gene therapy

Abstract

An organism comprising (a) a vector for phagocytic cell-specific expression of the liver and / or spleen, wherein the vector comprises a transgene operably linked to one or more expression regulatory sequences, and (b) an immune checkpoint inhibitor or a Tr1 cell inhibitor.

Description

[Technical Field]

[0001] The present invention relates to vectors for phagocytic cell-specific expression, particularly for phagocytic cell-specific expression in the liver and / or spleen, and to combinations of vectors with immune checkpoint inhibitors and / or Tr1 cell inhibitors. The present invention also relates to cells, and pharmaceutical compositions comprising such vectors, and to their use in the treatment of cancer, for example, liver metastases. [Background technology]

[0002] The liver is involved in several biological functions, including detoxification, protein and cell clearance, and metabolic functions. To protect the liver from immune responses that could damage it, the liver is characterized by an immunosuppressive environment that limits immunity. Due to this immunosuppressive environment, some tumor types tend to spread towards the liver and cause liver metastases.

[0003] The liver is one of the most common sites of cancer metastasis, accounting for nearly 25% of all cases. While various primary tumors can cause metastasis, colorectal adenocarcinoma is the most common, considering the total number of affected patients. Liver metastasis is associated with a poor prognosis and often constitutes a cause of death in cancer patients.

[0004] For anatomically resectable liver metastases, surgical resection remains the gold standard. Strategies to improve the chances of resection include neoadjuvant chemotherapy, portal vein embolization to increase future liver remnants, or a combination of two-stage resection versus one-stage resection of the primary tumor and liver lesion. However, the 5-year survival rate after therapeutic resection of liver lesions in patients with colorectal metastases is reported to be only 25%–58%, with a median survival time of 74 months. Therefore, improved treatments for cancers such as liver metastases are greatly needed. [Overview of the project]

[0005] The inventors have developed a lentiviral vector (LV) platform that enables the manipulation of hepatic resident macrophages (Kupffer cells) for the delivery of transgenes, such as interferon-alpha (IFNα), particularly to liver metastases. The inventors observed that gene-based IFNα delivery to different mouse models of liver metastases from colorectal adenocarcinoma and pancreatic ductal adenocarcinoma significantly delayed tumor growth. While not theoretically bound, the inventors observed that responses to IFNα were associated with tumor-associated macrophage (TAM) immune activation, enhanced MHCII-restricted antigen presentation by tumor-infiltrating dendritic cells, and reduced CD8 T cell exhaustion. Conversely, increased IL10 signaling, enhanced CTLA4 expression, and proliferation of Eomes CD4 T cells (a cell type exhibiting characteristics of type I regulatory T(Tr1) cells) were associated with resistance to IFNα gene therapy. Subsequently, the inventors observed that targeting regulatory T cell function through immune checkpoint blockade and IFNαLV delivery resulted in a potent synergistic effect that achieved a complete response in most mice.

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

[0007] A vector may contain a transgene operably ligated to one or more expression regulatory sequences.

[0008] In one embodiment, the present invention provides an product comprising (a) a vector for phagocytic cell-specific expression of the liver and / or spleen, wherein the vector comprises a transgene operably linked to one or more expression regulatory sequences, and (b) an immune checkpoint inhibitor or a Tr1 cell inhibitor.

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

[0010] In one aspect, the present invention provides an product comprising (a) a vector for phagocytic cell-specific expression of the liver and / or spleen, wherein the vector comprises a transgene operably linked to one or more expression regulatory sequences, and (b) an immune checkpoint inhibitor. In one aspect, the present invention provides an product comprising (a) a vector for phagocytic cell-specific expression of the liver and / or spleen, wherein the vector comprises a transgene operably linked to one or more expression regulatory 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 embodiment, the present invention provides a vector for therapeutic use, the vector being for phagocytic cell-specific expression in the liver and / or spleen, and the vector being used in combination with an immune checkpoint inhibitor or a Tr1 cell inhibitor.

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

[0014] In one embodiment, the present invention provides a vector for therapeutic use, the vector being for phagocytic cell-specific expression in the liver and / or spleen, the vector comprising a transgene operably linked to one or more expression regulatory sequences, and the vector being used in combination with an immune checkpoint inhibitor or a Tr1 cell inhibitor.

[0015] In one embodiment, the present invention provides a vector for therapeutic use, the vector for phagocytic expression in the liver and / or spleen, the vector comprises a transgene operably linked to one or more expression regulatory sequences, and the vector is used in combination with an immune checkpoint inhibitor. In one embodiment, the present invention provides a vector for therapeutic use, the vector for phagocytic expression in the liver and / or spleen, the vector comprises a transgene operably linked to one or more expression regulatory sequences, and the vector is used in combination with a Tr1 cell inhibitor.

[0016] In one embodiment, the present invention provides an immune checkpoint inhibitor or Tr1 cell inhibitor for therapeutic use, which is used in combination with a vector for phagocytic cell-specific expression in the liver and / or spleen.

[0017] In one embodiment, the present invention provides an immune checkpoint inhibitor or Tr1 cell inhibitor for therapeutic use, the immune checkpoint inhibitor or Tr1 cell inhibitor being used in combination with a vector for phagocytic cell-specific expression in the liver and / or spleen, the vector comprising a transgene operably linked to one or more expression regulatory sequences.

[0018] In one embodiment, the present invention provides an immune checkpoint inhibitor for therapeutic use, the immune checkpoint inhibitor being used in combination with a vector for phagocytic expression in the liver and / or spleen, the vector comprising a transgene operably linked to one or more expression regulatory sequences. In one embodiment, the present invention provides a Tr1 cell inhibitor for therapeutic use, the Tr1 cell inhibitor being used in combination with a vector for phagocytic expression in the liver and / or spleen, the vector comprising a transgene operably linked to one or more expression regulatory sequences.

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

[0020] In some embodiments, one or more expression regulatory sequences include (a) a phagocytic cell-specific promoter and / or enhancer, and / or (b) one or more miRNA target sequences.

[0021] In some embodiments, one or more expression regulatory sequences include a phagocytic cell-specific promoter and / or enhancer, and / or (b) one or more miRNA target sequences.

[0022] In some embodiments, one or more expression regulatory sequences include (a) a phagocytic cell-specific promoter and / or enhancer. In some embodiments, one or more expression regulatory sequences include one or more miRNA target sequences. In some embodiments, one or more expression regulatory sequences include (a) a phagocytic cell-specific promoter and / or enhancer, and (b) one or more miRNA target sequences.

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

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

[0025] In some embodiments, the phagocytic cells are hepatic phagocytic cells and / or splenic phagocytic cells.

[0026] In some embodiments, the phagocytic cells are macrophages. In some embodiments, the phagocytic cells are M2-like macrophages and / or MRC1+ macrophages, dendritic cells, or hepatic sinusoidal endothelial cells.

[0027] In some embodiments, the phagocytes are hepatic resident phagocytes. In some embodiments, the phagocytes are hepatic resident macrophages. In some embodiments, the phagocytes are Kupffer cells.

[0028] In some embodiments, the phagocytic cells are hepatic sinusoidal endothelial cells.

[0029] In some embodiments, the vector comprises a phagocytic cell-specific promoter and / or enhancer-transgene-one or more miRNA target sequences, oriented from 5' to 3'.

[0030] In some embodiments, the phagocytic cell-specific promoter and / or enhancer is selected from the group consisting of the MRC1 promoter and / or enhancer, ITGAM promoter and / or enhancer, CD86 promoter and / or enhancer, CD274 promoter and / or enhancer, CD163 promoter and / or enhancer, LYVE1 promoter and / or enhancer, STAB1 promoter and / or enhancer, ITGAX promoter and / or enhancer, SIRPA promoter and / or enhancer, TIE2 promoter and / or enhancer, CHIL3 promoter and / or enhancer, CD68 promoter and / or enhancer, CSF1R promoter and / or enhancer, VCAM1 promoter and / or enhancer, PTGS1 promoter and / or enhancer, and C1QA promoter and / or enhancer, fragments thereof, or combinations thereof.

[0031] In some embodiments, the phagocytic cell-specific promoter and / or enhancer is the 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 or fragment thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1.

[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, one or more miRNA target sequences suppress expression in non-phagocytes (e.g., non-hepatic phagocytes and / or non-splenic phagocytes). In preferred embodiments, one or more miRNA target sequences suppress expression in non-hepatic phagocytes (i.e., cells other than hepatic phagocytes).

[0035] In some embodiments, one or more miRNA target sequences suppress transgene expression in hepatocytes and / or hepatic sinusoidal endothelial cells and / or splenic phagocytes.

[0036] One or more miRNA target sequences may suppress expression in certain hepatocyte populations and / or spleen cell populations.

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

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

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

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

[0041] In some embodiments, the miR-126 target sequence includes or consists of SEQ ID NO: 3. In some embodiments, the miR-122 target sequence includes or consists of SEQ ID NO: 4.

[0042] In a preferred embodiment, the vector comprises (a) an MRC1 promoter and / or enhancer, or a fragment thereof, and (b) a transgene operably ligated to 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-α, interferon-β, interferon-γ, IL2, IL12, TNF-α, CXCL9, IL1-β, IL15, IL18, IL10, GMCSF, FLT3, IL7, or IL21.

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

[0047] In some embodiments, interferon-α comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 8.

[0048] In some embodiments, interferon-α contains 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, the melanoma-associated antigen (MAGE) family, the cancer germline (CAGE) family, B melanoma antigen (BAGE-1), synovial sarcoma x breakpoint 20 (SSX-2), the 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 containing a second transgene operably ligated to one or more expression regulatory sequences, and optionally a third vector containing a third transgene operably ligated to one or more expression regulatory sequences. In some embodiments, the transgene encodes a cytokine, and the product or combination further comprises a second vector containing a second transgene operably ligated to one or more expression regulatory sequences, and a third vector containing a third transgene operably ligated to one or more expression regulatory sequences. The one or more expression regulatory sequences may be as disclosed herein.

[0053] In some embodiments, the transgene encodes a cytokine, and the vector further comprises a second transgene operably ligated to one or more expression regulatory sequences, and optionally a third transgene operably ligated to one or more expression regulatory sequences. The one or more expression regulatory sequences may be as disclosed herein. The third transgene may be included in the 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 vector and / or the third vector and the second transgene and / or the third transgene may include further features or be operably linked to further features in the same manner as disclosed herein with respect to the vector and / or transgene of the present invention.

[0055] In some embodiments, the cytokine is interferon-α, interferon-β, interferon-γ, IL2, IL12, TNF-α, CXCL9, IL1-β, IL15, IL18, IL10, GMCSF, FLT3, IL7, or IL21. In some embodiments, the cytokine is interferon-α.

[0056] In some embodiments, the second transgene encodes a second cytokine, which is distinct from the second cytokine. In some embodiments, the second cytokine is IL12, interferon-α, interferon-β, interferon-γ, IL2, TNF-α, CXCL9, IL1-β, IL15, IL18, IL10, GMCSF, FLT3, IL7, or IL21. In some embodiments, the second cytokine is IL12.

[0057] In some embodiments, the cytokine is interferon-α, and the second cytokine is IL-12.

[0058] In some embodiments, the second transgene encodes a tumor antigen. In some embodiments, the tumor antigen is carcinoembryonic antigen (CEA), TRP2, the melanoma-associated antigen (MAGE) family, the cancer germline (CAGE) family, melanoma B antigen (BAGE-1), synovial sarcoma X breakpoint 20 (SSX-2), the sarcoma antigen (SAGE) family, LAGE1, NY-ESO-1, HER2, EGFR, MUC-1, or GAST.

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

[0060] In some embodiments, the cytokine is IL12, and the second cytokine is a tumor antigen. 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, melanoma B antigen (BAGE-1), synovial sarcoma X breakpoint 20 (SSX-2), sarcoma antigen (SAGE) family, LAGE1, NY-ESO-1, HER2, EGFR, MUC-1, or GAST.

[0061] In some embodiments, the tumor antigen is TRP2.

[0062] In some embodiments, the second transgene encodes a second cytokine, which is different from the second cytokine, and the third transgene encodes a tumor antigen.

[0063] In some embodiments, the cytokine is interferon-alpha, the second cytokine is IL-12, and the third transgene encodes a tumor antigen.

[0064] In some embodiments, the cytokine is interferon-alpha, the second cytokine is IL-12, and the tumor antigen is TRP2.

[0065] In some embodiments, the transgene is further operably linked to one or more regulatory elements.

[0066] In some embodiments, the transgene is further operably ligated to the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).

[0067] In some embodiments, the transgene is further operably ligated to an destabilization domain. In some embodiments, the destabilization domain is a dihydrofolate reductase destabilization domain.

[0068] In some embodiments, the vector is a viral vector. In some embodiments, the vector is an embedded viral vector. In some embodiments, the vector is a non-embedded viral vector.

[0069] In some embodiments, the vector is a lentiviral vector, a retroviral vector, an adenovirus vector, an adeno-associated virus vector, or a herpes simplex virus vector.

[0070] In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an embedded / deleted lentiviral vector.

[0071] In some embodiments, the viral vector is a viral vector particle.

[0072] In some embodiments, the viral vector particle is a VSV-G pseudotype. In some embodiments, the viral vector is a VSV-G pseudotype lentiviral vector particle.

[0073] In some embodiments, viral vector particles are produced in viral particle producer cells or packaging cells that have been genetically engineered to reduce the expression of CD47 and / or HLA on the cell surface. In some embodiments, viral vector particles substantially lack surface-exposed CD47 and / or HLA.

[0074] The vector can specifically express the transgene in phagocytic cells. In some embodiments, (i) the expression of the transgene in phagocytic cells transduced by the vector is greater than the expression of the transgene in other cells transduced by the vector, and / or (ii) the transgene is substantially not expressed in cells other than phagocytic cells when transduced by the vector, and / or (iii) the transgene is substantially not expressed in lung cells, bone marrow cells and / or blood cells when transduced by the vector, and / or (iv) the transgene is substantially expressed only in some liver cells and / or some spleen cells, and / or (v) the expression of the transgene in Kupffer cells is at least 10 times greater than the expression in hepatocytes when transduced by the vector, and / or (vi) the transgene is substantially not expressed in hepatocytes when transduced by the vector.

[0075] In some embodiments, the expression of the transgene in phagocytic cells transduced by the vector is greater than the expression of the transgene in other cells transduced by the vector. In some embodiments, the transgene is substantially not expressed in cells other than phagocytic cells when transduced by the vector. In some embodiments, the transgene is substantially not expressed in lung cells, bone marrow cells and / or blood cells when transduced by the vector. In some embodiments, the expression of the transgene in Kupffer cells is at least 10 times greater than the expression in hepatocytes when transduced by the vector. In some embodiments, the transgene is substantially not expressed in hepatocytes when transduced by the vector.

[0076] In some embodiments, when transduced by a vector, the transgene is expressed substantially only in liver cells and / or spleen cells, and optionally substantially not in liver cells.

[0077] In some embodiments, the transgene is substantially expressed only in liver cells and / or spleen cells.

[0078] In some embodiments, the immune checkpoint inhibitor is 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 cell death 1 receptor), PD-L1 (PD-1 ligand 1), PD-L2 (PD-1 ligand 2), TIM-3 (T cell immunoglobulin domain The inhibitory checkpoint molecules selected from the group consisting of mucin domains3), VISTA (V domain Ig inhibitor of T cell activation), B7-1 (CD80), B7-2 (CD86), TGFB (transforming growth factor β) pathway-related proteins, 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-related protein 2 (PVRL2)), LGALS3 (galectin 3), and CD47 (integrin-related protein) are inhibited. A combination of two or more immune checkpoint inhibitors may be used.

[0079] In some embodiments, immune checkpoint inhibitors inhibit PD-1.

[0080] In some embodiments, the cytokine is interferon-alpha, the second cytokine is IL-12, the tumor antigen is TRP2, and the immune checkpoint inhibitor inhibits PD-1.

[0081] In some embodiments, the TGFB pathway-related proteins are selected from the group consisting of TGFB1 (transforming growth factor β1), TGFB2 (transforming growth factor β2), TGFB3 (transforming growth factor β3), LTBP1 (latent 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-containing 32).

[0082] 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 anti-CTLA4 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-PDL2 antibody, and anti-LAG3 antibody.

[0083] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA4 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody.

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

[0085] In some embodiments, the transgene encodes interferon-alpha, and the immune checkpoint inhibitor is an anti-PD1 antibody. In some embodiments, the transgene encodes interferon-alpha, and the immune checkpoint inhibitor is an anti-CTLA4 antibody.

[0086] In some embodiments, the cytokine is interferon-alpha, the second cytokine is IL-12, the tumor antigen is TRP2, and the immune checkpoint inhibitor is an anti-PD1 antibody.

[0087] In some embodiments, the Tr1 cell inhibitor inhibits molecules selected from the group consisting of Cd4, Eomes, Gzmk, Lag3, Pdcd1, Ahr, Maf, Prdm1, Ctla4, and Il10ra.

[0088] In one embodiment, the present invention provides cells containing the product of the present invention.

[0089] In one embodiment, the present invention provides a cancer vaccine comprising a product of the present invention. In one embodiment, the present invention provides a product of the present invention for use in therapy.

[0090] In a preferred embodiment, the therapeutic use is for the treatment or prevention of cancer.

[0091] In one embodiment, the present invention provides a method for treating or preventing cancer, comprising administering to a subject in need of (a) a vector for phagocytic cell-specific expression of the liver and / or spleen, the vector comprising a transgene operably linked to one or more expression regulatory sequences, and (b) an immune checkpoint inhibitor or a Tr1 cell inhibitor.

[0092] The components of the combination may be administered, for example, simultaneously, sequentially, or separately.

[0093] In some embodiments, the cancer is a liver metastasis. The metastasis may originate, for example, from colorectal cancer or pancreatic ductal adenocarcinoma (PDAC).

[0094] In some embodiments, the cancer is a primary liver tumor.

[0095] In some embodiments, the product, combination, or component thereof is administered systemically. In some embodiments, the product, combination, or component thereof is administered by intravenous injection, portal vein injection, or hepatic artery injection.

[0096] In one embodiment, the present invention provides an product comprising: (a) a vector for phagocytic cell-specific expression of liver and / or spleen, the vector comprising a transgene operably linked to one or more expression regulatory sequences; and (b) a second vector for phagocytic cell-specific expression of liver and / or spleen, the second vector comprising a second transgene operably linked to one or more expression regulatory sequences, the transgene being different from the second transgene, the second vector.

