site into the vector. This resulted in selective transgenic expression of KC, particularly in regions adjacent to metastatic lesions. The enrichment of LV-based KC engineering in peripheral metastatic areas is likely due to remodeled local vascular systems and tumor-driven changes in KC phagocytic activity. Furthermore, previous reports have shown increased MRC1 expression in macrophages in the presence of tumors.

[0846] Selective exogenous expression of cytokines in KCs can limit hepatotoxicity from direct expression in hepatocytes or LSECs. Furthermore, it has been proposed that macrophages (including KCs) rearrange their genetic programs in the presence of tumors to promote tumor growth and immune evasion. Therefore, direct expression of IFNα in these cells could reshape their pro-tumorigenic genetic programs, leading to greater therapeutic benefits. On the other hand, KC-derived IFNα also reaches systemic circulation, establishing sustained plasma levels. Although previous studies have only reported prophylactic activity against translocational inoculation of recombinant type I interferon administered via implanted microosmotic pumps, this systemic exposure may contribute to the therapeutic benefits observed in this study. Intratissue expression via KC engineering bypasses the biodistribution and vascular barriers of systemic administration, potentially achieving more efficient concentrations on target cells within the TME. Although systemic IFNα administration has been associated with significant toxicity in preclinical models and humans, we did not gather evidence of tissue damage or autoimmunity in our study. This may be due to: (i) an elevated therapeutic index of locally generated IFNα in the liver matrix and preferential IFNα signaling in liver regions carrying liver metastases; (ii) stable expression of IFNα compared to the dynamic changes in peak and trough of systemically delivered cytokines in plasma (which are often associated with desensitization and toxicity); and (iii) plasma IFNα levels within physiological ranges and similar to those observed during viral infection.

[0847] Notably, LV-based cell engineering led to vector integration and sustained transgene expression. Importantly, our strategy ultimately achieved practical extinction within one year.

[0848] The termination of expression may be due to the metabolic turnover of engineered KC, which is faster in cells expressing exogenous IFNα than in those transduced with control LV, suggesting some form of anti-selection. Alternatively, it is possible to use an integrase-deficient (ID) LV, which persists in the nucleus as an adjunct form driving lower and more transient transgene expression. Therefore, the choice between LV or IDLV may depend on the desired level and duration of transgene output.

[0849] We observed therapeutic benefits of IFNα LV in all tested mouse liver metastasis models, with a superior response in AKPTF CRC, which better replicated the human disease in terms of gene mutations and histopathological features. The presence of multifocal gland-like structures in the AKPTF model, which may enhance the interaction between engineered macrophages and the tumor mesenchyme (TME), along with slower tumor growth, may prolong the therapeutic window and thus benefit the therapeutic activity of IFNα LV.

[0850] Although therapeutic activity was observed after KC engineering via a single dose of IFNα LV, the Eomes CD4 T cell population exhibiting tr1-like gene signatures counteracted the effects of IFNα in a subset of resistant mice. Consistent with previous reports, we showed that Eomes CD4 T cell development is dependent on type I IFN and IL10 stimulation. This observation highlights the complex and sometimes contradictory effects of IFNα, which can promote tumor growth and immune evasion in some cases. For example, in mouse models of chronic viral infection, IFNα exposure promotes differentiation of bone marrow-derived suppressor cells, which in turn suppress CD8 T cell responses or promote cancer stem cell phenotypes in mouse models of fibrosarcoma. On the other hand, forced expression of IFNαR1 in CD8 T cells enhances cytotoxic activity in subcutaneous MC38 mouse tumors, or restoration of IFNα signaling in cancer cells leads to CD8-dependent therapeutic activity in various syngeneic and xenograft tumor mouse models. In a similar manner, IL10 can promote or suppress tumor immunity depending on the presence of its targets and other stimuli. For example, by acting on dendritic cells (DCs), IL10 impairs activation and antigen presentation, while simultaneously preventing DC-induced CD8 T cell apoptosis by impairing DC function. By acting on CD8 T cells, IL10 prevents T cell desaturation and promotes T cell activation in renal cell carcinoma patients and mouse models of tumors. Consistent with these observations, we found that IFNαLV or IL10 blockade alone promotes tumor immunity and delays liver metastasis growth. Conversely, the combination of IFNαLV and IL10 blockade does not affect liver metastasis growth.

[0851] We found that exogenous IFNα released by KC strongly upregulated genes involved in MHCI-restricted antigen presentation in different populations of liver metastatic invasive APC, including TAM and DC from responder and resistant mice, suggesting that IFNα may exert its therapeutic activity at least in part by activating antigen presentation.

[0852] Interestingly, MHCII-restricted antigen presentation was suppressed in liver metastases from resistant mice, partly due to lower infiltration in different dendritic cell populations and downregulation of genes associated with MHCII-restricted antigen presentation. Consistent with this observation, MHCII-restricted antigen presentation may be essential for maintaining functional T cells in tumors and achieving a response to immunotherapy. Further investigation is needed to determine whether the reduced MHCII-restricted antigen presentation in resistant mice is upstream or downstream of enhanced IL10 signaling and Eomes CD4 T cell differentiation.

[0853] Tr1 cells and Tregs have been described as suppressing immunity through the expression of IL10 and CTLA4. CTLA4 may play a key role in reducing antigen presentation and T cell initiation through CD80 / CD86 segregation, which in turn leads to the activation of defective T cells. Notably, CTLA4 is highly upregulated in CD8 T cells and Eomes CD4 T cells in resistant mice, suggesting that it may play an important role in preventing immune activation in the presence of high IFNα signaling. Consistent with this concept, a dual intervention of IFNα LV delivery and CTLA4 blockade produced a strong therapeutic effect, achieving complete regression of liver metastases in most mice.

[0854] Our findings have clinical relevance to support their associations. These include differential prognostic values ​​for genetic signatures associated with specific subsets of innate or adaptive immune cells described in our study, such as pro-tumor macrophages, CD8 tissue-resident effector memory cells, serotonin T cells, and Eomes CD4 T cells. We present evidence of a correlation between the degree of IFNα signaling and Tr1 trait scores in clinical samples of liver metastases of CRC. Furthermore, we found expression of the Tr1 cell marker LAG3, which has high IFNα signaling scores, in CD4 T cells infiltrating human CRC liver metastases. Notably, Eomes CD4 T cells are associated with a subgroup of patients showing high IFNα signaling in liver metastases and may be present at low levels in patients showing low IFNα signaling. Thus, although previous studies have dissected and highlighted the complexity of CD4 T cells 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).

[0855] Overall, we have developed a novel off-the-shelf gene therapy tool that engineers KC through a single, well-tolerated systemic administration, which then rapidly delivers IFNα from the liver to liver metastases and releases tumor immunity against liver metastases in relevant mouse models.

[0856] Methods

[0857] Plasmid design

[0858] To construct the Mrc1.GFP lentiviral vector (LV), we used restriction enzyme sites XhoI and AgeI to insert a putative Mrc1 promoter sequence covering a 1883 bp DNA sequence (MM39 assembly: CHR2:14232425-14234307) into the previously described PGK.GFP LV by replacing the PGK promoter sequence. The bidirectional miRT LV was generated by inserting four tandem copies perfectly complementary to either miR-122-5p (miRT-122-5p: 5'-ACAAACACCATTGTCACACTCCA-3') or miR-126-3p (miRT-126-3p: 5'-CGCATTATTACTCACGGTACGA-3'), with the miRT sites separated by random 4 bp DNA adapter sequences. Four copies of the miRT sequence were then inserted downstream of the WPRE sequence of a bidirectional LV containing oppositely positioned minimal cytomegalovirus (mCMV) and human phosphoglycerate kinase 1 (PGK) promoters, driving the expression of truncated low-affinity nerve growth factor receptor (NDiGFR) and GFP, respectively. The miRT sequence was inserted using the restriction enzyme site KpnI. Four copies of miRT-122-5p and four copies of miRT-126-3p were inserted downstream of the WPRE sequence of the Mrc1.GFP LV transfer vector plasmid using the restriction enzyme sites KpnI to construct the Mrc1.GFP.miRT LV. The IFNα LV transfer vector plasmid was generated by replacing the GFP sequence of the Mrc1.GFP.miRT LV transfer vector plasmid with cDNA encoding the mouse IFNα1 protein using the restriction enzyme sites SalI and ScaI. The GFP sequence of the Mrc1.GFP LV transfer vector plasmid was depleted by digestion with restriction enzymes AgeI and SalI, and then control LV was generated by inserting four copies of miRT-122-5p and four copies of miRT-126-3p downstream of WPRE using the restriction enzyme site KpnI.