[0097] In one embodiment, the present invention provides a vector for therapeutic use, the vector being for phagocytic expression in the liver and / or spleen, the vector comprising a transgene operably linked to one or more expression regulatory sequences, the vector being used in combination with a second vector for phagocytic expression in the liver and / or spleen, the second vector comprising a second transgene operably linked to one or more expression regulatory sequences, the transgene being different from the second transgene.

[0098] In one embodiment, the present invention provides a method for treating or preventing cancer, comprising administering to a subject in need of the treatment of cancer the following: (a) a vector for phagocytic expression of liver and / or spleen, the vector comprising a transgene operably linked to one or more expression regulatory sequences; and (b) a second vector for phagocytic expression of liver and / or spleen, the second vector comprising a second transgene operably linked to one or more expression regulatory sequences, the transgene being different from the second transgene.

[0099] In one embodiment, the present invention provides an output comprising a vector for phagocytic cell-specific expression of the liver and / or spleen, the vector comprising (a) a transgene operably ligated to one or more expression regulatory sequences, and (b) a second transgene operably ligated to one or more expression regulatory sequences, wherein the transgene is different from the second transgene.

[0100] In one embodiment, the present invention provides a vector for therapeutic use, the vector for phagocytic cell-specific expression in the liver and / or spleen, the vector comprising (a) a transgene operably ligated to one or more expression regulatory sequences, and (b) a second transgene operably ligated to one or more expression regulatory sequences, wherein the transgene is different from the second transgene.

[0101] In one embodiment, the present invention provides a method for treating or preventing cancer, comprising administering to a subject in need of the vector for phagocytic cell-specific expression of the liver and / or spleen, wherein the vector comprises (a) a transgene operably ligated to one or more expression regulatory sequences, and (b) a second transgene operably ligated to one or more expression regulatory sequences, the transgene comprising a second transgene different from the second transgene.

[0102] In some embodiments, the transgene encodes a cytokine. In some embodiments, the cytokine is IL12, interferon α, interferon β, interferon γ, IL2, TNFα, CXCL9, IL1-β, IL15, IL18, IL10, GMCSF, FLT3, IL7, or IL21. In some embodiments, the cytokine is IL12.

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

[0104] In some embodiments, (a) the transgene encodes a cytokine (e.g., IL12, interferon α, interferon β, interferon γ, IL2, TNFα, CXCL9, IL1-β, IL15, IL18, IL10, GMCSF, FLT3, IL7, or IL21), and (b) the second transgene encodes a second cytokine (e.g., interferon α, interferon β, interferon γ, IL2, IL12, TNFα, CXCL9, IL1-β, IL15, IL18, IL10, GMCSF, FLT3, IL7, or IL21), and the cytokine is different from the second cytokine.

[0105] In some embodiments, the first transgene encodes IL12, and the second transgene encodes interferon-α.

[0106] In some embodiments, the second transgene encodes a tumor antigen. In some embodiments, the tumor antigen is carcinoembryonic antigen (CEA), TRP2, the melanoma-associated antigen (MAGE) family, the cancer germline (CAGE) family, melanoma B antigen (BAGE-1), synovial sarcoma X breakpoint 20 (SSX-2), the sarcoma antigen (SAGE) family, LAGE1, NY-ESO-1, HER2, EGFR, MUC-1, or GAST.

[0107] In some embodiments, (a) the transgene encodes a cytokine (e.g., IL12, interferon α, interferon β, interferon γ, IL2, TNFα, CXCL9, IL1-β, 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, melanoma B 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, the first transgene encodes IL12, and the second transgene encodes a tumor antigen.

[0109] In some embodiments, the organism or combination further comprises a third vector for phagocytic cell-specific expression of the liver and / or spleen, the third vector comprising a third transgene operably linked to one or more expression regulatory sequences, the third transgene being different from the transgene and the second transgene.

[0110] In some embodiments, the vector further comprises a third transgene operably ligated to one or more expression regulatory sequences, the third transgene being different from the transgene and the second transgene.

[0111] In some embodiments, the organism or combination further comprises a second vector for phagocytic cell-specific expression of the liver and / or spleen, the second vector comprising a third transgene operably linked to one or more expression regulatory sequences, the third transgene being different from the transgene and the second transgene.

[0112] The first, second, and third transgenes may each be independently selected from cytokines or tumor antigens, for example, the cytokines or tumor antigens disclosed herein.

[0113] In one embodiment, the present invention provides a method for treating or preventing cancer, comprising administering to a subject in need of the following: (a) a vector for phagocytic cell-specific expression of the liver and / or spleen, the vector comprising a transgene operably linked to one or more expression regulatory sequences; (b) a second vector for phagocytic cell-specific expression of the liver and / or spleen, the second vector comprising a second transgene operably linked to one or more expression regulatory sequences, wherein the transgene is different from the second transgene; and (c) a third vector for phagocytic cell-specific expression of the liver and / or spleen, the third vector comprising a third transgene operably linked to one or more expression regulatory sequences, wherein the third transgene is different from the transgene and the second transgene.

[0114] In one embodiment, the present invention provides a method for treating or preventing cancer, comprising administering a vector for phagocytic cell-specific expression of the liver and / or spleen to a subject in need thereof, the vector comprising (a) a transgene operably ligated to one or more expression regulatory sequences, (b) a second transgene operably ligated to one or more expression regulatory sequences, and (c) a third transgene operably ligated to one or more expression regulatory sequences.

[0115] In some embodiments, (a) the transgene encodes a cytokine (e.g., IL12, interferon α, interferon β, interferon γ, IL2, TNFα, CXCL9, IL1-β, IL15, IL18, IL10, GMCSF, FLT3, IL7, or IL21), and (b) the second transgene encodes a second cytokine (e.g., interferon α, interferon β, interferon γ, IL2, IL12, TNFα, CXCL9, IL1-β, IL15, IL18, IL (c) The third transgene encodes a tumor antigen (e.g., carcinoembryonic antigen (CEA), TRP2, melanoma-associated antigen (MAGE) family, cancer germline (CAGE) family, melanoma B antigen (BAGE-1), synovial sarcoma X breakpoint 20 (SSX-2), sarcoma antigen (SAGE) family, LAGE1, NY-ESO-1, HER2, EGFR, MUC-1, or GAST). Cytokines and tumor antigens may be as disclosed herein.

[0116] In some embodiments, the first transgene encodes IL12, the second transgene encodes interferon-alpha, and the third transgene encodes a tumor antigen.

[0117] In one embodiment, the present invention provides a method for treating or preventing cancer, comprising administering (i) a vector for phagocytic cell-specific expression of the liver and / or spleen, the vector comprising (a) a transgene operably ligated to one or more expression regulatory sequences, (b) a second transgene operably ligated to one or more expression regulatory sequences, and (c) a third transgene operably ligated to one or more expression regulatory sequences, and (ii) an immune checkpoint inhibitor to a subject requiring the same.

[0118] In some embodiments, the first transgene encodes interferon-alpha, the second transgene encodes IL-12, 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 explanation of the drawing]