[0859] Cell culture

[0860] HEK293T, MC38, and K8484 cells were cultured in adherent cell culture plates in Iscove-modified Dulbecco-modified Eagle medium (IMDM, Corning) supplemented with 10% fetal bovine serum (FBS; HyClone™), penicillin (100 IU / mL), and streptomycin (100 μg / mL). To generate MC38-mCherry cells expressing mCherry in almost all cells (99.97% of all cells), MC38 cells were transduced with an LV expressing a chimeric protein fused to the C-terminus of the CD81 transmembrane domain from the constitutively expressed human phosphoglycerate kinase 1 (PGK) promoter. To generate MC38-OVA cells, MC38 cells were transduced from an LV expressing the hPGK promoter with a full-length chicken ovalbumin (OVA), and 2.86 VCN was detected in MC38-OVA cells.

[0861] AKTPF organoids were cultured at 37°C in 30 µL of phenol red-free and growth factor-reduced substrate (BDBiosciences) in 48 wells, surrounded by a 2% GlutaMAX supplement. ™ 300 µL of Advanced Dulbecco-modified Eagle Medium (DMEM) / F-12 (ThermoFisher Scientific) containing supplements (Gibco), penicillin (100 IU / mL), streptomycin (100 µg / mL), 1% hepes buffer (Gibco), 1% N-2 supplement (Gibco), 2% B-27 supplement (Gibco), 1 mM N-acetylcysteine ​​(Sigma-Aldrich), and 50 ng / mL mouse epidermal growth factor (rmEGF; Gibco). To convert AKTPF organoids into adherent 2D cell cultures, AKTPF organoids were passed twice to NSG mice and once to c57Bl6 mice. For this purpose, AKTPF organoids were transplanted into NSG mice via intrasplenic injection and recovered after four weeks. Single cells were obtained by cutting tumors into small pieces and filtering them through a 45 µm cell filter. Then, 1,000,000 single cells were transplanted into NSG mice via intrasplenic injection. Four weeks after tumor cell recovery, the mice were supplemented with 10% FBS and 2% GlutaMAX. ™Tumor cells were cultured in DMEM / F-12 medium containing penicillin (100 IU / mL) and streptomycin (100 μg / mL) in cell culture-treated plates. After in vitro culture, 1,000,000 tumor cells were transplanted into c57Bl6 mice. Four weeks post-transplantation, tumor cells were extracted and cultured as previously described. The cells cultured as described above were used in experiments employing AKTPF cells.

[0862] To extract bone marrow-derived macrophages (BMDM), bone marrow was harvested from C57Bl6 mice by washing the femur and tibia with 10 mL of MACS buffer (Miltenyi Biotec). For erythrocyte lysis, 1 mL of dehydrated saline was added to the cell spheroids, and immediately followed by 50 mL of MACS buffer. The cells were then placed in a solution supplemented with 10% FBS and 2% GlutaMAX. ™ Macrophages were cultured in a medium consisting of supplements (Gibco), penicillin (100 IU / mL), streptomycin (100 μg / mL), and RPMI medium (Corning) containing 100 ng / mL mouse M-CSF (Miltenyi Biotec).

[0863] Seven days later, 1,000,000 BMDM cells were seeded into 24-well plates and transduced with LV at an MOI of 10. The next day, macrophage culture medium was added to the cells. For M2-like polarization, 50 ng / mL mouse IL-4 (Miltenyi) was added to the cell culture medium, or for M1-like polarization, 100 ng of lipopolysaccharide (LPS) from *E. coli* O55:B5 (Sigma-Aldrich) and 5 ng / mL mouse IFNγ (Miltenyi) were added. Flow cytometry (FC) analysis was performed six days after polarization induction.

[0864] LV production

[0865] In this study, third-generation VSV-G pseudotyped LV was used. LV stock solutions were produced at either laboratory-grade or process-developed (PDL) grade. For biodistribution studies in the presence of AKTPF-derived LMS, CD47-depleted LV was generated in CD47-negative HEK 293T cells. The titer of the LV stock solution was measured in HEK 293T cell transduction units (TU / mL).

[0866] Determining LV copy number by ddPCR

[0867] By using Maxwell ® 16 instruments (Promega) and Maxwell ®Genomic DNA was extracted from cell culture samples using a DNA purification kit (Promega). Genomic DNA was extracted from whole tissue samples using a DNeasy blood and tissue kit (Qiagen). LV copy number was determined using a QX200 Droplet Digital PCR System (Biorad). Digital droplet PCR was performed according to the manufacturer's instructions; in short, 5 ng to 20 ng of genomic DNA was added to the reaction, primers were used at a concentration of 900 nM, and the detection probe was used at 250 nM. Droplet quantification was acquired using a BioRad QX200 droplet reader and analyzed using QuantaSoft software (BioRad). For HIV genome detection, 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 a standard for mouse samples, the Sema3a gene was used: forward primer: 5'-ACCGATTCCAGATGATTGGC-3'; reverse primer: 5'-TCCATATTAATGCAGTGCTTG-3'; Hex channel detection probe: 5'-(HEX)-AGAGGCCTGTCCTGCAGCTCATGG-(BHQ-1)-3'. As a standard for human samples, the commercially available GAPDH expression assay (TaqMan) was used. ™ Gene expression assay (Invitrogen; Hs00894322_cn). The LV copy number per cell was calculated using the following formula:

[0868] LV copy number per cell = ((HIV concentration) / (normalized concentration)) 2

[0869] Gene expression by ddPCR

[0870] For gene expression analysis, RNeasy was used. ® RNA was extracted from frozen tissue using the Plus Mini Kit (Qiagen). Reverse transcription was performed using SuperScript™ IV VILO (Invitrogen) according to the manufacturer's instructions. Five to 20 ng of cDNA were used as input for gene expression analysis. The following TaqMan from Invitrogen was used. ™ Gene expression assay:

[0871] Table 1: TaqMan probes used in this project ™ Gene expression assays .

[0872]

[0873] As described above for LV copy number determination, ddPCR was used for data acquisition and analysis.

[0874] FC analysis and fluorescence-activated cell sorting

[0875] Based on the manufacturer's recommendations for fixed samples, use 7AAD nuclear staining or LIVE / DEAD. ™ A blue fixed dead cell staining kit (Invitrogen) was used to estimate cell viability. After single-cell dissociation (see below), an Fc blocker (BD Pharmagen) was added to the cells to prevent non-specific staining via binding to the FC receptor. For membrane-bound antigens, samples were stained on ice for 15 minutes. To stain intracellular proteins, True-Nuclear was used as directed by the manufacturer. ™The transcription factor buffer (BioLegend) was used to fix, permeate, and stain cells. For staining of TCRs specific to the SIINFEKL peptide loaded on MHC class I (H2kb), samples were stained with the SIINFEKL-loaded MHC class I pentamer (ProImmune) according to the manufacturer's instructions. We used the following gating strategy to define cell populations via flow cytometry: B cells (CD45+ B220+), CD4 T cells (CD45+ CD4+), CD45- cells in bone marrow (CD45-), CD8 T cells in tumors (CD45+ TCRb+ CD8+), CD8 T cells in blood (CD45+ B220- CD8+), CD86+ TAM (CD45+ CD11b+ F4 / 80+ CD86+), DCs in the liver (CD45+ F4 / 80- CD11chigh), EOMES+ CD4 T cells in tumors (CD45+ B220- CD11b- CD4+ EOMES+), granulocytes (CD45+ B220- Ly6g+), inflammatory monocytes in blood (CD45+ CD11b+ Ly6c+ Gr1-), and KCs in the liver (CD45+ B220- Ly6g+). Monocytes in the blood of nude mice (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 of 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 lungs (CD45+, Ly6g-, CD11b+, MRC1-), and 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 lungs (CD45+ Ly6g- CD11b+ MRC1+), neutrophils in the blood (CD45+ CD11b+The following antibodies were used for FC analysis: Ly6c+ Gr1+, parenchymal cells in the lungs (CD45-), parenchymal cells in the liver (CD45- CD31 / MRC1-), pentameric CD8 T cells in tumors (CD45+ CD8+ pentameric+), resident monocytes in the blood (CD45+ CD11b+ Ly6c- Gr1-), and T cells in the blood (CD45+ CD11b- CD3+).

[0876] Table 2: Antibodies for flow cytometry .

[0877]

[0878] By using FACSCanto II or FACSymphony ™ Samples were acquired using an A5 cell analyzer (BD Biosciences). For fluorescently activated cell sorting, BD FACSAria fusions were used.