[0119] [Figure 1] Development of an LV platform enabling in vivo liver macrophage manipulation. (A) Schematic diagram of Mrc1.GFP and Mrc1.GFP.miRT LV. (B) Schematic diagram of the experiments shown in panels C-F. (C) Graph of LV copies per cell in the indicated organ by digital droplet PCR (ddPCR) analysis. (D and E) Graph of GFP expression of the indicated cell type in the indicated organ by flow cytometry (FC) analysis. (n=5 mice / group, statistical analysis by Mann-Whitney test comparing only Mrc1.GFP LV vs. Mrc1.GFP.miRT LV, and p-values ​​adjusted for multiple testing using Bonferroni correction). In E, Mrc1.GFP LV and Mrc1.GFP.miRT LV were used at 3 × 10¹⁰ TU / kg. (F) This figure shows representative immunofluorescence (IF) images obtained by confocal microscopy (CM) and relative GFP quantification of livers with metastases from MC38 cells, mCherry (red), GFP (green), F4 / 80 (gray), and nuclei (blue, left panel) in the left panel, or AKTPF cells, GFP (green), F4 / 80 (gray), and nuclei (blue, right panel) in the right panel, indicating metastases (Met), perimetastatic regions (dotted lines), and intact liver. In NSG mice, MC38 cells were injected at 3 × 10¹⁰ TU / kg 10 days after LV delivery in the left panel, and Mrc1.GFP.miRT LV was used at 5 × 10⁹ TU / kg in the right panel (n=5 mice / group; statistical analysis by bootstrap t-test). [Figure 2]In vivo LV-modified KCs enable rapid, sustained, and well-tolerated IFNα production. (A) Schematic diagram of KCs modified with IFNα LV (top) or control LV (bottom). (B) Graph of plasma IFNα levels by ELISA analysis at the indicated time point at LV injection (n=10, 10, and 5 mice / group for control LV, IFNα LV, or non-transduced UT, respectively). (C) Graph of LV copies per cell by ddPCR analysis (n=8, 8, and 5 mice / group for control LV, IFNα LV, or UT, respectively; Kruskal-Wallis statistical analysis using Dunn test, p-value adjusted by Bonferroni correction). (D) Graph of blood cell counts of B cells (left panel), eosinophils (center panel), and neutrophils (right panel) at the indicated time point at LV injection (same n-values ​​as in B; statistical analysis by Mann-Whitney U test). (E) Graphs of serum levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) on day 126 after LV injection (n=9, 10, and 5 mice / group, respectively, for control LV, IFNαLV, or UT, statistical analysis by Kruskal-Wallis test). (F) Graphs of histopathological analysis of the indicated organs on day 366 after LV injection (same n value as C). [Figure 3-1]Gene-based forced IFNα expression by KC inactivates T cell activation and reduces liver metastatic proliferation. (A) Schematic diagram of the experiments in panels B-N. (B and H) Graphs of plasma IFNα levels by ELISA at the indicated time points during tumor challenge. Low dose and high dose: 1.5 × 10⁹ or 1.5 × 10¹⁰ TU / kg, respectively (in control LV, IFNα LV, or UTB, B: left panel, n=10, 10, 5; B: right panel, n=10, 10, 5; H: n=11, 10, 3 mice / group). (C, E, I, and N) Graphs of tumor growth by magnetic resonance imaging (MRI). (From left to right: C: left panel, n=9, 8, 9, 8; C: right panel, n=10, 10, 10, 10; E: n=10, 10, 10, 9; I: n=9, 10, 9, 10, and N: n=7, 8 mice / group; Mann-Whitney statistical analysis) (D and J) These are representative MRI images of mice with control LV (left panel) and treated IFNαLV (right panel). In D, MC38 liver metastases 20 days post-tumor transplantation are shown, and in J, AKTPF liver metastases, complete responders (CR), healthy liver (white), and metastases (Met, yellow) 28 days post-tumor transplantation are shown, indicated by dotted lines in D and arrows in J. (F, G, K, and L) F and G show the proportion of invasive cell types shown in MC38.OVA, and K and L show the proportion of AKTPF liver metastases, based on FC analysis (n=10, 7 mice / group, respectively, for control LV and IFNαLV; statistical analysis by Mann-Whitney test). (M) This figure shows representative IF images obtained by CM, as well as relative CD8 T cell quantification of AKTPF liver metastases, CD4 (green), CD8 (red), E-cadherin (gray), and nucleus (blue, same n as L; statistical analysis by Mann-Whitney test). [Figure 3-2]Gene-based forced IFNα expression by KC inactivates T cell activation and reduces liver metastatic proliferation. (A) Schematic diagram of the experiments in panels B-N. (B and H) Graphs of plasma IFNα levels by ELISA at the indicated time points during tumor challenge. Low dose and high dose: 1.5 × 10⁹ or 1.5 × 10¹⁰ TU / kg, respectively (in control LV, IFNα LV, or UTB, B: left panel, n=10, 10, 5; B: right panel, n=10, 10, 5; H: n=11, 10, 3 mice / group). (C, E, I, and N) Graphs of tumor growth by magnetic resonance imaging (MRI). (From left to right: C: left panel, n=9, 8, 9, 8; C: right panel, n=10, 10, 10, 10; E: n=10, 10, 10, 9; I: n=9, 10, 9, 10, and N: n=7, 8 mice / group; Mann-Whitney statistical analysis) (D and J) These are representative MRI images of mice with control LV (left panel) and treated IFNαLV (right panel). In D, MC38 liver metastases 20 days post-tumor transplantation are shown, and in J, AKTPF liver metastases, complete responders (CR), healthy liver (white), and metastases (Met, yellow) 28 days post-tumor transplantation are shown, indicated by dotted lines in D and arrows in J. (F, G, K, and L) F and G show the proportion of invasive cell types shown in MC38.OVA, and K and L show the proportion of AKTPF liver metastases, based on FC analysis (n=10, 7 mice / group, respectively, for control LV and IFNαLV; statistical analysis by Mann-Whitney test). (M) This figure shows representative IF images obtained by CM, as well as relative CD8 T cell quantification of AKTPF liver metastases, CD4 (green), CD8 (red), E-cadherin (gray), and nucleus (blue, same n as L; statistical analysis by Mann-Whitney test). [Figure 4-1]Manipulation of KC with IFNαLV enables preferential IFNα signaling in the peripheral regions of metastases. (A) A parallel comparison of representative liver sections containing metastatic lesions (Met) from the indicated treatment cohort, analyzed by spatial transcriptome analysis showing H&E staining (left) or by using spatial transcriptome analysis (right). Spatial spots are indicated by the color associated with the spatial compartment. (B) A heatmap showing gene set enrichment analysis (GSEA) normalized enrichment scores (NES) for selected gene ontology (GO) terms across separate spatial compartments, by spatial transcriptome analysis (Visium). Gene sets are grouped into cytokine-related effects (red), immune activation status (blue), tumor-related (black), and liver function (olive; n=3, 3, and 2 mice / group in partial responder, resistance, or control, respectively). (C) A figure showing the multiplier change relative to the mean gene expression level for indicated genes belonging to indicated gene categories in the spatial compartment and treatment cohort (same n value as B). [Figure 4-2]Manipulation of KC with IFNαLV enables preferential IFNα signaling in the peripheral regions of metastases. (A) A parallel comparison of representative liver sections containing metastatic lesions (Met) from the indicated treatment cohort, analyzed by spatial transcriptome analysis showing H&E staining (left) or by using spatial transcriptome analysis (right). Spatial spots are indicated by the color associated with the spatial compartment. (B) A heatmap showing gene set enrichment analysis (GSEA) normalized enrichment scores (NES) for selected gene ontology (GO) terms across separate spatial compartments, by spatial transcriptome analysis (Visium). Gene sets are grouped into cytokine-related effects (red), immune activation status (blue), tumor-related (black), and liver function (olive; n=3, 3, and 2 mice / group in partial responder, resistance, or control, respectively). (C) A figure showing the multiplier change relative to the mean gene expression level for indicated genes belonging to indicated gene categories in the spatial compartment and treatment cohort (same n value as B). [Figure 5-1]IFNα promotes APC immune activation and enhanced MHCII-restricted antigen presentation in responder mice. (A) Figure of GSEA analysis of scRNA sequencing data showing NES for selected GO terms, calculated based on differentially expressed genes in antigen-presenting cells (APCs) in the shown comparison (n: same as A; statistical analysis by adaptive multilevel split Monte Carlo method; *:padj<0.05; **:padj<0.01; ***:padj<0.001; ****:padj<0.0001). (B) UMAP display of APC scRNA sequencing data for the shown groups (partial responder, resistant, or control, n=3, 3, and 2 mice / group, respectively). (C) Figure of selected gene expression showing mean expression (color scale) and percentage (size of shape) of cells expressing the shown genes belonging to the shown gene categories in IFNαTAM and TAM. (D) A figure of GSEA analysis showing the NES of selected GO terms on genes expressed differently in IFNαTAM vs. TAM (n=8 mice / group; statistical analysis similar to A). (E) A graph of the proportion of cells in the indicated population belonging to the APC compartment (partial responder, resistant, or control, n=3, 3, and 2 mice / group, respectively). (F) Expression profiles of genes belonging to the indicated category, showing mean expression (color scale) and the proportion of cells expressing the indicated gene (shape size). (G) A figure of combined gene expression scores of genes belonging to the indicated category in different cell populations from the indicated cohort. [Figure 5-2]IFNα promotes APC immune activation and enhanced MHCII-restricted antigen presentation in responder mice. (A) Figure of GSEA analysis of scRNA sequencing data showing NES for selected GO terms, calculated based on differentially expressed genes in antigen-presenting cells (APCs) in the shown comparison (n: same as A; statistical analysis by adaptive multilevel split Monte Carlo method; *:padj<0.05; **:padj<0.01; ***:padj<0.001; ****:padj<0.0001). (B) UMAP display of APC scRNA sequencing data for the shown groups (partial responder, resistant, or control, n=3, 3, and 2 mice / group, respectively). (C) Figure of selected gene expression showing mean expression (color scale) and percentage (size of shape) of cells expressing the shown genes belonging to the shown gene categories in IFNαTAM and TAM. (D) A figure of GSEA analysis showing the NES of selected GO terms on genes expressed differently in IFNαTAM vs. TAM (n=8 mice / group; statistical analysis similar to A). (E) A graph of the proportion of cells in the indicated population belonging to the APC compartment (partial responder, resistant, or control, n=3, 3, and 2 mice / group, respectively). (F) Expression profiles of genes belonging to the indicated category, showing mean expression (color scale) and the proportion of cells expressing the indicated gene (shape size). (G) A figure of combined gene expression scores of genes belonging to the indicated category in different cell populations from the indicated cohort. [Figure 6-1]The therapeutic response to IFNα is associated with T cell activation and is offset by Eomes CD4 T cell infiltration. (A) A figure of GSEA analysis showing NES for selected GO terms on differentially expressed genes in the shown comparison (n=3, 3, and 2 mice / group in partial responders, resistance, or controls, respectively; statistical analysis similar to Figure 5A). (B) UMAP representation of AKTPF liver metastasis-derived cells annotated as T cells and NK cells (n value similar to A). (C) A figure of selected gene expression showing the mean expression (color scale) and percentage (size of shape) of cells expressing the indicated genes belonging to the indicated gene categories in pooled Eomes CD4 T cells and all other T and NK cells. (D) A graph of the percentage of the indicated cell population for the indicated groups (n similar to A; statistical analysis similar to Figure 5D). (E) Gene expression profiles showing the average expression (color scale) and the percentage of cells expressing the indicated gene (size of shape) for all CD8 T cell subtypes pooled together with the exhaustion-related gene signatures and effector / memory-like genes, respectively, highlighted in yellow or green. [Figure 6-2]The therapeutic response to IFNα is associated with T cell activation and is offset by Eomes CD4 T cell infiltration. (A) A figure of GSEA analysis showing NES for selected GO terms on differentially expressed genes in the shown comparison (n=3, 3, and 2 mice / group in partial responders, resistance, or controls, respectively; statistical analysis similar to Figure 5A). (B) UMAP representation of AKTPF liver metastasis-derived cells annotated as T cells and NK cells (n value similar to A). (C) A figure of selected gene expression showing the mean expression (color scale) and percentage (size of shape) of cells expressing the indicated genes belonging to the indicated gene categories in pooled Eomes CD4 T cells and all other T and NK cells. (D) A graph of the percentage of the indicated cell population for the indicated groups (n similar to A; statistical analysis similar to Figure 5D). (E) Gene expression profiles showing the average expression (color scale) and the percentage of cells expressing the indicated gene (size of shape) for all CD8 T cell subtypes pooled together with the exhaustion-related gene signatures and effector / memory-like genes, respectively, highlighted in yellow or green. [Figure 7-1]IFNα from engineered KCs, combined with functional inhibition of regulatory T cells, eradicates liver metastases. (A) Figure showing patient stratification into low and high IFNα signaling cohorts (n=21) based on IFNα signature scores obtained by bulk RNA sequencing of tumors. (B) Graph of Tr1 cell signature scores detected in bulk RNA sequencing data from human patient CRC-derived liver metastases stratified by endogenous IFNα signaling score (n=21 patients per group, statistical analysis by Mann-Whitney test). (C) IF images of CRC liver metastases from two patients showing CD4 (green), LAG3 (red), and nucleus (blue) (patient #31, high IFNα signaling; patient #31, low IFNα signaling). (D) Graphs showing the percentage of EOMES CD4+ T cells infiltrating AKTPF liver metastases treated as shown, based on FC analysis (from left to right, n=7, 8, 5, 10 mice / group; statistical analysis by Mann-Whitney test, p-values ​​adjusted for Bonferroni correction). (E and H) Graphs of tumor growth by MRI analysis. (from left to right, E: n=9, 9, 9, 10; H: n=7, 8, 9, 9 mice / group; E: statistical analysis by ANCOVA, H: p-values ​​adjusted for Mann-Whitney test and Bonferroni correction). (F) Schematic diagrams of the experiments shown in G and H. (G) Graphs of tumor growth evaluated by tumor weight. (n=13, 8, 10, 9 mice / group, controls consist of 10 control LV mice and 3 UT mice; statistical analysis by Mann-Whitney test, p-values ​​adjusted for Bonferroni correction). [Figure 7-2]IFNα from engineered KCs, combined with functional inhibition of regulatory T cells, eradicates liver metastases. (A) Figure showing patient stratification into low and high IFNα signaling cohorts (n=21) based on IFNα signature scores obtained by bulk RNA sequencing of tumors. (B) Graph of Tr1 cell signature scores detected in bulk RNA sequencing data from human patient CRC-derived liver metastases stratified by endogenous IFNα signaling score (n=21 patients per group, statistical analysis by Mann-Whitney test). (C) IF images of CRC liver metastases from two patients showing CD4 (green), LAG3 (red), and nucleus (blue) (patient #31, high IFNα signaling; patient #31, low IFNα signaling). (D) Graphs showing the percentage of EOMES CD4+ T cells infiltrating AKTPF liver metastases treated as shown, based on FC analysis (from left to right, n=7, 8, 5, 10 mice / group; statistical analysis by Mann-Whitney test, p-values ​​adjusted for Bonferroni correction). (E and H) Graphs of tumor growth by MRI analysis. (from left to right, E: n=9, 9, 9, 10; H: n=7, 8, 9, 9 mice / group; E: statistical analysis by ANCOVA, H: p-values ​​adjusted for Mann-Whitney test and Bonferroni correction). (F) Schematic diagrams of the experiments shown in G and H. (G) Graphs of tumor growth evaluated by tumor weight. (n=13, 8, 10, 9 mice / group, controls consist of 10 control LV mice and 3 UT mice; statistical analysis by Mann-Whitney test, p-values ​​adjusted for Bonferroni correction). [Figure 8-1]Generation of an LV platform enabling in vivo liver macrophage manipulation. (A) Schematic diagram showing the putative Mrc1 promoter in mouse (created with USCS Genome Browser). (B) Representative FACS plot showing GFP expression in transduced bone marrow-derived macrophages (BMDM) as shown. (C) Graph of the percentage of GFP+BMDM by flow cytometry (FC) analysis (n=3 cell cultures / group). (D) Graph of the mean fluorescence intensity of PDL1 (left panel) and MRC1 (right panel) in BMDM analyzed by using polarized, untreated (NT), FC as shown (n=3 cell cultures / group). (E) Graph of LV copies per cell calculated by using ddPCR analysis (n=3 cell cultures / group). (F) Schematic diagram of bidirectional LV designs without (top) and with (bottom) miRT sites delivered systemically (iv) to mice. (G) Graph of LV copies per cell calculated using ddPCR analysis (from left to right, n=8, 8, 8, 3 mice / group, statistical analysis by Kruskal-Wallis test). (H) Graph of the average number of GFP+ hepatocytes per frame detected in 5-6 CM images of the liver (n=8 mice / group, statistical analysis by Mann-Whitney test). (I) Representative immunofluorescence (IF) image of the liver obtained by confocal microscopy (CM), showing GFP (green), F4 / 80 (red), and nucleus (blue). Enlarged view of the indicated area showing GFP and nucleus (top), and F4 / 80 and nucleus (bottom). (J and K) Graph of the percentage of GFP-positive cells and dlNGFR-positive cells (J) and MFI (K) from liver KC and LSEC analyzed using FC. (n is the same as A; statistical analysis by Kruskal-Wallis with Dunn test, p-value adjusted by Bonferroni correction). (L) This figure shows GFP expression in different organs upon delivery of PBS(UT) or Mrc1.GFP.miRT LV to mice challenged with liver metastases. Single channel of the image shown in F. Liver with metastases from MC38 cells (top) or AKTPF cells (bottom).(M) These are representative CM images of organs from mice treated with PBS (UT, top) or Mrc1.GFP.miRT LV (bottom), with GFP (green), F4 / 80 (gray), and the nucleus (blue). [Figure 8-2]Generation of an LV platform enabling in vivo liver macrophage manipulation. (A) Schematic diagram showing the putative Mrc1 promoter in mouse (created with USCS Genome Browser). (B) Representative FACS plot showing GFP expression in transduced bone marrow-derived macrophages (BMDM) as shown. (C) Graph of the percentage of GFP+BMDM by flow cytometry (FC) analysis (n=3 cell cultures / group). (D) Graph of the mean fluorescence intensity of PDL1 (left panel) and MRC1 (right panel) in BMDM analyzed by using polarized, untreated (NT), FC as shown (n=3 cell cultures / group). (E) Graph of LV copies per cell calculated by using ddPCR analysis (n=3 cell cultures / group). (F) Schematic diagram of bidirectional LV designs without (top) and with (bottom) miRT sites delivered systemically (iv) to mice. (G) Graph of LV copies per cell calculated using ddPCR analysis (from left to right, n=8, 8, 8, 3 mice / group, statistical analysis by Kruskal-Wallis test). (H) Graph of the average number of GFP+ hepatocytes per frame detected in 5-6 CM images of the liver (n=8 mice / group, statistical analysis by Mann-Whitney test). (I) Representative immunofluorescence (IF) image of the liver obtained by confocal microscopy (CM), showing GFP (green), F4 / 80 (red), and nucleus (blue). Enlarged view of the indicated area showing GFP and nucleus (top), and F4 / 80 and nucleus (bottom). (J and K) Graph of the percentage of GFP-positive cells and dlNGFR-positive cells (J) and MFI (K) from liver KC and LSEC analyzed using FC. (n is the same as A; statistical analysis by Kruskal-Wallis with Dunn test, p-value adjusted by Bonferroni correction). (L) This figure shows GFP expression in different organs upon delivery of PBS(UT) or Mrc1.GFP.miRT LV to mice challenged with liver metastases. Single channel of the image shown in F. Liver with metastases from MC38 cells (top) or AKTPF cells (bottom).(M) These are representative CM images of organs from mice treated with PBS (UT, top) or Mrc1.GFP.miRT LV (bottom), with GFP (green), F4 / 80 (gray), and the nucleus (blue). [Figure 8-3]Generation of an LV platform enabling in vivo liver macrophage manipulation. (A) Schematic diagram showing the putative Mrc1 promoter in mouse (created with USCS Genome Browser). (B) Representative FACS plot showing GFP expression in transduced bone marrow-derived macrophages (BMDM) as shown. (C) Graph of the percentage of GFP+BMDM by flow cytometry (FC) analysis (n=3 cell cultures / group). (D) Graph of the mean fluorescence intensity of PDL1 (left panel) and MRC1 (right panel) in BMDM analyzed by using polarized, untreated (NT), FC as shown (n=3 cell cultures / group). (E) Graph of LV copies per cell calculated by using ddPCR analysis (n=3 cell cultures / group). (F) Schematic diagram of bidirectional LV designs without (top) and with (bottom) miRT sites delivered systemically (iv) to mice. (G) Graph of LV copies per cell calculated using ddPCR analysis (from left to right, n=8, 8, 8, 3 mice / group, statistical analysis by Kruskal-Wallis test). (H) Graph of the average number of GFP+ hepatocytes per frame detected in 5-6 CM images of the liver (n=8 mice / group, statistical analysis by Mann-Whitney test). (I) Representative immunofluorescence (IF) image of the liver obtained by confocal microscopy (CM), showing GFP (green), F4 / 80 (red), and nucleus (blue). Enlarged view of the indicated area showing GFP and nucleus (top), and F4 / 80 and nucleus (bottom). (J and K) Graph of the percentage of GFP-positive cells and dlNGFR-positive cells (J) and MFI (K) from liver KC and LSEC analyzed using FC. (n is the same as A; statistical analysis by Kruskal-Wallis with Dunn test, p-value adjusted by Bonferroni correction). (L) This figure shows GFP expression in different organs upon delivery of PBS(UT) or Mrc1.GFP.miRT LV to mice challenged with liver metastases. Single channel of the image shown in F. Liver with metastases from MC38 cells (top) or AKTPF cells (bottom).(M) These are representative CM images of organs from mice treated with PBS (UT, top) or Mrc1.GFP.miRT LV (bottom), with GFP (green), F4 / 80 (gray), and the nucleus (blue). [Figure 9-1] In vivo LV-modified KC enables rapid, sustained, and well-tolerated IFNα production. (A) Graphs of inflammatory monocytes, resident monocytes, CD8 T cells, CD4 T cells, eosinophils, platelets, and erythrocytes, as well as hemoglobin levels, at indicated time points at the time of LV injection (n=10, 10, and 5 mice / group, respectively, for control LV, IFNαLV, or UT mice; statistical analysis by Mann-Whitney test). (B) Heatmaps showing the multiplier change relative to the column mean of autoreactive antibodies against indicated targets detected in the serum of mice at 52 days (n=3 and 4 mice / group, respectively, for IFNαLV or control LV) and 366 days (n=8, 8, and 5 mice / group, respectively, for IFNαLV, control LV, or UT) after LV injection. Positive control: Plasma from lupus mice (18-week-old female NZB / NZW mice, statistical analysis by Mann-Whitney test, p-value adjusted by Bonferroni correction). (C) Graph of histopathological analysis of the indicated organs at 366 days after LV injection, scored as absent, minimal, mild, moderate, prominent, and severe (n=8, 8, and 5 mice / group in control LV, IFNαLV, or UT mice, respectively). [Figure 9-2]In vivo LV-modified KC enables rapid, sustained, and well-tolerated IFNα production. (A) Graphs of inflammatory monocytes, resident monocytes, CD8 T cells, CD4 T cells, eosinophils, platelets, and erythrocytes, as well as hemoglobin levels, at indicated time points at the time of LV injection (n=10, 10, and 5 mice / group, respectively, for control LV, IFNαLV, or UT mice; statistical analysis by Mann-Whitney test). (B) Heatmaps showing the multiplier change relative to the column mean of autoreactive antibodies against indicated targets detected in the serum of mice at 52 days (n=3 and 4 mice / group, respectively, for IFNαLV or control LV) and 366 days (n=8, 8, and 5 mice / group, respectively, for IFNαLV, control LV, or UT) after LV injection. Positive control: Plasma from lupus mice (18-week-old female NZB / NZW mice, statistical analysis by Mann-Whitney test, p-value adjusted by Bonferroni correction). (C) Graph of histopathological analysis of the indicated organs at 366 days after LV injection, scored as absent, minimal, mild, moderate, prominent, and severe (n=8, 8, and 5 mice / group in control LV, IFNαLV, or UT mice, respectively). [Figure 9-3]In vivo LV-modified KC enables rapid, sustained, and well-tolerated IFNα production. (A) Graphs of inflammatory monocytes, resident monocytes, CD8 T cells, CD4 T cells, eosinophils, platelets, and erythrocytes, as well as hemoglobin levels, at indicated time points at the time of LV injection (n=10, 10, and 5 mice / group, respectively, for control LV, IFNαLV, or UT mice; statistical analysis by Mann-Whitney test). (B) Heatmaps showing the multiplier change relative to the column mean of autoreactive antibodies against indicated targets detected in the serum of mice at 52 days (n=3 and 4 mice / group, respectively, for IFNαLV or control LV) and 366 days (n=8, 8, and 5 mice / group, respectively, for IFNαLV, control LV, or UT) after LV injection. Positive control: Plasma from lupus mice (18-week-old female NZB / NZW mice, statistical analysis by Mann-Whitney test, p-value adjusted by Bonferroni correction). (C) Graph of histopathological analysis of the indicated organs at 366 days after LV injection, scored as absent, minimal, mild, moderate, prominent, and severe (n=8, 8, and 5 mice / group in control LV, IFNαLV, or UT mice, respectively). [Figure 10-1]Gene-based forced IFNα expression by KC inactivates T cell activation and reduces liver metastatic proliferation. (A) Graphs of LV copies per cell in the liver analyzed using ddPCR (left panel, n=9, 10, 3; right panel, 8, 7, 5 mice / group for control LV, IFNαLV, or UT, respectively). (B) Graphs of B cell counts in the blood (left panel, n=10, 9, 5; right panel, n=9, 9, 5 mice / group for control LV, IFNαLV, or UT, respectively; statistical analysis by Mann-Whitney test). (C) Graphs of tumor volume measured by calipers at the indicated time (left panel, n=1, 4; right panel, n=1, 5 mice / group for IFNαLV complete responder or UT cohort, respectively). (D) Graphs of plasma IFNα levels measured using ELISA (day 5, n=9, 6; day 11, n=9, 7 mice / group, left panel) and LV copies per cell measured using ddPCR analysis in the liver (n=10, 10 mice / group, right panel). (E) Representative H&E staining images of human liver sections containing CRC metastases (left) and mouse liver sections containing AKTPF metastases (right). Note the tumor glands (NG), tumor endothelium (NE), and dirty central necrosis (CDN) highlighted with black arrows. (F) Representative CM images of mouse liver containing AKTPF metastases stained for CD4 (green), CD8 (red), CD11c (green), F4 / 80 (red), CD31 (green), α-SMA (red), E-cadherin (gray), and nucleus (blue), as shown in the figure. (G) Graphs of tumor growth obtained using MRI analysis (n=10 and 9 mice / group, respectively, in control LV or IFNαLV mice; statistical analysis by Mann-Whitney test), (H) Graphs of plasma IFNα levels obtained using ELISA (n=10, 9, and 5 mice / group, respectively, in control LV, IFNαLV, or UT mice, left) and LV copies per cell obtained by ddPCR analysis in the liver (n=10, 10, and 5 mice / group, respectively, right).