[0879] Mouse strain

[0880] C57Bl / 6N mice (in all experiments using IFNα LV or control LV), NUDE mice (in all experiments using Mrc1.GFP LV or Mrc1.GFP.miRT LV unless otherwise instructed), or NSG mice were purchased from Charles River Laboratory. All experiments and procedures were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the San Raffaele Hospital animal facilities (IACUC numbers: 1007, 1098, 1108, and 1227) and authorized by the Italian Ministry of Health and local authorities in accordance with Italian law.

[0881] For endpoint analysis, mice were euthanized via cervical dislocation. The liver was perfused by injecting 10 mL of PBS containing 5 mM EDTA (Invitrogen) into the inferior vena cava and cutting the portal vein to allow a solution containing most circulating blood cells to exit the liver. For FC analysis instead of immunofluorescence (IF) analysis, 10 mL of IMDM (Corning) containing 0.35 mg / mL collagenase (Sigma-Aldrich) was injected into the inferior vena cava.

[0882] Intravenous injection of LV

[0883] For systemic LV injection, LV was diluted in PBS to obtain the required TU per mouse, ranging in volume from 200 µL to 300 µL. For intravenous injection (iv), mice were warmed under an infrared / red light and LV was delivered into the tail vein. LV was delivered 7 days after tumor placement unless otherwise instructed. In experiments using K8484, LV was delivered 3 days after tumor implantation. Unless otherwise instructed, IFNα LV and control LV were administered at 5... 10 9 Use TU / kg; unless otherwise specified, bidirectional LV (i.e., miRT LV), Mrc1.GFP LV, and Mrc1.GFP.miRT LV are used at 1.5 TU / kg. 10 10 TU / kg was used. In experiments involving MC38.OVA cells, TU was administered at 1.5 mmol / kg 3 days after tumor implantation. 10 10 IFNα LV and control LV were delivered at TU / kg. In all experiments, untransduced (UT) mice were injected with PBS ranging from 200 µL to 300 µL.

[0884] Monoclonal antibody injection

[0885] Monoclonal antibodies were administered via intraperitoneal injection in 100 µL of PBS. Dosage was as follows: MAB anti-mouse IL-10R (α-IL10R, BioXCell) and isotype control MAB rat IgG1κ anti-horseradish peroxidase (BioExcell) were administered initially at a dose of 1 mg / mouse, followed by 0.5 mg / mouse every 4 days; MAB anti-mouse CTLA4 (α-CTLA4, BioXCell) and isotype control MAB polyclonal hamster serum (BioXCell) were administered three times weekly at a dose of 0.1 mg / mouse. The monoclonal antibody treatment regimen was initiated concurrently with LV delivery.

[0886] Mouse experimental liver metastasis model

[0887] We delivered AKTPF organoids or AKTPF cells via intrasplenic injection. Briefly, the fur on the left upper ventral side of the mice was removed by shaving followed by application of depilatory cream (Balea). Immediately before surgery, mice were injected with 50 µL of carbofen (2.5 mg / mL) for pain management. During surgery, mice were anesthetized with 3% isoflurane (Iso-Vet) at a flow rate of 1.5 L / min. AKTPF cells were resuspended in 50 µL of Matrigel (BDBiosciences) or Geltrex (Thermo Scientific) for AKTPF organoids and carefully injected into the spleen using a pre-cooled syringe. For injections of AKTPF cells derived from organoids, the former were divided two days prior to injection. AKTPF cells derived from organoids or from 2D cultured AKTPF cells were dissociated into single cells, and 30,000 cells were injected into each mouse. The peritoneal wall was sutured using absorbable sutures, while the skin was closed using stainless steel wound clamps. Following surgery, mice were treated with antibiotics for one week by adding Baytril (Bayer) at a concentration of 0.5 mg / mL to their drinking water. For intrahepatic transplantation of MC38 and K8484 cells, the fur in the abdominal region of the mice was removed as described above. Pain management, anesthesia, and surgical procedures were performed as described 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 preferentially injected into the left lobe of the liver in 5 µL of PBS. Following surgery, mice were treated with antibiotics as described above. Liver metastatic growth was measured using magnetic resonance imaging (MRI) as described below, or by tumor weight (i.e., dissecting liver metastases after the experiment and measuring their weight on a digital balance with an accuracy of 10 mg).