(I) Graph of LV copies per cell obtained using ddPCR analysis in the liver (control LV or IFNαLV, n=11, 6 mice / group). (J) Graph of plasma IFNα levels obtained using ELISA (control LV, n=7; IFNαLV, n=8 mice / group). [Figure 10-2]Gene-based forced IFNα expression by KC inactivates T cell activation and reduces liver metastatic proliferation. (A) Graphs of LV copies per cell in the liver analyzed using ddPCR (left panel, n=9, 10, 3; right panel, 8, 7, 5 mice / group for control LV, IFNαLV, or UT, respectively). (B) Graphs of B cell counts in the blood (left panel, n=10, 9, 5; right panel, n=9, 9, 5 mice / group for control LV, IFNαLV, or UT, respectively; statistical analysis by Mann-Whitney test). (C) Graphs of tumor volume measured by calipers at the indicated time (left panel, n=1, 4; right panel, n=1, 5 mice / group for IFNαLV complete responder or UT cohort, respectively). (D) Graphs of plasma IFNα levels measured using ELISA (day 5, n=9, 6; day 11, n=9, 7 mice / group, left panel) and LV copies per cell measured using ddPCR analysis in the liver (n=10, 10 mice / group, right panel). (E) Representative H&E staining images of human liver sections containing CRC metastases (left) and mouse liver sections containing AKTPF metastases (right). Note the tumor glands (NG), tumor endothelium (NE), and dirty central necrosis (CDN) highlighted with black arrows. (F) Representative CM images of mouse liver containing AKTPF metastases stained for CD4 (green), CD8 (red), CD11c (green), F4 / 80 (red), CD31 (green), α-SMA (red), E-cadherin (gray), and nucleus (blue), as shown in the figure. (G) Graphs of tumor growth obtained using MRI analysis (n=10 and 9 mice / group, respectively, in control LV or IFNαLV mice; statistical analysis by Mann-Whitney test), (H) Graphs of plasma IFNα levels obtained using ELISA (n=10, 9, and 5 mice / group, respectively, in control LV, IFNαLV, or UT mice, left) and LV copies per cell obtained by ddPCR analysis in the liver (n=10, 10, and 5 mice / group, respectively, right).(I) Graph of LV copies per cell obtained using ddPCR analysis in the liver (control LV or IFNαLV, n=11, 6 mice / group). (J) Graph of plasma IFNα levels obtained using ELISA (control LV, n=7; IFNαLV, n=8 mice / group). [Figure 11] Manipulation of KC by IFNαLV enables preferential IFNα signaling in the peripheral regions of metastases. (A) Graph of tumor gene expression analysis using ddPCR analysis (n=10, 7 mice / group, for control LV or IFNαLV, respectively). (B) Schematic diagram showing tumor volume at day 28 for three different cohorts: control (red), partial responder (blue), or resistance (green). (C) UMAP display based on spatial transcriptome analysis spots of AKTPF liver metastases (left), and representative H&E image (right) overlaid with transcriptome analysis spots highlighted by color according to UMAP clustering. (D) Figure of gene expression of selected genes related to CRC (cancer cell gene signature) or liver function (liver gene signature). Mean gene expression is shown on a color scale, and the proportion of cells expressing the indicated gene is represented by the size of the shape (n=8). (E) Bottom right: Schematic diagram showing spatial compartments A-H. The approximate distance to the tumor-liver boundary is shown in millimeters, and the color associated with each spatial compartment is also indicated. Upper right and upper left: Sections analyzed using spatial transcriptome, excluding the section shown in Figure 4A. [Figure 12-1]IFNα promotes APC immune activation and enhanced MHCII-restricted antigen presentation in responder mice. (A) UMAP display showing cells from AKTPF liver metastases divided into different treatment cohorts (n similar to A), as shown in Figure 4B. (B) Graph showing the percentage of cells belonging to the identified cell population for each sample (n=3, 3, and 2 mice / group for control, partial responder, or resistance, respectively). UMAP display showing all cells from all groups (n similar to A), showing gene expression levels indicated on a scale from gray (low expression) to blue (high expression). (D) Heatmap showing logarithmic changes in the expression of the top 20 upregulated genes in each cluster (n=8). (E) Figure of gene expression of selected genes associated with the indicated features in different clusters of the APC compartment, showing the mean expression (color scale) and percentage (shape size; n=8) of cells expressing the indicated genes. [Figure 12-2] IFNα promotes APC immune activation and enhanced MHCII-restricted antigen presentation in responder mice. (A) UMAP display showing cells from AKTPF liver metastases divided into different treatment cohorts (n similar to A), as shown in Figure 4B. (B) Graph showing the percentage of cells belonging to the identified cell population for each sample (n=3, 3, and 2 mice / group for control, partial responder, or resistance, respectively). UMAP display showing all cells from all groups (n similar to A), showing gene expression levels indicated on a scale from gray (low expression) to blue (high expression). (D) Heatmap showing logarithmic changes in the expression of the top 20 upregulated genes in each cluster (n=8). (E) Figure of gene expression of selected genes associated with the indicated features in different clusters of the APC compartment, showing the mean expression (color scale) and percentage (shape size; n=8) of cells expressing the indicated genes. [Figure 13-1]The therapeutic response to IFNα is associated with T cell activation and is offset by Eomes CD4 T cell infiltration. (A) A heatmap showing the logarithmic changes in the expression of the top 20 upregulated genes in each cluster identified in the T cell and NK cell compartments (n=8). (B) A diagram of gene expression of selected genes associated with the shown features in different clusters (similar display to Figure 12E; n=8). (C) A diagram of GSEA analysis showing NES for selected GO terms for genes differentially expressed in CD8 T cells in the shown comparison (similar n to Figure 4A; similar statistical analysis to Figure 5A). [Figure 13-2] The therapeutic response to IFNα is associated with T cell activation and is offset by Eomes CD4 T cell infiltration. (A) A heatmap showing the logarithmic changes in the expression of the top 20 upregulated genes in each cluster identified in the T cell and NK cell compartments (n=8). (B) A diagram of gene expression of selected genes associated with the shown features in different clusters (similar display to Figure 12E; n=8). (C) A diagram of GSEA analysis showing NES for selected GO terms for genes differentially expressed in CD8 T cells in the shown comparison (similar n to Figure 4A; similar statistical analysis to Figure 5A). [Figure 14-1]IFNα from engineered KCs, combined with functional inhibition of regulatory T cells, eradicates liver metastases. (A) Graph of the correlation between IFNα signature score and Tr1 signature score (n=42; statistical analysis by Spearman's correlation coefficient). (B) IF images of human liver sections containing metastases, stained for CD4 (green), LAG3 (red), and nucleus (blue). Liver, metastases (Met), tumor glands (NG), tumor endothelium (NE), and dirty central necrosis (CDN) are shown. CD4+LAG3+ cells are indicated by arrows. (C) Graphs of plasma IFNα levels by ELISA (left panel, left to right, n=10, 9, 9, 9 mice / group) and LV copies per cell by ddPCR in the liver (right panel, left to right, n=9, 8, 9, 10 mice / group). (D) Graphs of MFI of PD1 expression on CD8 (left panel) or CD4 (right panel) T cells in blood circulation, analyzed using FC (from left to right, n=10, 9, 10, 10 mice / group; statistical analysis by Mann-Whitney test, p-value adjusted by Bonferroni correction). (E) Graphs of plasma IFNα levels (from left to right, left panel, n=10, 8, 10, 9, 3 mice / group) analyzed using ELISA and LV copies per cell in the liver (from left to right, right panel, n=10, 8, 10, 9, 3 mice / group) analyzed using ddPCR. (F) Graphs of plasma IFNα levels (from left to right, left panel, n=10, 9, 9, 9 mice / group) analyzed using ELISA and LV copies per cell in the liver (from left to right, right panel, n=6, 7, 9, 9 mice / group) analyzed using ddPCR. [Figure 14-2]IFNα from engineered KCs, combined with functional inhibition of regulatory T cells, eradicates liver metastases. (A) Graph of the correlation between IFNα signature score and Tr1 signature score (n=42; statistical analysis by Spearman's correlation coefficient). (B) IF images of human liver sections containing metastases, stained for CD4 (green), LAG3 (red), and nucleus (blue). Liver, metastases (Met), tumor glands (NG), tumor endothelium (NE), and dirty central necrosis (CDN) are shown. CD4+LAG3+ cells are indicated by arrows. (C) Graphs of plasma IFNα levels by ELISA (left panel, left to right, n=10, 9, 9, 9 mice / group) and LV copies per cell by ddPCR in the liver (right panel, left to right, n=9, 8, 9, 10 mice / group). (D) Graphs of MFI of PD1 expression on CD8 (left panel) or CD4 (right panel) T cells in blood circulation, analyzed using FC (from left to right, n=10, 9, 10, 10 mice / group; statistical analysis by Mann-Whitney test, p-value adjusted by Bonferroni correction). (E) Graphs of plasma IFNα levels (from left to right, left panel, n=10, 8, 10, 9, 3 mice / group) analyzed using ELISA and LV copies per cell in the liver (from left to right, right panel, n=10, 8, 10, 9, 3 mice / group) analyzed using ddPCR. (F) Graphs of plasma IFNα levels (from left to right, left panel, n=10, 9, 9, 9 mice / group) analyzed using ELISA and LV copies per cell in the liver (from left to right, right panel, n=6, 7, 9, 9 mice / group) analyzed using ddPCR. [Figure 15-1]The combination of IL12 and IFNα promotes CD8 T cell activation and liver metastasis clearance. (a) Schematic diagram of the experiment. (b) Graph of tumor weight 22 days after tumor cell inoculation in the indicated group. (c) Graph of digital droplet PCR analysis showing OVA expression in total tumor lysates in the indicated group. (d) Graph of flow cytometry blood analysis at day 15 showing the percentage of tetrameric (anti-OVA) CD8 T cells out of the total number of circulating CD8 T cells. (e and f) Graphs of the percentage of CD8 T cells out of total CD45 cells infiltrating MC38.OVA liver metastases. PEX was identified as shown in the Methods section. (g) Flow cytometry analysis of liver showing terminally exhausted (TEX) CD8 T cells out of total tetrameric CD8 T cells. (h) Flow cytometry analysis of liver showing PEX CD8 T cells out of total tetrameric CD8 T cells. (i) Flow cytometry analysis of liver cells showing the median fluorescence intensity of PD1 markings in tetrameric CD8 T cells. TEX was identified as shown in the Methods section. [Figure 15-2]The combination of IL12 and IFNα promotes CD8 T cell activation and liver metastasis clearance. (a) Schematic diagram of the experiment. (b) Graph of tumor weight 22 days after tumor cell inoculation in the indicated group. (c) Graph of digital droplet PCR analysis showing OVA expression in total tumor lysates in the indicated group. (d) Graph of flow cytometry blood analysis at day 15 showing the percentage of tetrameric (anti-OVA) CD8 T cells out of the total number of circulating CD8 T cells. (e and f) Graphs of the percentage of CD8 T cells out of total CD45 cells infiltrating MC38.OVA liver metastases. PEX was identified as shown in the Methods section. (g) Flow cytometry analysis of liver showing terminally exhausted (TEX) CD8 T cells out of total tetrameric CD8 T cells. (h) Flow cytometry analysis of liver showing PEX CD8 T cells out of total tetrameric CD8 T cells. (i) Flow cytometry analysis of liver cells showing the median fluorescence intensity of PD1 markings in tetrameric CD8 T cells. TEX was identified as shown in the Methods section. [Figure 16-1]The combination of IL12, IFNα, and anti-PD1 monoclonal antibody promotes CD8 T cell activation and liver metastasis clearance. (a) Schematic diagram of the experiment. (b) Graph of tumor weight 22 days after tumor cell inoculation in the indicated group. (c) Graph of digital droplet PCR analysis showing OVA expression in total tumor lysates in the indicated group. (d) Table showing treatment groups that do not show tumor (complete responder) or show OVA expression (all OVA-expressing cells removed from the tumor). (e) Graph of flow cytometry blood analysis on day 14 showing the percentage of tetrameric (anti-OVA) CD8 T cells out of the total number of circulating CD8 T cells. (f) Graph of the percentage of CD8 T cells out of all CD45 cells infiltrating MC38.OVA liver metastases. (g) Graph of the percentage of PEX tetrameric CD8 T cells out of all tetrameric CD8 T cells infiltrating MC38.OVA liver metastases. (h) Flow cytometry analysis of liver cells showing terminally exhausted (TEX) CD8 T cells among all tetrameric CD8 T cells. (i) Flow cytometry analysis of liver cells showing PEX CD8 T cells among all tetrameric CD8 T cells. (j) Flow cytometry analysis of liver cells showing median fluorescence intensity (MFI) of PD1 marking in tetrameric CD8 T cells. [Figure 16-2]The combination of IL12, IFNα, and anti-PD1 monoclonal antibody promotes CD8 T cell activation and liver metastasis clearance. (a) Schematic diagram of the experiment. (b) Graph of tumor weight 22 days after tumor cell inoculation in the indicated group. (c) Graph of digital droplet PCR analysis showing OVA expression in total tumor lysates in the indicated group. (d) Table showing treatment groups that do not show tumor (complete responder) or show OVA expression (all OVA-expressing cells removed from the tumor). (e) Graph of flow cytometry blood analysis on day 14 showing the percentage of tetrameric (anti-OVA) CD8 T cells out of the total number of circulating CD8 T cells. (f) Graph of the percentage of CD8 T cells out of all CD45 cells infiltrating MC38.OVA liver metastases. (g) Graph of the percentage of PEX tetrameric CD8 T cells out of all tetrameric CD8 T cells infiltrating MC38.OVA liver metastases. (h) Flow cytometry analysis of liver cells showing terminally exhausted (TEX) CD8 T cells among all tetrameric CD8 T cells. (i) Flow cytometry analysis of liver cells showing PEX CD8 T cells among all tetrameric CD8 T cells. (j) Flow cytometry analysis of liver cells showing median fluorescence intensity (MFI) of PD1 marking in tetrameric CD8 T cells. [Figure 17-1] Expression of IL12 and IFNα from liver macrophages in combination with melanoma-associated antigens promotes melanoma liver metastasis clearance. (a) Schematic diagram of the experiment. (b) Graph of magnetic resonance imaging (MRI) analysis 13 days after melanoma inoculation in the indicated group. (c) Graph of magnetic resonance imaging (MRI) analysis 19 days after melanoma inoculation in the indicated group. (d) Representative images of mouse livers from the indicated treatment group. (e) Graph of the percentage of CD4 T cells among all CD45 cells infiltrating B16 liver metastases. (f) Graph of the percentage of CD8 T cells among all CD45 cells infiltrating B16 liver metastases. (f) Graph of liver flow cytometry analysis showing the percentage of PD1-expressing CD4 T cells among all CD4 T cells. [Figure 17-2]Expression of IL12 and IFNα from liver macrophages in combination with melanoma-associated antigens promotes melanoma liver metastasis clearance. (a) Schematic diagram of the experiment. (b) Graph of magnetic resonance imaging (MRI) analysis 13 days after melanoma inoculation in the indicated group. (c) Graph of magnetic resonance imaging (MRI) analysis 19 days after melanoma inoculation in the indicated group. (d) Representative images of mouse livers from the indicated treatment group. (e) Graph of the percentage of CD4 T cells among all CD45 cells infiltrating B16 liver metastases. (f) Graph of the percentage of CD8 T cells among all CD45 cells infiltrating B16 liver metastases. (f) Graph of liver flow cytometry analysis showing the percentage of PD1-expressing CD4 T cells among all CD4 T cells. [Figure 18-1] a and b are UMAP projections of single-cell RNA sequencing (scRNA-seq) for the entire dataset for the indicated tissues. c and d are UMAP projections of scRNA-seq for APC subclusters for the indicated tissues. e and f are figures of selected gene expression belonging to the indicated categories and tissues in APCs. (n=2, 3, 3, 3 mice / group for liOVA, OVA.Ifna, OVA.Il12, and OVA.Combo; statistical analysis by Wilcoxon test with Bonferroni correction compared to liOVA; *:padj<0.05; **:padj<0.005; ***:padj<0.0005). g is a graph of combined gene expression scores for genes belonging to the indicated categories in different cell populations from the indicated tissues and cohorts. [Figure 18-2]a and b are UMAP projections of single-cell RNA sequencing (scRNA-seq) for the entire dataset for the indicated tissues. c and d are UMAP projections of scRNA-seq for APC subclusters for the indicated tissues. e and f are figures of selected gene expression belonging to the indicated categories and tissues in APCs. (n=2, 3, 3, 3 mice / group for liOVA, OVA.Ifna, OVA.Il12, and OVA.Combo; statistical analysis by Wilcoxon test with Bonferroni correction compared to liOVA; *:padj<0.05; **:padj<0.005; ***:padj<0.0005). g is a graph of combined gene expression scores for genes belonging to the indicated categories in different cell populations from the indicated tissues and cohorts. [Figure 19] a and b are UMAP projections of scRNA-seq for the entire dataset for the indicated groups and tissues. c and d are GSEAs of scRNA-seq data showing NES for selected GO terms calculated based on differentially expressed genes in KC, macrophages, and monocytes in the indicated comparisons. (n=2, 3, 3, 3 mice / group for liOVA, OVA.Ifna, OVA.Il12, and OVA.Combo; statistical analysis by adaptive multilevel split Monte Carlo method; *:padj<0.05; **:padj<0.005; ***:padj<0.0005). [Figure 20-1]a is a UMAP projection of scRNA-seq of subclustered liver T cells and NK cells. b is a figure of gene set enrichment analysis (GSEA) of scRNA-seq data showing normalized enrichment scores (NES) for selected gene ontology (GO) terms, calculated based on differentially expressed genes in T cells and NK cells in the shown comparison (n=2, 3, 3, 3 mice / group for liOVA, OVA.Ifna, OVA.Il12 and OVA.Combo; statistical analysis by adaptive multilevel split Monte Carlo method; *:padj<0.05; **:padj<0.005; ***:padj<0.0005). c is a graph of combined gene expression scores for genes belonging to the shown categories in different cell populations from the shown tissues and cohorts. d is a figure of the expression of selected genes belonging to the indicated categories in liver CD8+ T cells (similar number of mice as in b, statistical analysis by Wilcoxon test with Bonferroni correction compared to liOVA; *:padj<0.05;**:padj<0.005;***:padj<0.0005). e is a UMAP projection of liver scRNA-seq showing cells with OVA-specific TCRs. f is a figure of GSEA of scRNA-seq data showing NES for selected GO terms calculated based on differentially expressed genes in OVA-specific CD8+ T cells in the indicated comparison (similar number of mice and statistics as in b). g is a graph of combined gene expression scores of genes belonging to the indicated categories in different cell populations from the indicated tissues and cohorts. h is a figure of chronotypes shared between liver and tumor CD4+ T cells, grouped by TCR chronotype. i is a UMAP projection of liver scRNA sequencing showing CD4+ T cell chronotypes shared between liver and tumor tissue. j is a graph showing the proportion of cells within the CD4+ T cell population in the liver. k is a graph showing the number of CD4+ T cells divided by the TCR chronotype frequency. [Figure 20-2]a is a UMAP projection of scRNA-seq of subclustered liver T cells and NK cells. b is a figure of gene set enrichment analysis (GSEA) of scRNA-seq data showing normalized enrichment scores (NES) for selected gene ontology (GO) terms, calculated based on differentially expressed genes in T cells and NK cells in the shown comparison (n=2, 3, 3, 3 mice / group for liOVA, OVA.Ifna, OVA.Il12 and OVA.Combo; statistical analysis by adaptive multilevel split Monte Carlo method; *:padj<0.05; **:padj<0.005; ***:padj<0.0005). c is a graph of combined gene expression scores for genes belonging to the shown categories in different cell populations from the shown tissues and cohorts. d is a figure of the expression of selected genes belonging to the indicated categories in liver CD8+ T cells (similar number of mice as in b, statistical analysis by Wilcoxon test with Bonferroni correction compared to liOVA; *:padj<0.05;**:padj<0.005;***:padj<0.0005). e is a UMAP projection of liver scRNA-seq showing cells with OVA-specific TCRs. f is a figure of GSEA of scRNA-seq data showing NES for selected GO terms calculated based on differentially expressed genes in OVA-specific CD8+ T cells in the indicated comparison (similar number of mice and statistics as in b). g is a graph of combined gene expression scores of genes belonging to the indicated categories in different cell populations from the indicated tissues and cohorts. h is a figure of chronotypes shared between liver and tumor CD4+ T cells, grouped by TCR chronotype. i is a UMAP projection of liver scRNA sequencing showing CD4+ T cell chronotypes shared between liver and tumor tissue. j is a graph showing the proportion of cells within the CD4+ T cell population in the liver. k is a graph showing the number of CD4+ T cells divided by the TCR chronotype frequency. [Figure 21]a is a figure showing chronotype sharing between liver and tumor OVA-specific and bystander CD8+ T cells, grouped by TCR chronotype. b is a figure showing the expression of selected genes belonging to the indicated categories in liver OVA-specific CD8+ T cells (for liOVA, OVA.Ifna, OVA.Il12, and OVA.Combo, n=2, 3, 3, 3 mice / group; statistical analysis by Wilcoxon test with Bonferroni correction compared to liOVA. *:padj<0.05;**:padj<0.005;***:padj<0.0005). c is a figure showing the expression of selected genes belonging to the indicated categories in liver shared vs. non-shared CD4+ T cells in animals treated with OVA.Combo or OVA.Il12 (number of mice and statistical analysis). [Figure 22] a is a schematic diagram of the antigen prediction pipeline used for neoantigen identification in the AKTPF LM model. b is a schematic diagram of TA33 LV. c is a schematic diagram of the experiments shown in panels d-h. d-h are graphs of treatment of mice with established AKTPF LM by TA33 with TA33.Combo 7 days after tumor challenge (TA33 1 × 10⁷ TU / mouse, TA33.Combo total dose 1.2 × 10⁸ TU / mouse). d is a graph of quantification of LM volume by MRI 27 days after tumor injection (for untreated control, TA33 or TA33.Combo treated mice, n=6, 6, 9 mice / group; horizontal line represents median, Kruskal-Wallis statistical analysis with Dunn test). e and f are graphs of FC analysis of liver (for untreated control, TA33 or TA33.Combo treated mice, n=6, 6, 8 mice / group; similar statistics as in I). g and h are graphs of the IFNg ELISPOT assay performed on CD8+ T cells isolated from the spleen of the indicated mice (n=3, 3 mice / group). [Figure 23]a is a graph of plasma levels of IFN-γ and IL-12 measured by ELISA on day 7 post-treatment (n=6, 6, 9 mice / group for untreated control, TA33, or TA33.Combo treated mice; horizontal line represents median). b is a graph of blood FC analysis performed on day 14 post-tumor injection (same number of mice as in a, horizontal line represents median, Kruskal-Wallis statistical analysis using Dunn test). c is a graph of the correlation between circulating Ly6c+CD44+CD8 T cells and tumor volume measured by MRI on day 27 (same n number of mice as in a, statistical analysis by Spearman correlation). [Figure 24] Figures a-g show the treatment of mice with established B16-F10 LM 5 days after tumor challenge with Trp2.Combo (control mice remained untreated, and the total dose of OVA.Combo was 1.2 × 10⁸ TU / mouse). Three and ten days after LV injection, mice were either injected with 0.2 mg of a-PD1 or left untreated. Figure b is a graph of quantification of LM volumetric MRI at the indicated time points (n=5, 8, 6, 7 mice / group for untreated control, control + a-PD1, Trp2.Combo, or Trp2.Combo + a-PD1 treated mice; horizontal line represents median, Kruskal-Wallis statistical analysis using Dunn test). Figure c is a representative MRI image of the liver from untreated control or Trp2.Combo + a-PD1 treated mice. Figures d-g are graphs of FC analysis of the indicated tissues. (For untreated controls, control + a-PD1, Trp2.Combo, or Trp2.Combo + a-PD1 treated mice, n=3, 5, 6, or 7 mice / group; horizontal lines represent the median; Kruskal-Wallis statistical analysis using Dunn's test). [Figure 25]a is a graph of blood FC analysis (n=5, 8, 6, 7 mice / group for untreated control, control+a-PD1, Trp2.Combo, or Trp2.Combo+a-PD1 treated mice; horizontal line represents median; Kruskal-Wallis statistical analysis using Dunn test). b is a graph of the correlation between circulating Ly6c+CD44+CD8+ T cells and tumor volume, measured by MRI on day 13 (same mouse n as c; statistical analysis using Spearman correlation). [Modes for carrying out the invention]

[0120] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including” or “includes,” or “containing” or “contains,” and are inclusive or non-exclusive, and do not exclude additional, unlisted members, elements, or steps. The terms “comprising,” “comprises,” and “comprised of” also include the term “consisting of.”

[0121] phagocytes This invention relates to phagocyte-specific transgene expression, particularly to phagocyte-specific transgene expression in the liver and / or spleen.

[0122] As used herein, “phagocyte” refers to a specialized cell capable of phagocytosis. Phagocytosis can consist of recognizing particles larger than 0.5 μm and engulfing them into plasma membrane-derived vesicles known as phagosomes. Phagocytes can engulf microbial pathogens and apoptotic cells. Therefore, phagocyte activity is essential not only for microbial removal but also for tissue homeostasis (Rosales, C. and Uribe-Querol, E., 2017. BioMed Research International, 2017).

[0123] Preferably, the phagocytes targeted in the present invention are liver phagocytes and / or spleen phagocytes.

[0124] As used herein, “liver phagocytes” may refer to phagocytes primarily present in liver tissue, and “spleen phagocytes” may refer to phagocytes primarily present in spleen tissue.

[0125] Preferably, the phagocytic cells may be monocytes, macrophages, neutrophils, dendritic cells, eosinophils, fibroblasts, epithelial cells and / or endothelial cells.

[0126] Preferably, the phagocytic cells may be macrophages, dendritic cells, and / or hepatic sinusoidal endothelial cells. For example, the phagocytic cells may be hepatic macrophages and / or splenic macrophages, hepatic dendritic cells and / or splenic dendritic cells, and / or hepatic sinusoidal endothelial cells.

[0127] Preferably, the phagocytes may be specialized phagocytes such as monocytes, macrophages, neutrophils, dendritic cells, and eosinophils (e.g., liver specialized phagocytes and / or spleen specialized phagocytes). In some embodiments, the phagocytes are macrophages and / or dendritic cells.

[0128] Preferably, the phagocytes may be non-specialized phagocytes such as fibroblasts, epithelial cells, and / or endothelial cells. In some embodiments, the phagocytes are endothelial cells.

[0129] "Specialized phagocytic cells" 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. Furthermore, fibroblasts, epithelial cells, and endothelial cells can also perform phagocytosis. These "non-specialized" phagocytic cells cannot ingest microorganisms but are important for eliminating apoptotic bodies (Rosales, C. and Uribe-Querol, E., 2017. BioMed Research International, 2017).

[0130] macrophages In some embodiments, the phagocytic cells are macrophages (e.g., liver macrophages and / or spleen macrophages).

[0131] Macrophages are innate immune cells that protect tissues from pathogens or other biological substances. In adult mammals, macrophages are found in all tissues, and within tissues, they exhibit great anatomical and functional diversity. Within tissues, they are organized in defined patterns, with each cell occupying its own territory. Macrophages play a role in almost every aspect of the organism's biology, from development and homeostasis to repair through immune responses to pathogens. In particular, tumors are rich in macrophages, and they play a crucial role in tumor development, progression, and metastasis (Ta, W., Chawla, A. and Pollard, JW, 2013. Nature, 496, pp. 445-455).

[0132] Liver macrophages may include hepatic resident macrophages, infiltrating macrophages (e.g., bone marrow (BM)-derived macrophages), avascular peritoneal macrophages, and spleen-derived monocytes. Spleen macrophages may include marginal zone macrophages (MZMΦ), marginal metallophilic macrophages (MMMΦ), and red pulp macrophages (RpMΦ).

[0133] In some embodiments, the phagocytic cells are M2-like macrophages and / or MRC1+ macrophages (e.g., M2-like macrophages and / or MRC1+ macrophages from the liver and / or spleen).

[0134] Macrophages can be classified into M1-like (classically activated macrophages) and M2-like (alternatively activated macrophages) according to their activation state and function. M1 activation is induced by intracellular pathogens, bacterial cell wall components, lipoproteins, and cytokines such as interferon-γ and tumor necrosis factor-α. M1-like macrophages are characterized by the secretion of inflammatory cytokines and the production of nitric oxide (NO), resulting in an effective pathogen killing mechanism.

[0135] M2 activation is induced by fungal cells, parasites, immune complexes, complement, apoptotic cells, macrophage colony-stimulating factor, IL-4, IL-13, IL-10, and tumor growth factor β. M2-like macrophages have high phagocytic capacity and produce extracellular matrix (ECM) components, angiogenic and chemotactic factors, and IL-10. In addition to pathogen defense, M2-like macrophages can eliminate apoptotic cells, mitigate inflammatory responses, and promote wound healing. M2-like macrophages are commonly known as anti-inflammatory, anti-inflammatory, wound-healing, tissue-repairing, and nutritive or regulatory macrophages (Roszer, T., 2015. Mediators of inflammation, 2015).

[0136] M2-like macrophages can be identified based on gene transcription or protein expression of a set of M2 markers, as described in Roszer, T., 2015, Mediators of inflammation, 2015. These markers include transmembrane glycoproteins, scavenger receptors, enzymes, growth factors, hormones, cytokines, and cytokine receptors. Preferably, M2-like macrophages express one or more M2 macrophage markers such as MRC1 (CD206), CD163, CD209, arginase-1, Chi3l3, FIZZ1, MGL-1, and Dectin-1.

[0137] In some embodiments, the phagocytic cells are MRC1+ macrophages.

[0138] C-type mannose receptor 1 (MRC1) is also known as CD206, CLEC13D, and CLEC13DL. MRC1 is a C-type lectin primarily found on the surface of macrophages, immature dendritic cells, and hepatic sinusoidal endothelial cells, and mediates glycoprotein endocytosis. Exemplary human MRC1 sequences are described under acceptance number UniProtKB P22897. Exemplary mouse MRC1 sequences are described under acceptance number UniProtKB Q61830.

[0139] In mice and humans, M2-like polarized macrophages, including tumor-associated macrophages (TAMs), or several 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 several dendritic cell (DC) populations and hepatic sinusoidal endothelial cells (LSECs) (Pandey, E., ASNour, and ENHarris, Front Physiol, 2020.11: p.873).

[0140] In some embodiments, the phagocytic cells are resident macrophages (e.g., liver resident macrophages or spleen resident macrophages).

[0141] Most tissues in the body contain a population of tissue-resident macrophages. Tissue-resident macrophages are known to play a role as immune sentinels on the front lines of tissue defense, where they are discretely positioned and transcriptionally programmed for encounters with pathogens or environmental challenges (Davies, LC, et al., 2013. Nature Immunology, 14(10), p.986).

[0142] Liver resident macrophages (also called "liver macrophages") include Kupffer cells and motile liver macrophages. Kupffer cells are maintained in the adult independently of the bone marrow and function to remove microorganisms and cell debris from the blood and to remove aged red blood cells. Kupffer cell phenotypic markers may include F4 / 80 hi , CD11b lo , CD169 + , CD68 + , galectin-3 + , and CD80 lo / - . Motile liver macrophages have an immune surveillance function, and phenotypic markers may include F4 / 80 + , CD11b + , and CD80 hi (Davies, L.C., et al., 2013. Nature immunology, 14(10), p.986).

[0143] Spleen resident macrophages include marginal zone macrophages (MZMΦ), marginal metallophilic macrophages (MMMΦ), and red pulp macrophages (RpMΦ). Histologically, the spleen is divided into white pulp and red pulp (Rp), separated by the marginal zone (MZ). RpMΦ form extensive networks within the Rp and are characterized in mice by the expression of F4 / 80 high CD68 + CD11b low / - and strong autofluorescence. Within the MZ, two macrophage populations can be distinguished. MZMΦ typically express on their surface type I scavenger receptors called C-type lectin SIGN-related 1 (SIGNR1) and macrophage receptor with collagenous structure (MARCO). MMMΦ are defined by the expression of, among other molecules, sialic acid-binding Ig-like lectin-1 (Siglec-1, sialoadhesin, CD169) and MOMA-1.

[0144] In some embodiments, the phagocytic cells are invasive macrophages (e.g., hepatic invasive macrophages or splenic invasive macrophages), such as bone marrow (BM)-derived macrophages.

[0145] In some embodiments, the phagocytic cells are avascular peritoneal macrophages (PMs).

[0146] PMs are self-renewing macrophages located in the peritoneal cavity and exist as two distinct PM subsets: large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs). LPMs originate from embryonic precursors and are produced in F4 / 80. high CD11b high MHCII low It represents the richest subset under steady-state conditions that exhibit the phenotype. On the other hand, SPM is F4 / 80 low CD11b low MHCII high This is a minor subset with a distinct phenotype, derived from BM-derived myeloid progenitor cells, and primarily appears during infection.

[0147] In some embodiments, the phagocytic cells are monocyte-derived macrophages (e.g., liver monocyte-derived macrophages and / or spleen monocyte-derived macrophages).

[0148] Monocytes circulate in the bloodstream and are recruited to mucosal tissue or inflammatory sites, where they can differentiate into monocyte-derived macrophages or monocyte-derived dendritic cells. MerTK, CD68, CD163, and the transcription factor MAFB are considered robust markers for macrophages, while dendritic cells express CD1a, CD1b, FcεRI, and CD226. Macrophages are large cells containing many phagocytic vesicles. In contrast, dendritic cells are smaller and exhibit dendritic projections on their surface (Segura, E. and Coillard, A., 2019. Frontiers in Immunology, 10, p.1907).

[0149] In some embodiments, the phagocytic cells are tumor-associated macrophages (e.g., liver tumor-associated macrophages and / or spleen tumor-associated macrophages).

[0150] Tumor-associated macrophages (TAMs) are a class of macrophages that are abundant in the microenvironment of solid tumors. TAMs contribute to tumor progression at different levels by promoting genetic instability, fostering cancer stem cells, supporting metastasis, and suppressing protective adaptive immunity. Depending on the disease stage, the tissues involved, and the host microbiome, TAMs can have a dual supportive and inhibitory effect on cancer (Mantovani, A., et al., 2017. Nature reviews Clinical oncology, 14(7), p.399).

[0151] In some embodiments, the phagocytic cells are MRC1+ liver macrophages (e.g., Kupffer cells) and / or MRC1+ spleen macrophages.

[0152] In some embodiments, the phagocytic cells are Kupffer cells.

[0153] Dendritic cells In some embodiments, the phagocytic cells are dendritic cells (e.g., liver dendritic cells and / or spleen dendritic cells).

[0154] Dendritic cells (DCs) are antigen-presenting cells in the mammalian immune system. Their main function is to process antigenic substances and present them to T cells of the immune system on their cell surface.

[0155] In a normal liver, DCs are typically found only around the portal vein triad and, like DCs in other peripheral sites, can efficiently capture, process, and transport antigens to local lymphoid tissue. Compared to LSECs and KCs, newly isolated liver DCs are primarily immature cells that express surface MHC but express few costimulatory molecules necessary for T cell activation (Lau, A.Hand Thomson, AW, 2003. Gut, 52(2), pp.307-314).

[0156] Both conventional / bone marrow DCs (cDCs) and plasmacytoid DCs (pDCs) are present in the human spleen at different maturation stages and in different subsets (Velasquez-Lopera, MM, et al., 2008. Clinical & Experimental Immunology, 154(1), pp.107-114).

[0157] endothelial cells In some embodiments, the phagocytes are endothelial cells (e.g., liver endothelial cells and / or spleen endothelial cells). For example, the phagocytes may be hepatic sinusoidal endothelial cells (LSECs).

[0158] LSECs possess one of the highest endocytogenic capacities in the human body and can remove soluble macromolecules and small particles via endocytosis receptors. Features used to identify LSECs include: (a) their high and rapid endocytogenic capacity, (b) a window lacking a diaphragm and organized into a cluster of pores, and (c) VEGFR3 + CD34 - VEGFR2 + VE-Cadherin + Factor VIII + CD45 - or CD31 + , LYVE-1 + L-SIGN + Stabilin-1 + CD34 - PROX-1 -Surface markers such as (Poisson, J., et al., 2017. Journal of hepatology, 66(1), pp.212-227).

[0159] In some embodiments, the phagocytic cell is LSEC.

[0160] In another aspect, the present invention provides a vector for LSEC-specific expression, the vector comprising a transgene operably ligated to one or more expression regulatory sequences.

[0161] vector In one embodiment, the present invention provides an organism comprising a vector for phagocytic cell-specific expression, particularly for phagocytic cell-specific expression of the liver and / or spleen.

[0162] Phagocyte-specific expression The vector may be a phagocyte-specific expression vector, particularly 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” may refer to the preferential or dominant expression of the transgene (e.g., as polypeptide or RNA) in phagocytes compared to other cells (e.g., blood, lung, and bone marrow cells). In some embodiments, at least 50% of the transgene expression occurs in phagocytes. In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the transgene expression occurs in phagocytes. In some embodiments, the transgene is substantially expressed only in phagocytes.

[0163] for example, (i) The expression of the transgene in phagocytic cells transduced by the vector may be greater than the expression of the transgene in other cells transduced by the vector, and / or (ii) When transduced by a vector, the introduced gene does not need to be substantially expressed in cells other than phagocytic cells, and / or (iii) When transduced by a vector, the introduced gene does not need to be substantially expressed in lung cells, bone marrow cells and / or blood cells, and / or (iv) The transgene may be substantially expressed only in some liver cells and / or some spleen cells, and / or (v) The expression of the transgene in Kupffer cells may be at least 10 times greater than that in hepatocytes when transduced by the vector, and / or (vi) When transduced by a vector, the introduced gene does not need to be substantially expressed in hepatocytes.

[0164] The expression of a transgene can be determined by any suitable method known to those skilled in the art. For example, if the transgene is a reporter gene (e.g., GFP), flow cytometry analysis may be used to determine the expression level in different cell types. Alternatively, if the transgene is a reporter gene (e.g., GFP), immunofluorescence analysis (e.g., by confocal imaging) may be used to determine the expression level in different cell types.

[0165] Preferably, the expression of the transgene in phagocytic cells transduced by the vector may be greater than the expression of the transgene in other cells transduced by the vector. For example, the expression of the transgene in phagocytic cells transduced by the vector may be at least 10 times, at least 20 times, at least 50 times, or at least 100 times greater than the expression in other cells transduced by the vector.