[0888] Magnetic resonance imaging for liver metastasis volume estimation

[0889] A 7-Tesla preclinical scanner (Bruker, BioSpec 70 / 30 USR, Paravision 6.0.1) equipped with a 450 / 675 mT / m gradient (switching rate: 3400-4500 T / m / s; rise time 140 µs) and a circularly polarized mouse body volume coil with an inner diameter of 40 mm was used. During acquisition, mice were anesthetized by inhaling 3% isoflurane (Iso-Vet) at a flow rate of 1.5 L / min under a dedicated temperature control device to prevent hypothermia. Respiratory rate and body temperature were continuously monitored (SA Instruments, Inc., Stony Brook, NY, USA). To aid in visualization of liver lesions, a hepatocyte-specific contrast agent, Gd-EOB-DTPA (Bayer Schering Pharma), was used at a concentration of 0.05 μmol / g body weight. During the hepatobiliary phase enhanced by Gd-EOB-DTPA (10 minutes after administration), axial fat-saturated T2-weighted images (RARE-T2, rapid acquisition with relaxation enhancement, TR=3000ms, TE=40ms, voxel size=0.125 × 0.100 × 0.8mm, mean=4) and axial fat-saturated T1-weighted sequences (RARE-T1: TR=540ms, TE=7.2ms, voxel size=0.125 × 0.100 × 0.8mm, mean=4) were acquired. Volumetric measurements were performed using Medical Image Processing, Analysis, and Visualization (MIPAV) software.

[0890] Subcutaneous injection of MC38 cells

[0891] One million MC38 cells were subcutaneously injected into the flank of mice with 100 µL of PBS. Tumor growth was monitored by measuring the size of the subcutaneous lesions (larger diameter x and smaller diameter y) using calipers. Tumor volume was calculated using the following formula:

[0892] Volume = 3 / 4 π (0.5 diameter(x)^2 0.5 Diameter(y) / 2

[0893] Blood collection and analysis

[0894] Blood was drawn from the tail vein or the retroorbital venous plexus. Whole blood was analyzed using a hemocytometer using ProCyte DX™ (IDEXX). For FC analysis, erythrocyte lysis was performed using Hybri-Max™ (Sigma) erythrocyte lysis buffer. To retrieve the absolute number of hematopoietic cell populations, the percentage of CD45+ cells identified by FC analysis was multiplied by the absolute count of white blood cells detected by hemocytometer analysis. For plasma collection, plasma was collected in a Microvette. ®Blood in Sarstedt tubes was centrifuged at 3,000 rpm for 10 minutes at room temperature, and the precipitated red and white blood cells were discarded. For serum collection, blood collected in standard Eppendorf tubes was incubated at room temperature for 40 minutes and then centrifuged at 3,000 rpm for 10 minutes at room temperature. Fractions containing platelets, red blood cells, and white blood cells were discarded. IFNα levels in plasma were quantified using the mouse IFNα all-subtype ELISA KIT with high sensitivity (pbl Assay Science) according to the manufacturer's instructions. To estimate serum transaminases, ALT (Instrumentation Laboratory) and AST (Instrumentation Laboratory) quantification kits were used in an ILab Aries chemical analyzer (Instrumentation Laboratory), employing a kinetic UV (UV) method optimized by the International Federation for Clinical Chemistry and Laboratory Medicine. Simultaneously, quality control analyses were performed at SeraChem control levels 1 and 2 (#0018162412 and #0018162512). The quantification of autoreactive antibodies in serum was performed. The autoantigen microarray was fabricated at the Microarray & Immune Phenotyping coreFacility at the University of Texas Southwestern Medical Center, Dallas, TX, USA. Based on published literature, 120 autoantigens were selected from previously known autoantibodies in various immune-related diseases, cancers, allergic diseases, etc. Eight 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, and anti-Ig control 1:16) were also imprinted on the array as positive controls. Mouse serum samples were first treated with DNase I to remove cell-free DNA and then applied to the autoantigen array at a 1:50 dilution. Autoantibodies binding to antigens on the array were detected using cy3-labeled anti-mouse IgG and cy5-labeled anti-mouse IgM. The array slides were scanned using a Genepix 4400A scanner with a laser wavelength of 532 nm for cy3 and 635 nm for cy5 to generate TIFF images. The images were analyzed using Genepix Pro 7.0 software to generate Genepix Reporting Permit (GPR) files (Molecular Devices, Sunnyvale, California, USA).Net fluorescence intensity (NFI) for each antigen was generated by subtracting the signals from the local background and negative control (phosphate-buffered saline or simplified to PBS). The NFI was normalized by the absolute amount of IgG detected in each sample (based on a 1:2 anti-Ig control). Each individual value was then normalized to the mean detected in all experimental mice included in this study (excluding positive controls), resulting in a value describing the fold change compared to the mean.