[0166] Preferably, when transgenes are introduced by a vector, they are not substantially expressed in cells other than phagocytic cells. For example, the percentage of non-phagocytic cells that express the transgene may be 5% or less, 2% or less, 1% or less, or 0%. For example, the expression of the transgene in non-phagocytic cells may be undetectable.

[0167] Preferably, when transgenes are introduced by a vector, they are substantially not expressed in lung cells, bone marrow cells, and / or blood cells. For example, the percentage of lung cells, bone marrow cells, and / or blood cells expressing the transgene may be 5% or less, 2% or less, 1% or less, or 0%. For example, the expression of the transgene in lung cells, bone marrow cells, and / or blood cells may be undetectable.

[0168] Preferably, the transgene is substantially expressed only in liver cells and / or spleen cells. For example, the proportion of cell types other than liver cells and / or spleen cells that express the transgene may be 5% or less, 2% or less, 1% or less, or 0%. For example, the expression of the transgene in cell types other than liver cells and / or spleen cells may be undetectable.

[0169] Preferably, the expression of the transgene in Kupffer cells may be at least 10 times greater than the expression in hepatocytes when transduced by a vector. For example, the expression of the transgene in Kupffer cells may be at least 10 times, at least 20 times, or at least 50 times greater than the expression in hepatocytes.

[0170] Preferably, the transgene may not be substantially expressed in hepatocytes after transduction by the vector. For example, the percentage of hepatocytes expressing the transgene may be 5% or less, 2% or less, 1% or less, or 0%. For example, the expression of the transgene in hepatocytes may be undetectable.

[0171] Preferably, the expression of the transgene in Kupffer cells may be at least 10 times greater than the expression in LSEC when transduced by a vector. For example, the expression of the transgene in Kupffer cells may be at least 10 times, at least 20 times, or at least 50 times greater than the expression in LSEC.

[0172] Preferably, the transgene may not be substantially expressed in LSECs when transduced by the vector. For example, the percentage of LSECs expressing the transgene may be 5% or less, 2% or less, 1% or less, or 0%. For example, the expression of the transgene in LSECs may be undetectable.

[0173] If the vector is an integration vector (e.g., possessing integrase activity), copies of the vector can be specifically integrated into phagocytes, particularly liver phagocytes and / or spleen phagocytes. For example, (i) The integration of the vector in the liver and spleen may be greater than the integration of the vector in other organs (e.g., lymph nodes, brain, small intestine, blood, bone marrow), and / or (ii) Vector integration may substantially occur in the liver, spleen, optionally in the blood, and optionally in the bone marrow, and / or (iii) Vector integration does not necessarily have to occur substantially in the lymph nodes, brain, or small intestine.

[0174] Vector incorporation can be determined by any suitable method known to those skilled in the art, for example, by viral copy number analysis, e.g., quantitative digital droplet PCR of different organs.

[0175] Preferably, vector integration in the liver and spleen is greater than vector integration in other organs such as lymph nodes, brain, small intestine, blood, and bone marrow. For example, the viral copy number in the liver and spleen may be at least 10 times, at least 20 times, at least 50 times, or at least 100 times greater than in other organs.

[0176] Preferably, vector integration substantially occurs in the liver, spleen, optionally blood, and optionally bone marrow. For example, vector integration in the liver and spleen, optionally blood, and optionally bone marrow may be at least detectable.

[0177] Preferably, vector integration does not occur substantially in lymph nodes, the brain, or the small intestine. For example, vector integration in lymph nodes, the brain, and the small intestine may be undetectable. All of these biological compartments host resident macrophage populations that can potentially express the transgene upon systemic delivery of the vector.

[0178] Viral vector Preferably, the vector of the present invention is a viral vector. The vector of the present invention is a lentiviral vector, but it is intended that other viral vectors may be used.

[0179] 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, adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, alphavirus vectors, flavivirus vectors, rhabdovirus vectors, measles virus vectors, Newcastle disease virus vectors, poxvirus vectors, and picornavirus vectors.

[0180] The vector of the present invention may be in the form of a viral vector particle. Preferably, the viral vector of the present invention is in the form of a lentiviral vector particle.

[0181] The vector can be an embedded viral vector or a non-embedded viral vector. An "embedded viral vector" can be integrated into the host cell genome after transduction into the host cell. A "non-embedded viral vector" cannot be integrated into the host cell genome after transduction into the host cell, or exhibits very weak integration ability.

[0182] Methods for preparing and modifying viral vectors, such as lentiviral vectors, and viral vector particles are well known in the art. Preferred methods are described in Merten, OW, et al., 2016. Molecular Therapy-Methods & Clinical Development, 3, p.16017; Nadeau, I. and Kamen, A., 2003. Biotechnology advances, 20(7-8), pp.475-489; Ayuso, E., et al., 2010. Current gene therapy, 10(6), pp.423-436; and Goins, WF, et al., 2008. Methods Mol Biol. 433, pp.97-113.

[0183] Retrovirus and lentiviral vectors The vector of the present invention may be a retroviral vector or a lentiviral vector. The vector of the present invention may also be a retroviral vector particle or a lentiviral vector particle.

[0184] Retroviral vectors may be derived from or derive from any suitable retrovirus. Numerous 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 (avian Examples include erythroblastosis virus (AEV).

[0185] Retroviruses can be broadly classified into two categories: "simple" and "complex." Retroviruses can also be further classified into seven groups. Five of these groups represent retroviruses with carcinogenic potential. The remaining two groups are lentiviruses and spumaviruses.

[0186] The basic structure of retroviral and lentiviral genomes shares many common features, such as the 5'LTR and 3'LTR. Between or within these are the packaging signals that enable the packaging of the genome, primer binding sites, integration sites that allow integration into the host cell genome, and the gag, pol, and env genes that encode packaging components—these are polypeptides necessary for the assembly of the viral particle. Lentiviruses possess additional features, such as the rev and RRE sequences in HIV, which enable the efficient transport of the incorporated proviral RNA transcript from the nucleus to the cytoplasm of the infected target cell.

[0187] In proviruses, these genes are flanked at both ends by regions called long terminal repeats (LTRs). LTRs are responsible for provirus integration and transcription. LTRs also function as enhancer-promoter sequences, regulating the expression of viral genes.

[0188] The LTR itself is an identical sequence that can be classified into three elements: U3, R, and U5. U3 originates from a sequence specific to the 3' end of the RNA. R originates from a sequence repeated at both ends of the RNA. U5 originates from a sequence specific to the 5' end of the RNA. The sizes of the three elements can vary considerably among different retroviruses.

[0189] In defective retroviral vector genomes, gag, pol, and env may be absent or non-functional.

[0190] In a typical retroviral vector, at least a portion of one or more protein-coding regions essential for replication may be removed from the virus. This results in a replication defect in the viral vector. A portion of the viral genome may also be replaced by a library encoding candidate modifiers operably linked to regulatory and reporter regions in the vector genome, in order to generate a vector containing candidate modifiers that can be transduced into target host cells and / or incorporate their genome into the host genome.

[0191] Lentiviral vectors are part of a larger group of retroviral vectors. Briefly, lentiviruses can be classified into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototype "slow virus" visna / maedi virus (VMV), as well as related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[0192] 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, cannot infect non-dividing or slowly dividing cells, such as the cells that make up muscle, brain, lung, and liver tissue.

[0193] As used herein, “lentiviral vector” is a vector comprising at least one component moiety that can be derived from a lentivirus. Preferably, the component moiety is involved in a biological mechanism by which the vector infects cells, expresses genes, or replicates.

[0194] Lentiviral vectors can be "primate" vectors. Lentiviral vectors can be "non-primate" vectors (i.e., derived from viruses that do not primarily infect primates, especially humans). Examples of non-primate lentiviruses can be any member of the Lentiviridae family that does not naturally infect primates.

[0195] Examples of lentivirus-based vectors include those based on HIV-1 and HIV-2, which are listed below.

[0196] HIV-1 vectors contain cis-acting elements, also found in simple retroviruses. Sequences extending within the gag open reading frame have been shown to be crucial for HIV-1 packaging. Therefore, HIV-1 vectors often contain the relevant portion of the gag with a mutated translation start codon. Furthermore, most HIV-1 vectors also contain a portion of the env gene, including the RRE. rev binds to the RRE, which enables the transport of full-length or single-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, the need for rev and RRE may be reduced by using constitutive transport elements derived from certain simple retroviruses, such as Mason-Pfizer salvir. The viral protein Tat is required for efficient transcription from the HIV-1 LTR promoter.

[0197] Most HIV-2-based vectors are structurally very similar to HIV-1 vectors. Like HIV-1-based vectors, HIV-2 vectors also require RRE (Rapid Regeneration) for efficient transport of full-length or single-spliced ​​viral RNA.

[0198] Optionally, the viral vector used in this invention has a minimal viral genome.

[0199] It should be understood that the “minimal viral genome” is manipulated to remove non-essential elements and retain essential elements in order to provide the functionality necessary for infecting, transducing, and delivering the desired nucleotide sequence to target host cells. Further details of this strategy can be found in International Publication No. 1998 / 017815.

[0200] Optionally, plasmid vectors used to produce a viral genome within host cells / packaging cells contain sufficient lentiviral genetic information to enable the packaging of the RNA genome into viral particles that can infect target cells in the presence of packaging components but cannot independently replicate and produce infectious viral particles within the final target cells. Optionally, the vectors lack functional gag-pol and / or env genes and / or other genes essential for replication.

[0201] However, plasmid vectors used to produce viral genomes within host / packaging cells also contain transcriptional regulatory sequences operably ligated to the lentiviral genome to guide genome transcription in the host / packaging cells. These regulatory sequences may be native sequences related to the transcribed viral sequence (i.e., the 5'U3 region), or they may be heterologous promoters, such as another viral promoter (e.g., the CMV promoter).

[0202] The vector may be a self-inactivating (SIN) vector lacking viral enhancer and promoter sequences. SIN vectors can be generated and transduced into non-dividing cells in vivo with efficacy similar to that of wild-type vectors. Transcriptional inactivation of the long-chain terminal repeat (LTR) in the SIN provirus should prevent recruitment by the reproducible virus. This should also allow for regulated gene expression from the internal promoter by eliminating any cis-effect of the LTR.

[0203] The vector may be integrase-deficient (i.e., integrase-scarce). An embedded-deficient lentiviral vector can be produced, for example, by packaging the vector with a catalytically inactive integrase (such as HIV integrase with a D64V mutation in the catalytic site), by modifying or deleting the essential att sequence from the vector LTR, or by any combination of the above.

[0204] In some embodiments, the vector is an integrase-deficient lentiviral vector. In some embodiments, the vector is an integrase-capable lentiviral vector.

[0205] The vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vector (LV), upon systemic delivery, can efficiently and specifically target the liver and is preferentially internalized by hepatic and splenic phagocyte populations, but is also transduced into other cell types, including endothelial cells and hepatocytes (Milani, M., et al., Sci Transl Med, 2019.11(493)). Therefore, the VSV-G pseudotyped LV constitutes an excellent tool for delivering target genes to hepatocyte populations.

[0206] Preferably, the vector is a VSV-G pseudotype. In some embodiments, the vector is a VSV-G pseudotype lentiviral vector particle.

[0207] Gene transfer into specialized phagocytic cells and antigen-presenting cells (APCs) is constrained by the presence of the CD47 molecule on LV particles. LV lacking CD47 exhibits retained infectivity and substantially increased susceptibility to phagocytosis. CD47-free LV more efficiently transduces specialized phagocytic cells both in vitro and in vivo, and induces substantially higher cytokine responses upon systemic administration to mice compared to LV with CD47. CD47-free LV can increase the efficiency of gene transfer into human primary monocytes and exhibits increased susceptibility to phagocytosis both in vitro by primary human macrophages and in vivo upon systemic administration to mice compared to previously available LV. For example, VSV-G pseudotype LV lacking the CD47 molecule on its surface is taken up more efficiently by liver and spleen specialized phagocytic cells than VSV-G pseudotype LV with CD47.

[0208] 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. As a result, cells and enveloped viruses containing HLA proteins from allogeneic sources can be targeted and neutralized by the immune system. A reduction or absence of surface-exposed HLA molecules is advantageous for viruses intended for use as vaccines, as it makes the virus less likely to be neutralized by antibodies that bind to HLA.

[0209] A preferred method for producing a CD47-free and / or HLA-free vector is described in International Patent Publication No. 2019 / 219836.

[0210] In some embodiments, the vector substantially lacks surface-exposed CD47 and / or HLA molecules. In some embodiments, the vector is a VSV-G pseudotyped lentiviral vector particle substantially lacking surface-exposed CD47 and / or HLA molecules.

[0211] As used herein, the term “substantially lacking” means that there is a substantial reduction in the number of molecules expressed on the surface of the vector compared to the number of molecules expressed on the surface of a vector produced in cells that have not been genetically engineered to reduce molecular expression (but under otherwise substantially identical conditions), so that the vector exhibits a therapeutically useful increase in its ability to transduce macrophages, phagocytic cells, antigen-presenting cells and / or monocytes, and / or induce a cytokine response upon systemic administration.

[0212] In some embodiments, the vector does not contain surface-exposed CD47 molecules and / or HLA molecules. In some embodiments, the vector is a VSV-G pseudotyped lentiviral vector particle that does not contain surface-exposed CD47 molecules and / or HLA molecules.

[0213] Adenovirus vector The vector of the present invention may be an adenovirus vector. The vector of the present invention may be an adenovirus vector particle.

[0214] Adenoviruses are double-stranded, linear DNA viruses that do not undergo RNA intermediates. There are over 50 different human serotypes of adenoviruses, classified into six subgroups based on genetic sequence homology. The natural targets of adenoviruses are the respiratory and gastrointestinal epithelium, generally causing only mild symptoms. Serotypes 2 and 5 (with 95% sequence homology) are the most commonly used in adenovirus vector systems and are typically associated with upper respiratory tract infections in young people.

[0215] Adenoviruses are used as vectors for gene therapy and heterologous gene expression. Their large (36kb) genomes can accommodate up to 8kb of foreign insertion DNA and replicate efficiently in complementary cell lines, up to 10 12 Adenoviruses can produce extremely high titers. Therefore, adenoviruses are one of the best systems for studying gene expression in primary non-replicating cells.

[0216] The expression of viruses or exogenous genes from adenovirus genomes does not require replicating cells. Adenovirus vectors enter cells via receptor-mediated endocytosis. Once inside the cell, adenovirus vectors are hardly integrated into the host chromosome. Instead, they function episomal (independent of the host genome) as linear genomes in the host nucleus. Therefore, the use of recombinant adenoviruses mitigates the problems associated with random integration into the host genome.

[0217] Adeno-associated virus vector The vector of the present invention may be an adeno-associated virus (AAV) vector. The vector of the present invention may also be in the form of AAV vector particles.

[0218] AAV vectors or AAV vector particles may contain an AAV genome or a fragment or derivative thereof. The AAV genome is a polynucleotide sequence capable of encoding functions necessary for the production of AAV particles. These functions include those that operate in the AAV replication and packaging cycle in host cells, including capsid formation of the AAV genome into AAV particles. Naturally occurring AAV is replication-deficient and relies on the provision of helper functions in trans for the completion of the replication and packaging cycle. Therefore, AAV genomes are typically replication-deficient.

[0219] The AAV genome may be in a single-stranded form, either positive-sense or negative-sense, or alternatively, a double-stranded form. The use of a double-stranded form allows for the avoidance of the DNA replication process in target cells, thereby potentially accelerating transgene expression.

[0220] Naturally occurring AAVs can be classified according to various biological systems. AAV genomes can originate from any naturally occurring serotype, isolate, or clade of AAV.

[0221] AAVs are referred to in terms of their serotypes. Serotypes correspond to variant subspecies of AAV, which have specific reactivity that can be used to distinguish them from other variant subspecies due to the expression profile of the capsid surface antigen. Typically, AAV vector particles with a particular AAV serotype do not efficiently cross-react with neutralizing antibodies specific to any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11.

[0222] AAVs can also be referred to in terms of clades or clones. This refers to the phylogenetic relationships of naturally occurring AAVs, typically a phylogenetic group of AAVs that can be traced back to a common ancestor and include all of its descendants. Furthermore, AAVs can be referred to in terms of specific isolates, i.e., genetic isolates of a particular AAV found in nature. The term genetic isolate refers to a population of AAVs that has undergone limited genetic mixing with other naturally occurring AAVs, thereby defining a genetically distinct population.

[0223] Typically, the AAV genome of a naturally occurring serotype, isolate, or clade of AAV contains at least one terminal inversion (ITR) sequence. The ITR sequence acts cis-wise to provide a functional origin for replication, enabling vector incorporation and excision from the cell's genome. The ITR may be the only sequence required cis-wise adjacent to the therapeutic gene. Preferably, one or more ITR sequences are adjacent to the transgene.

[0224] The AAV genome can also contain packaging genes such as the rep and / or cap genes that encode the packaging functions of the AAV particles. A promoter can be operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19, and p40 promoters. For example, the p5 and p19 promoters are generally used to express the rep gene, and the p40 promoter is generally used to express the cap gene. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52, and Rep40 or variants thereof.

[0225] The cap gene encodes one or more capsid proteins such as VP1, VP2, and VP3 or variants thereof.

[0226] The AAV genome can be the complete genome of a naturally occurring AAV. For example, an AAV vector or vector particle may be prepared using a vector containing the complete AAV genome.

[0227] Preferably, the AAV genome is derivatized for the purpose of administration to a patient. Such derivatization is standard in the art, and the present invention encompasses the use of any known derivative of the AAV genome and derivatives that can be generated by applying techniques known in the art. The AAV genome can be a derivative of any naturally occurring AAV. Preferably, the AAV genome is a derivative of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11.

[0228] Derivatives of the AAV genome include any shortened or modified form of the AAV genome that enables the expression of the transgene from the AAV vector of the present invention in vivo. Typically, it is possible to significantly truncate the AAV genome while retaining the above function, including a minimal viral sequence. This can reduce the risk of recombination of the vector with the wild-type virus and avoid inducing a cellular immune response due to the presence of viral gene proteins in the target cells.