[0895] Organ processing for FC analysis

[0896] For FC analysis, organs were cut into small pieces and incubated with a tissue digestion solution consisting 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). The tissue digestion solution was then incubated at 37°C with stirring at 350 rpm for 10 min. The tissue was then further dissociated by pipetting and filtered through a 0.4 µm filter (Corning).

[0897] Processing of organs for imaging

[0898] For IF, tissues were incubated at 4°C in 4% paraformaldehyde in PBS solution (PFA; ChemCruz). ®Incubate for 4 to 12 hours (depending on tissue size). Then, replace PFA with a solution of 10% sucrose (Sigma-Aldrich) and 0.02% NaN3 in H2O. After incubation at room temperature for 8 to 15 hours, increase the sucrose solution to 20%, and after an additional 8 to 15 hours, increase it to 30%. Then embed the organ in Killik, OCT composite embedding medium (Bio-Optica) for cryostat. Prepare 20 mm thick sections and place them on glass slides using a cryostat. Dry the sections at room temperature for 30 minutes. For antigen retrieval, incubate the slides in a water bath preheated to 95°C in the following solutions for 20 minutes: (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 1 mM EDTA in H2O with 0.05% tween, pH adjusted to pH 9. The slides were then cooled at room temperature for 15 minutes in the indicated solution and washed three times with PBS. Blocking was performed using a blocking buffer consisting of 5% normal donkey serum, 1% BSA (Sigma-Aldrich), and 0.3% Triton™ X-100 (Sigma) in PBS. For staining with mouse primary antibody, mouse-to-mouse IgG blocking solution (Vector Laboratories) was added to the blocking buffer according to the manufacturer's instructions. After blocking for 1 hour at RT, the blocking buffer was replaced with blocking buffer containing the indicated concentration of primary antibody and incubated overnight at 4°C. The sections were then washed five times with washing buffer (PBS containing 0.3% Triton™ X-100). The sections were stained with secondary antibody in the indicated concentration of blocking buffer. Incubation was performed in the dark at room temperature for 1 hour, followed by six washing steps with washing buffer. To stain cell nuclei, the sections were covered for 2 minutes with a 1 / 2000 dilution of Hoechst 33342 solution (Life Technology) in PBS. Wash the slides three additional times with PBS and use Fluoromount-G. ® (SouthernBiotech) Fixation. Images were acquired at 10x or 20x magnification using an SP8 Lightning confocal microscope (Leica Microsystems). The antibody combinations for primary and secondary antibodies, as well as the antigen retrieval protocols, are shown in the table below.

[0899] For histopathological evaluation of side effects, the indicated organs were collected from euthanized mice, fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) according to OECD Good Laboratory Practice (GLP) principles, data integrity principles, and applicable GLP SR-TIGET SOP. Histopathological changes were assessed by an experienced pathologist and graded on a scale of 1 to 5 as minimal (1), mild (2), moderate (3), significant (4), or severe (5); minimal refers to the least identifiable degree, and severe refers to the greatest possible degree. Slides were reviewed independently by an experienced pathologist, and consensus was reached on findings and scores.

[0900] Antibodies against human antigens LAG3 (Biotechne; clone: ​​874512; host: mouse) and CD4 (Roche-Ventana; clone: ​​SP35; host: rabbit) were used, followed by donkey anti-mouse Alexa 555 and goat anti-rabbit Alexa 488 secondary antibodies as described above, to perform IF analysis of formalin-fixed and paraffin-embedded (FFPE) tumor specimens containing CRC-LMS. Representative images were captured at 500x magnification using a Nikon 80i Eclipse fluorescence microscope.

[0901] To prepare H&E staining for human and mouse livers containing metastatic tumors, samples were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with H&E. Slides were analyzed by experienced pathologists and digitized at 20x magnification using a Leika AperioScanscope XT scanner.

[0902] Table 3: Antibodies and antibody combinations used for IF staining .