[0229] Typically, the derivative contains at least one terminal inverted repeat (ITR), optionally more than one ITR, such as two or more ITRs. One or more ITRs can be derived from AAV genomes having different serotypes, or can be chimeric ITRs or mutant ITRs. Suitable mutant ITRs are those having a deletion of the trs (terminal resolution site). This deletion enables the generation of a single-stranded genome containing both the coding and complementary sequences, i.e., a self-complementary AAV genome, by continuous replication of the genome. This enables the avoidance of DNA replication in target cells, and thus enables the acceleration of transgene expression.

[0230] The AAV genome can contain one or more ITR sequences from any natural-derived serotype, isolate or clade of AAV or its variants. The AAV genome can contain at least one, e.g., two AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 ITRs, or variants thereof.

[0231] One or more ITRs can be adjacent to the transgene at either end. The inclusion of one or more ITRs can assist, for example, in the formation of concatemers of the AAV vector in the nucleus of the host cell after conversion of the single-stranded vector DNA to double-stranded DNA by the action of the host cell DNA polymerase. The formation of such episomal concatemers protects the AAV vector during the lifespan of the host cell, thereby enabling the extension of transgene expression in vivo.

[0232] Preferably, the ITR element is the only sequence retained from the native AAV genome in the derivative. Preferably, the derivative does not have to contain the rep and / or cap genes of the native genome, nor any other sequences of the native genome. This can reduce the possibility of vector integration into the host cell genome. Furthermore, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (e.g., regulatory elements) into the vector, in addition to the transgene.

[0233] Therefore, in derivatives of the present invention, the following portions may be removed: one terminal inversion (ITR) sequence, a replication (rep) gene, and a capsid (cap) gene. However, derivatives may further include one or more rep and / or cap genes or other viral sequences of the AAV genome. Since naturally occurring AAV is frequently integrated into specific sites on human chromosome 19 and exhibits negligible random integration, the retention of integration ability in AAV vectors may be acceptable in therapeutic settings.

[0234] The present invention further encompasses providing sequences of the AAV genome in an order and configuration different from that of the natural AAV genome. The present invention also encompasses the substitution of one or more AAV sequences or genes with chimeric genes composed of sequences from another virus or more than one virus. Such chimeric genes may consist of sequences from two or more related viral proteins of different viral species.

[0235] AAV vector particles can be capsid-formed by capsid proteins. Preferably, AAV vector particles may be in a transcapsidized form in which an AAV genome or derivative having one serotype ITR is packaged into a capsid of a different serotype. AAV vector particles also include a mosaic form in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral capsid. AAV vector particles also include a chemically modified form having ligands adsorbed to the capsid surface. For example, such ligands may include antibodies that target specific cell surface receptors.

[0236] If the derivative contains capsid proteins, i.e., VP1, VP2, and / or VP3, the derivative may be a chimeric, shuffled, or capsid-modified derivative of one or more naturally occurring AAVs. In particular, the present invention encompasses providing capsid protein sequences derived from different serotypes, clades, clones, or isolates of AAV within the same vector (i.e., a pseudotype vector). The AAV vector may be in the form of pseudotype AAV vector particles.

[0237] Chimeric, shuffled, or capsid-modified derivatives are typically selected to provide AAV vectors with one or more desired functionalities. Therefore, these derivatives may exhibit increased gene delivery efficiency and / or decreased immunogenicity (humoral or cellular) compared to AAV vectors containing naturally occurring AAV genomes. Increased gene delivery efficiency can be achieved, for example, by improved receptor or co-receptor binding on the cell surface, improved internalization, improved intracellular and nuclear transport, improved shedding of the viral particle, and improved conversion of single-stranded genomes to double-stranded forms.

[0238] Chimeric capsid proteins include those produced by recombination between two or more capsid-coding sequences of naturally occurring AAV serotypes. This can be carried out, for example, by a marker-rescue approach in which a non-infectious capsid sequence of one serotype is co-transfected with a capsid sequence of a different serotype, and targeted selection is used to select the capsid sequence with the desired properties. The capsid sequences of the different serotypes may be modified by homologous recombination within the cell to produce novel chimeric capsid proteins.

[0239] Chimeric capsid proteins also include those produced by manipulating the capsid protein sequence 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.

[0240] Shuffled or chimeric capsid proteins can also be generated by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be constructed by randomly fragmenting the sequence of the relevant AAV gene, for example, the sequence encoding the capsid proteins of several different serotypes, and then reconstructing the fragments in a self-priming polymerase reaction, which can also cause cross-reactivity in regions of sequence homology. A library of hybrid AAV genes thus constructed by shuffling the capsid genes of several serotypes can be screened to identify viral clones with desired functionality. Similarly, error-prone PCR may be used to randomly mutate AAV capsid genes to create a diverse library of variants, which can then be selected for desired properties.

[0241] The capsid gene sequence can also be genetically modified to introduce specific deletions, substitutions, or insertions from the natural wild-type sequence. In particular, the capsid gene can be modified by inserting sequences of unrelated proteins or peptides within the open reading frame of the capsid code sequence, or at the N-terminus and / or C-terminus of the capsid code sequence. The unrelated proteins or peptides may advantageously act as ligands for specific cell types, thereby conferring improved binding to target cells or improving the specificity of targeting the vector to a particular cell population. The unrelated proteins may also assist in the purification of viral particles as part of the production process, i.e., they may be epitopes or affinity tags. The insertion site is typically selected so as not to interfere with other functions of the viral particle, such as internalization and transport.

[0242] Capsid proteins may be artificial or mutant capsid proteins. As used herein, the term “artificial capsid” means that the capsid particle contains an amino acid sequence that is not naturally occurring or that has been manipulated (e.g., modified) from a naturally occurring capsid amino acid sequence. In other words, an artificial capsid protein, when the artificial capsid amino acid sequence is aligned with the parental capsid amino acid sequence, contains mutations or variations in the amino acid sequence compared to the parental capsid sequence from which it is derived.

[0243] Herpes simplex virus vector The vector of the present invention may be a herpes simplex virus vector. The vector of the present invention may be a herpes simplex virus vector particle.

[0244] Herpes simplex virus (HSV) is a neurotropic DNA virus with desirable properties as a gene delivery vector. HSV is highly infectious, and therefore, HSV vectors are efficient vehicles for the delivery of exogenous genetic material to cells. Viral replication is readily disrupted by null mutations in the earliest genes, which can be complemented in vitro and trans, allowing for the direct production of high-titer pure preparations of non-pathogenic vectors. The genome is large (152 Kb), and many of the viral genes are unnecessary for in vitro replication, allowing for substitution with large or multiple transgenes. Latent infection with wild-type virus results in the persistence of episomal viruses in sensory neuronal nuclei throughout the host's lifetime. The vectors are non-pathogenic, cannot be reactivated, and cannot persist for long periods. Latent activity promoter complexes can be used in vector design to achieve long-term stable transgene expression in the nervous system.

[0245] HSV vectors transduce a wide range of tissues due to their broad expression patterns of cell receptors recognized by the virus. As our understanding of the processes involved in cell entry deepens, it has become possible to target the targeting properties of HSV vectors.

[0246] Other viral vectors Other suitable viral vectors include those described in Lundstrom, K., 2018. Diseases, 6(2), p.42.

[0247] The vector of the present invention may be an alphavirus vector. The vector of the present invention may be an alphavirus vector particle. The vector of the present invention may be a flavivirus vector. The vector of the present invention may be a flavivirus vector particle.

[0248] 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), which have a positive-strand genome. Alphaviruses are mainly applied in preclinical gene therapy research for cancer treatment. Alphavirus vectors can be delivered in the form of naked RNA, plasmid DNA vectors, and recombinant replication-deficient or replication-competent particles.

[0249] The vector of the present invention may be a rhabdovirus vector. The vector of the present invention may be a rhabdovirus vector particle. The vector of the present invention may be a measles virus vector. The vector of the present invention may be a measles virus vector particle.

[0250] Rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus) and measles virus have a negative-strand genome. Among rhabdoviruses, recombinant vesicular stomatitis virus (VSV) is applied in preclinical gene therapy research. Measles virus (e.g., MV-Edm) has found many gene therapy applications.

[0251] The vector of the present invention may be a Newcastle disease virus vector. The vector of the present invention may be a Newcastle disease virus vector particle.

[0252] The ssRNA paramyxovirus Newcastle disease virus (NDV) replicates specifically in tumor cells and is thus frequently applied in cancer gene therapy.

[0253] The vector of the present invention may be a poxvirus vector. The vector of the present invention may be a poxvirus vector particle.

[0254] A characteristic feature of poxviruses is their dsDNA genome, which can readily accommodate foreign DNA exceeding 30kb. Poxviruses have found several applications as gene therapy vectors. For example, vaccinia virus vectors have shown potential in cancer treatment. Vaccinia virus is a large enveloped poxvirus with a linear double-stranded DNA genome of approximately 190kb. Vaccinia virus can accommodate foreign DNA up to approximately 25kb, which is also useful for delivering large genes. Many attenuated vaccinia virus strains suitable for gene therapy applications, such as the MVA and NYVAC strains, are known in the art.

[0255] The vector of the present invention may be a picornavirus vector. The vector of the present invention may be a picornavirus vector particle.

[0256] Picornaviruses are non-enveloped ssRNA viruses. Coxsackieviruses, belonging to the Picornaviridae family, are used as oncolytic vectors.

[0257] Expression regulatory sequences The vector of the present invention may contain one or more expression regulatory sequences. Preferably, the transgene is operably ligated to one or more expression regulatory sequences.

[0258] As used herein, “expression regulatory sequence” is any nucleotide sequence that controls the expression of a transgene, for example, to promote and / or increase its expression in some cell types and / or decrease its expression in other cell types.

[0259] The regulatory sequences and the transgene may be in any preferred arrangement in the vector, provided that the regulatory sequences are operably ligated to the transgene. As used herein, the term “operably ligated” means that the parts (e.g., the transgene and one or more regulatory sequences) are ligated together in such a manner that both can perform their functions substantially without interference.

[0260] The expression regulatory sequence may be a phagocyte-specific expression regulatory sequence, particularly a liver and / or spleen-specific expression regulatory sequence (e.g., so that the vector specifically expresses the transgene in phagocytes, especially liver and / or spleen phagocytes). The expression regulatory sequence includes a promoter, an enhancer, and a 5' untranslated region and a 3' untranslated region (e.g., a miRNA target sequence).

[0261] One or more expression regulatory sequences may include (a) a phagocytic cell-specific promoter and / or enhancer, and / or (b) one or more miRNA target sequences.

[0262] In some embodiments, one or more expression regulatory sequences include a phagocytic cell-specific promoter and / or enhancer, and optionally, one or more miRNA target sequences.

[0263] The vector may, for example, contain a phagocytic cell-specific promoter and / or enhancer-transgene-one or more miRNA target sequences from 5' to 3'.

[0264] MRC1-derived regulatory sequence Preferably, the vector of the present invention may include one or more MRC1-derived expression control sequences.

[0265] As used herein, “MRC1-derived regulatory sequence” refers to an regulatory sequence that contains any of the regulatory features present in the MRC1 gene. Examples of the human MRC1 gene include NCBI gene ID:4360 and GeneCard GCID:GC10P017809. Another name for it is CLEC13D. In assembly GRCh38.p13, the human MRC1 gene is located at Chr10:17809348..17911164. The MRC1 gene is conserved in chimpanzees, rhesus monkeys, dogs, cattle, mice, rats, chickens, zebrafish, and frogs.

[0266] The regulatory features present in the MRC1 gene may be identified by any preferred method known to those skilled in the art. For example, regulatory elements can be identified in GeneHancer, a genome-wide enhancer-pairs-gene and promoter-pairs-gene related database. Regulatory features present in the MRC1 gene include the MRC1 promoter, the MRC1 enhancer, and the MRC1 5'UTR and MRC1 3'UTR. The mannose receptor regulatory sequence is located at least partially immediately upstream of the transcription start site (Eichbaum, Q., et al., Blood, 1997.90(10):p.4135-43).

[0267] Phagocyte-specific promoter The vector of the present invention may include a phagocyte-specific promoter, particularly a phagocyte-specific promoter of the liver and / or spleen. Preferably, the transgene is operably ligated to a phagocyte-specific promoter, particularly a phagocyte-specific promoter of the liver and / or spleen.

[0268] A "promoter" is a region of DNA that initiates the transcription of a gene. Promoters are located upstream of the DNA (towards the 5' region of the sense strand) and near the gene's transcription start site.

[0269] As used herein, “phagocyte-specific promoter” may be a promoter that enables phagocyte-specific expression of an transgene operably linked to the promoter.

[0270] Exemplary phagocytic cell-specific promoters include the MRC1 promoter, ITGAM promoter, CD86 promoter, CD274 promoter, CD163 promoter, LYVE1 promoter, STAB1 promoter, ITGAX promoter, SIRPA promoter, TIE2 promoter, CHIL3 promoter, CD68 promoter, CSF1R promoter, VCAM1 promoter, PTGS1 promoter, and C1QA promoter.

[0271] Manipulated promoter variants derived from any of these promoters may be used, provided that the variants retain the ability to drive phagocytic-specific expression of the transgene operably linked to the promoter. Those skilled in the art will be able to obtain such variants using methods known in the art. The variants may have at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity with any of the promoters.

[0272] Fragments of any of these promoters (or their variants) may be used, provided that the fragments retain the ability to drive phagocytic-specific expression of the transgene operably ligated to the promoter. Those skilled in the art will be able to access such fragments using methods known in the art. The fragments may be, for example, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, or at least 1000 nucleotides in length.

[0273] In some embodiments, the phagocytic cell-specific promoter is selected from the group consisting of the MRC1 promoter, ITGAM promoter, CD86 promoter, CD274 promoter, CD163 promoter, LYVE1 promoter, STAB1 promoter, ITGAX promoter, SIRPA promoter, TIE2 promoter, CHIL3 promoter, CD68 promoter, CSF1R promoter, VCAM1 promoter, PTGS1 promoter, and C1QA promoter, or their variants and / or fragments.

[0274] In a preferred embodiment, the phagocytic cell-specific promoter is the MRC1 promoter, or a variant and / or fragment thereof.

[0275] MRC1 Promoter In one embodiment, the present invention provides a vector comprising an MRC1 promoter. Preferably, the transgene is operably ligated to the MRC1 promoter.

[0276] The MRC1 promoter may be identified using any preferred method, for example, by using a promoter prediction tool or by using a sequence immediately upstream of the MRC1 open reading frame. Preferably, the MRC1 promoter may be a sequence of about 0.2–5kb, 0.5–5kb, 1–2kb, or about 1.8kb immediately upstream of the MRC1 open reading frame.

[0277] In some embodiments, the MRC1 promoter includes or consists of a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 1. Preferably, the MRC1 promoter includes or consists of a nucleotide sequence or fragment thereof 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.

[0278] In some embodiments of the present invention, the MRC1 promoter includes or consists of the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof.

[0279] Exemplary human MRC1 promoter: (Sequence ID 1)

[0280] In some embodiments of the present invention, the MRC1 promoter includes or comprises a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 2. Preferably, the MRC1 promoter includes or comprises a nucleotide sequence or fragment thereof 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: 2.

[0281] In some embodiments of the present invention, the MRC1 promoter includes or consists of the nucleotide sequence of SEQ ID NO: 2 or a fragment thereof.

[0282] Exemplary mouse MRC1 promoter: (Sequence 2)

[0283] In some embodiments of the present invention, the MRC1 promoter includes or comprises a nucleotide sequence or fragment thereof that is at least 40% identical to SEQ ID NO: 1 and SEQ ID NO: 2. Preferably, the MRC1 promoter includes or comprises a nucleotide sequence or fragment thereof that is at least 50%, at least 60%, or at least 70% identical to SEQ ID NO: 1 and SEQ ID NO: 2.

[0284] In some embodiments, the MRC1 promoter includes or consists of a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 31. Preferably, the MRC1 promoter includes or consists of a nucleotide sequence or fragment thereof 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: 31.

[0285] In some embodiments of the present invention, the MRC1 promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 31 or a fragment thereof.

[0286] Exemplary XhoI-human.MRC1.promoter (Sequence ID 31)

[0287] Inducible promoter Preferably, the phagocytic cell-specific promoter may be an inducible promoter.

[0288] As used herein, “inducible promoter” is a promoter that is active only under specific conditions. For example, the expression of a transgene may be induced by a small molecule or drug (e.g., bound to a promoter, regulatory sequence, or transcriptional repressor or activator molecule), or by using 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 photo-inducible promoters. Preferably, the promoter is chemically inducible.

[0289] Any suitable method for manipulating the inducible phagocytic cell-specific promoter may be used.

[0290] Alternatively, a phagocyte-specific promoter may be a constitutive promoter. As used herein, “constitutive promoter” is a promoter that is always active.

[0291] Phagocyte-specific enhancer The vector of the present invention may include a phagocytic cell-specific enhancer. Preferably, the transgene is operably linked to the phagocytic cell-specific enhancer.

[0292] An "enhancer" is a region of DNA that can be bound by a protein (activator) to increase the likelihood of transcription of a particular gene occurring. Enhancers are cis-acting. They can be located up to 1 Mbp (1,000,000 bp) away from the gene and either upstream or downstream of the start site.

[0293] As used herein, “phagocyte-specific enhancer” may be an enhancer that enables phagocyte-specific expression of a transgene operably linked to the enhancer.

[0294] Examples of exemplary phagocytic cell-specific enhancers include MRC1 enhancer, ITGAM enhancer, CD86 enhancer, CD274 enhancer, CD163 enhancer, LYVE1 enhancer, STAB1 enhancer, ITGAX enhancer, SIRPA enhancer, TIE2 enhancer, CHIL3 enhancer, CD68 enhancer, CSF1R enhancer, VCAM1 enhancer, PTGS1 enhancer, and C1QA enhancer.

[0295] Manipulated enhancer variants derived from any of these enhancers may be used, provided that the variants retain the ability to drive phagocytic cell-specific expression of the transgene operably linked to the enhancer. Those skilled in the art will obtain such variants using methods known in the art. The variants may have at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity with any of the enhancers.

[0296] Fragments of any of these enhancers (or their variants) may be used, provided that the fragments retain the ability to drive phagocytic cell-specific expression of the transgene operably ligated to the enhancer. Those skilled in the art will be able to access such fragments using methods known in the art. The fragments may be at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, or at least 1000 nucleotides in length.