[0903]

[0904]

[0905] Single cell RNA (scRNA) sequencing

[0906] Following liver perfusion, liver metastases were immediately isolated and dissociated into single cells as described above. The single cells were resuspended in MACS buffer containing 7-AAD (BioLegend). Live cells were sorted by gating 7-AAD-negative cells. The sorted cells were further processed for scRNA sequencing. ScRNA sequencing was performed using the Next GEM SingleCell 3' GEM Kit v3.1 from Chromium 10X, according to the manufacturer's recommendations (User Guide Chromium NextGEM Single Cell 3' Reagent Kits v3.1). In each reaction, we loaded 10,000 cells belonging to the same sample. We sequenced 8 samples, obtaining 100bp paired-end reads on a NovaSeq 6000 Illumina device, totaling 4.75. 10 9 Each readout was processed. Base call files obtained as results from Illumina sequencing were converted to FASTQ files and processed using the Cell Ranger single-cell software suite (10X Chromium v3.1.0) with default settings. Specifically, the demultiplexed samples were aligned to the mouse mm10 reference genome using the STAR alignment tool (generating alignment files in BAM format), and UMI gene counting quantification (based on reference annotation) was performed. The latter gene-cell matrix was then imported into R and processed using the Seurat package (http: / / satijalab.org / seurat v4.0.3). As a first step in the analysis, duplexes were estimated using the DoubletFinder (v3) software. More precisely, following the authors' recommended "best practices" for scRNA-seq processing, the following parameters were selected to annotate duplexes in each sample:

[0907] Table 4: Parameters used for DoubletFinder v3 .

[0908]

[0909] The samples were merged into a single Seurat dataset using the Seurat package (http: / / satijalab.org / seurat v4.0.3), preserving information about the original samples and corresponding treatment groups. Preprocessing steps on the resulting data were then initiated by removing cells with low ordinal quality, cells with feature counts below 1,000 and above 6,000, and cells with mitochondrial gene fractions above 10%. Cells annotated as duplexes with DoubletFinder (v3) were then excluded from the Seurat analysis. RNA UMI counts were normalized using a global scaling normalization method, and scaling was performed using a variance stabilization transformation (SCTransform) based on the percentage of mitochondrial genes, the absolute RNA count per cell, and the difference between the S and G2 / M cell cycle scores calculated for each cell. Principal component analysis (PC) was performed on 50 principal components (PCs) to reduce dimensionality, and UMAP representations and clustering (at a resolution of 1.2) were computed on these dimensionality reductions. The FindAllMarkers Seurat function was used to obtain the marker genes for each cluster, thus annotating and manually curating the clusters, including small populations of undefined cells that were then removed from the dataset. Sub-clustering analyses were performed accordingly for "T and NK cells" and "APCs". First, T and NK cells were separated using a subset function, followed by SCTransform based on the RNA counting matrix, principal component analysis with 35 PCs, and cluster identification at a resolution of 0.8. At this resolution, populations of CD4 T cells, CD8 T cells, γδ T cells, NK cells, ILCs, and NKT cells, as well as undefined cells, were identified. CD4, CD8, and NKT cells were further refined by 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, respectively, with resolutions of 0.6, 0.3, and 0.3. Similarly, specificity analyses were performed in APC compartments 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 subsets of T and NK cells and APCs, with the same parameters as previously described. The top upregulated markers for each population were calculated using the FindAllMarkers function, and heatmaps were generated based on the to...

Claims

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

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

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

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

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

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

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

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

9. A product, or carrier, immune checkpoint inhibitor, or Tr1 cell inhibitor for use according to any of the preceding claims, wherein the transgene encodes a tumor antigen, optionally wherein the tumor 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. A product, or vector, immune checkpoint inhibitor, or Tr1 cell inhibitor for use according to any of the preceding claims, wherein the vector is a viral vector, optionally wherein the vector is a lentiviral vector, a retroviral vector, an adenovirus vector, an adeno-associated virus vector, or a herpes simplex virus vector.

11. A product or carrier, immune checkpoint inhibitor or Tr1 cell inhibitor for use according to any of the preceding claims, 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 attenuation factor; 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 (Ig inhibitor of V domain for T cell activation), B7-1 (CD80), B7-2 (CD86), TGFB (transforming growth factor β) pathway-associated protein, Il13 (interleukin-13), IL4 (interleukin-4), FGL (fibrinogen-like 1), TIGIT (T cell immune receptor with Ig and ITIM domains), CD96 (TACT protein), Ceacam-1 (carcinoembryonic antigen-associated cell adhesion molecule 1), CD155 (PVR protein), CD112 (PVR-associated protein 2 (PVRL2)), LGALS3 (galactolectin 3), and CD47 (integrin-associated protein).

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

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

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

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

Images