[0297] In some embodiments, the phagocytic cell-specific enhancer is selected from the group consisting of MRC1 enhancer, ITGAM enhancer, CD86 enhancer, CD274 enhancer, CD163 enhancer, LYVE1 enhancer, STAB1 enhancer, ITGAX enhancer, SIRPA enhancer, TIE2 enhancer, CHIL3 enhancer, CD68 enhancer, CSF1R enhancer, VCAM1 enhancer, PTGS1 enhancer, and C1QA enhancer, or their variants and / or fragments.

[0298] In a preferred embodiment, the phagocytic cell-specific enhancer is an MRC1 enhancer, or a variant and / or fragment thereof.

[0299] The vector of the present invention may include a phagocytic cell-specific promoter and / or a phagocytic cell-specific enhancer, i.e., a phagocytic cell-specific promoter and / or enhancer. Preferably, the transgene is operably linked to the phagocytic cell-specific promoter and / or enhancer.

[0300] In some embodiments, the phagocytic cell-specific promoter and / or enhancer is selected from the group consisting of the MRC1 promoter and / or enhancer, ITGAM promoter and / or enhancer, CD86 promoter and / or enhancer, CD274 promoter and / or enhancer, CD163 promoter and / or enhancer, LYVE1 promoter and / or enhancer, STAB1 promoter and / or enhancer, ITGAX promoter and / or enhancer, SIRPA promoter and / or enhancer, TIE2 promoter and / or enhancer, CHIL3 promoter and / or enhancer, CD68 promoter and / or enhancer, CSF1R promoter and / or enhancer, VCAM1 promoter and / or enhancer, PTGS1 promoter and / or enhancer, and C1QA promoter and / or enhancer, or variants and / or fragments thereof.

[0301] The phagocytic cell-specific promoter and phagocytic cell-specific enhancer may be any combination of the above, for example, the MRC1 promoter and the ITGAM enhancer.

[0302] In a preferred embodiment, the phagocytic cell-specific promoter and / or enhancer is the MRC1 promoter and / or enhancer, or its variant and / or fragment.

[0303] An exemplary MRC1 enhancer may include the following:

[0304] Mouse Mrc1 Enhancer 1 ACAGAACCAGCAGTATAGGGAAGGCCGTGGTGTTGTGGGACTCACATGATATTATTTATGATATCTTGGAAATTAGAGCAAAGACAGGTTAGGCATTGTGGTCAGAGGAGCTGGGTTATGACACCGAGGAAACAAGCTGACCCTTGAATTAAAACATATTGACGCCATAGCAATAAGAGGATGGAACCACATTGCCCTCTGCTGTTGGGGAATCATGGCCGCTGCCCCCATTCTGCAGTTAAGAGACCCGGTACTGCCCTCTGCTGGCTGGATGCACATGTTTCCACATTCTGGATTAGTATCCTTTTGAATTTAAATTTAAAAACAGTCTCCTGCTGCCTGCCAGTGACTCACTGTGGCCTCTTTATGTTGTTAGTAGCTTTGTTTTACTCTGGCAGATAGAAAATATGTTACAGGTCGCCATCTTGGTTCCGGGACTCAGCA (SEQ ID NO: 17)

[0305] Human MRC1 enhancer 1 AGCCCCACCATGTTATTGATGGCCAAACAATACGCATGCTGACAGCCATTATCTGTGGCCTCTGATGCTATTAGCCAAACCATGTTATTGATGGTCAAACAATACGCATGCTGACAGCCATTATCTGGGACTCAGAAAGTTCTGCATATTCAAGTCAGGCCAGAGGATCCGAGTTCTAATGTTAAGAGAAACCAACACACCAACAAGCAAATAAACAAACCTACCCTTGAACCAAAATATACATCAATACCTCCGTTGCAAATGGATAAATGGAACTGCATTGCCCTCTGCTGTTGGGGAATCTTGGCAACCATTTCAACTCTATGGCTGGAGATGACTTACTGCTCTGTTTATTTTCCATCCTCCTGCTTAGATTATTGCTTTCAAAGTTTCCAGAATAGAAGAAGTCAGTGGTGGCCAGTTGTCCTTTAATGGTCTCTTATCTACCAATGGCTAGTATCCTTTTTGCATTATCG TAGCTCTACTCTTGTAGATGTTAAATT (SEQ ID NO: 18)

[0306] Mouse Mrc1 enhancer 2 ACATGGGAGGCAAGGCGGAAGGAGCATGAGGCTGACCTAGCAGGCAGGAAGCACAGAAATCACATTTTGAGCTACATAGAAGAAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAATCAGGAAGTGAGACTAGTCTATAAAACTGCAAAGCCTACTCCCACTGACATACTTCCTTTAGCAATGCACAGCTGCCACAACCCTCCCAAATCCTGCCACCAACTGGAGACCAAGGGTTACAATAAGTAGACCTAAGGGAGGGGTACTTTTCTTTTCAACCACTGCAGTGGAGCACACCTCTATGTCCAACATGAAGGAAATAGAGGCTGGAAGACCAGAAATTCAAGGTCACCCACCAGCTCATCGCCAGTTGCAGATCAGTTTGAGCTACAGGCTATCTGCCTCAAATATAAAACTAAACAGAAAGTCAATAAAAAGGCCACACTTGGGGAAGTGGATAATAGGGTCAAATATTAGTAAACACCTCTTCTTCCCCATTGTTAAAGCCTGCTCCCTCCAGTTCCTCTGACTTTACTGTTACATAACAGATCTTGGACCTGTGACTGCTGTGTTTACAACATACTCAGTGACCCCTAACTTCTAATCATGAAACACATTTACCCGGTTCCAGGATGCCATCTCTCCACCTACAGCTCACCATGGAAGCATTTTGCCTCTTAGCAAAGGTCTTTGGTTTCTCGTGGGTGGCA (SEQ ID NO: 19)

[0307] Human MRC1 enhancer 2 (Sequence ID 20)

[0308] Mouse Mrc1 Enhancer 3 (Sequence ID 21)

[0309] Human MRC1 Enhancer 3 (Sequence ID 22)

[0310] Mouse Mrc1 Enhancer 4 AATAAACGTCTAGGAACATTTACCCTAAAGTACTGCCCTCTCTATGTGAACAAACTTAAGCCTGTGTTCTTTCCTTTTTGTGAACAGACGCGAGGCAATTTTTAATCTATAATGAAGATCACAAGCGCTGCGTGGACGCTCTAAGTGCCATCTCAGTTCA GACGGCAACTTGCAACCCGGAAGCTGAATCCCAGAAATTCCGCTGGGTGTCAGATTCTCAGATCATGAGTGTTGCTTTCAAATTATGTTTGGGAGTGCCATCAAAAACTGACTGGGCTTCCGTCACCCTGTATGCCTGTGATTCGAAAAGTGAATATCAGA (Sequence No. 23)

[0311] Human MRC1 Enhancer 4 TGGAAGAGTTGGAAACTTTTGACCTAAAAGATCGTCCTTGTTACATGAATCCACTTAGCCATGCTTGCTTTCTTCTTCTTTTCCTGCTTCTTTCTTTTTAAACAGACACCAGGCAATTTTTAATCTATAATGAAGATCACAAGCGCTGCGTGGAT GCAGTGAGTCCCAGTGCCGTCCAAACCGCAGCTTGCAACCAGGATGCCGAATCACAGAAATTCCGATGGGTGTCCGAATCTCAGATTATGAGTGTTGCATTTAAATTATGCCTGGGAGTGCCATCAAAAACGGACTGGGTTGCTATCACTCTCTAT (Sequence ID 24)

[0312] Mouse Mrc1 Enhancer 5 TGTCAGGTTCTCTGGAGCACCCTCTCACCTGTTCAGACTAATTTCCTAAGTTCGGCGGGTCCCGGACCAAGATGGCGACCCGCTACATTTCATTCTTACATGCAGGGGATGAGCGCACTGTTTCACCACTTTGATTGCCTTTTTTGAGCATGGTAGATATTCAGTAAGCAACCCATGGATTGAATTCTACTTTATGTTTAATGCAGGACGAAAGGCGGGATGTGTTGCCATGAAAACCGGAGTGGCAGGTGGCTTATGGGATGTTTTGAGTTGTGAAGAAAAGGCAAAATTTGTGTGCAAACATTGGGCAGAAGGAGTGACTCGCCCACCAGAGCCCACAACAACTCCTGAACCCAAATGTCCAGAAAACTGGGGTACCACCAGTAAAACCAGCATGTGTTTCAAAGT AAGGATCACTCGCCAAAT (SEQ ID NO: 25)

[0313] Human MRC1 enhancer 5 CATCCTCATTTTATTTTATGTACTTCTTTGTTCGTTAAAGCTGGCATTCCTTACAGTTCTATGAGGCAGGTCTTGGTATTTGCATTTGGAGAGGAGAAAGCAAGTTCAGAGCGTTTGAGTAACTTACCTAAAATCTCTAGTTGAGACGTGTCTCATTTTGAAATCTGTGAAAAACTTTGGTCCTGGAAAACCTACGTAGACCTTGGGAAGAAGGAAGGAAAAAGGGAAGGAAGGAGGGAGGGAGAGAGAAGCAGTAAACTATTTTTGCCATTATGGTGAATTTGATAATATAAAATATTTTATCATTAAATGCCTGTGTAGGGGGCACTTTGCCAAATGTTAGAAATATAAAGTGTTACAAACCCCCCTGCATCTGAGATCATAATTGGGCATCAGAACCCTGATGCTCGGTTCTGAGTGCCTTCTGTGAGCACGGCAGGCCTTCAGCAGGCACCTGTCAAGTGAATTCTACTTCATATATTTAATGCAGGGCGAAAGCCAGGGTGTGTTGCCATGAGAACCGGGATTGCAGGGGGCTTATGGGATGTTTTGAAATGTGATGAAAAGGCAAAATTTGTGTGCAAGCACTG (SEQ ID NO: 26)

[0314] Mouse Mrc1 enhancer 6 GAGTGATTGTGCATGAACTTGTGGAGACCTCAATTGTTCTTGCAACTTGTCTCTTCTATTACTATTGCAAAAGGAATGGCTAAGTCTTTCTTGAAAGAATTCATATAGTTCTCTTTCAGAGACCTGCAGCAGTTACCACTTTGGGGAACTAGAGAAAAGTTATTTTTAAGTTTCTCTGGAATGAAAGGCACAATTCTATAATTTGGCCTTATTGCTTAATCCACCAGTTTTAAGTTCCTTGTTTGTAAAATATGAATGTTAGTAACTCTTCTTCTTTAAAATCTCGTTATATCATCAAGCTTG (SEQ ID NO: 27)

[0315] Human MRC1 enhancer 6 (Sequence No. 28)

[0316] Mouse Mrc1 Enhancer 7 (Sequence ID 29)

[0317] Human MRC1 Enhancer 7 (Sequence ID 30)

[0318] In some embodiments, the MRC1 enhancer comprises or consists of a nucleotide sequence or fragment thereof that is at least 70% identical to any one of SEQ ID NOs: 17-30. Preferably, the MRC1 enhancer comprises or consists of a nucleotide sequence or fragment thereof that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 17-30.

[0319] In some embodiments of the present invention, the MRC1 enhancer comprises or consists of one nucleotide sequence or fragment thereof from sequence numbers 17 to 31.

[0320] In some embodiments of the present invention, the MRC1 enhancer comprises or consists of a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 32. Preferably, the MRC1 enhancer comprises or consists of a nucleotide sequence or fragment thereof 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: 32.

[0321] In some embodiments of the present invention, the MRC1 enhancer comprises or consists of the nucleotide sequence of SEQ ID NO: 32 or a fragment thereof.

[0322] Exemplary XhoI-human.MRC1.enhancer: CTCGAGAGCCCCACCATGTTATTGATGGCCAAACAATACGCATGCTGACAGCCATTATCTGTGGCCTCTGATGCTATTAGCCAAACCATGTTATTGATGGTCAAACAATACGCATGCTGACAGCCATTATCTGGGACTCAGAAAGTTCTGCATATTCAAGTCAGGCCAGAGGATCCGAGTTCTAATGTTAAGAGAAACCAACACACCAACAAGCAAATAAACAAACCTACCCTTGAACCAAAATACATCATCAAT ACCTCCGTTGCAAATGGATAAATGGAACTGCATTGCCCTCTGCTGTTGGGGAATCTTGGCAACCATTTCAACTCTATGGCTGGAGATGACTTACTGCTCTGTTTATTTTCCATCCTCCTGCTTAGAT TATTGCTTTCAAAGTTTCCAGAATAGAAGAAGTCAGTGGTGGCCAGTTGTCCTTTAATGGTCTCTTATCTACCAATGGCTAGTATCCTTTTTGCATTATCGTAGCTCTACTCTTGTAGATGTTAAATT (Sequence ID 32)

[0323] miRNA target sequences The vector of the present invention may contain one or more miRNA target sequences. Preferably, the transgene is operably ligated to one or more miRNA target sequences.

[0324] MicroRNA (miRNA) genes are scattered across all human chromosomes except the Y chromosome. They can be located either in non-coding regions of the genome or within introns of protein-coding genes. Approximately 50% of miRNAs appear in clusters that are transcribed as polycistronic primary transcripts. Like protein-coding genes, miRNAs are typically transcribed from the polymerase II promoter, producing a so-called primary miRNA transcript (pri-miRNA). This pri-miRNA is then processed through a series of endonuclease cleavage steps carried out by two enzymes belonging to the RNAse type III family, Drosha and Dicer. From the pri-miRNA, a stem-loop approximately 60 nucleotides long, called a pre-miRNA, is cleaved by a specific nuclear complex consisting of the Drosha and DiGeorge syndrome key region gene (DGCR8), which cleaves both strands near the bases of the primary stem-loop, leaving a 5' phosphate and a 2 bp long 3' overhang. Next, pre-miRNA is actively transported from the nucleus to the cytoplasm by RAN-GTP and Exportin. Then, Dicer performs double-strand breaks at the stem-loop ends not defined by dross cleavage, resulting in mature miRNA and miRNA. * This generates a 19-24 bp double helix, consisting of a double helix called the opposite strand. Consistent with the law of thermodynamic asymmetry, only one strand of the double helix is ​​selectively loaded into the RNA-induced silencing complex (RISC) and accumulated as a mature microRNA. This strand is typically one whose 5' end is not tightly paired with its complement, as demonstrated by the single nucleotide mismatch introduced at the 5' end of each strand of the siRNA double helix. However, there are some miRNAs that support the accumulation of both strands of the double helix to an equal degree.

[0325] MicroRNAs induce RNAi very similarly to small interfering RNAs (siRNAs), which are widely used for experimental gene knockdown. The main difference between miRNAs and siRNAs lies in their biosynthesis. When loaded into RISC, the guide strand of the small RNA molecule interacts with the mRNA target sequence, which is preferentially found in the 3' untranslated region (3'UTR) of protein-coding genes. The 2nd to 8th nucleotides from the 5' end of the miRNA, the so-called seed sequence, have been shown to be essential for inducing RNAi. If the entire guide strand sequence is perfectly complementary to the mRNA target, as is typically seen in siRNAs and plant miRNAs, the mRNA is endonuclease-likely cleaved by the involvement of the Argonaut (Ago) protein, also known as the "slicer" of the small RNA double helix incorporated into the RNA-induced silencing complex (RISC). DGRC (DiGeorge syndrome key region gene 8) and TRBP (TAR(HIV)RNA-binding protein 2) are double-stranded RNA-binding proteins that promote mature miRNA biosynthesis by Drosha and Dicer RNase III enzymes, respectively. The guide strand of the miRNA double helix is ​​incorporated into the effector complex RISC, which recognizes specific targets through incomplete base pairing and induces post-transcriptional gene silencing. Several mechanisms have been proposed for this regulatory mechanism, including the ability of miRNAs to induce repression of translation initiation, characterize target mRNAs for degradation by deadenylation, or sequester targets in the cytoplasmic P-isomer.

[0326] On the other hand, if only the seed is perfectly complementary to the target mRNA, but the remaining bases show incomplete pairing, RNAi acts through multiple mechanisms that result in translational repression. Eukaryotic mRNA degradation occurs primarily through shortening of the poly(A) tail at the 3' end of the mRNA, decapsulation at the 5' end, subsequent digestion by 5'-3' exonucleases, and accumulation of miRNAs in a separate cytoplasmic region rich in components of the mRNA degradation pathway, the so-called P-form.

[0327] Transgene 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 prevent or reduce transgene expression in that cell by binding to their corresponding miRNA target sequences located on a vector or polynucleotide (Brown, BD et al. (2007) Nat Biotechnol 25:1457-1467).

[0328] Suitable miRNA target sequences for suppressing transgene expression in specific cells are known to those skilled in the art. Suitable miRNA target sequences can be identified using any suitable method, for example, by performing microarrays containing known miRNAs from miRbases.

[0329] Including more than one copy of a miRNA target sequence in the vector can increase the effectiveness of the system. It is also conceivable that different miRNA target sequences may be included. For example, the transgene can be operably ligated to more than one miRNA target sequence, which may or may not be different. The miRNA target sequences may be in tandem, but other arrangements are also conceivable. The vector may contain, for example, one, two, three, four, five, six, seven, or eight copies of the same or different miRNA target sequences. Preferably, the vector contains four miRNA target sequences for each miRNA target sequence.

[0330] The target sequence may be fully or partially complementary to the miRNA. As used herein, the term “fully complementary” may mean that the target sequence has a nucleic acid sequence that is 100% complementary to the sequence of the miRNA that recognizes it.

[0331] As used herein, the term “partially complementary” may mean that a target sequence is only partially complementary to the sequence of the miRNA that recognizes it, thereby the partially complementary sequence is still recognized by the miRNA. In other words, a partially complementary target sequence in the context of the present invention is effective in recognizing the corresponding miRNA and resulting in the prevention or reduction of transgene expression in cells expressing that miRNA.

[0332] Copies of the miRNA target sequence can be separated by a spacer sequence. The spacer sequence may contain, for example, at least one, at least two, at least three, at least four, or at least five nucleotide bases.

[0333] Selective transgene expression can be driven in Kupffer cells (KCs) and, to a lesser extent, in MRC1+ spleen macrophages and hepatic sinusoidal endothelial cells (LSECs) using vectors that drive transgene expression from an M2-like macrophage-specific promoter (e.g., the MRC1 promoter). The specificity of the vector can be further increased using miRNA target sequences. One or more miRNA target sequences may suppress transgene expression in several hepatic cell populations and / or several spleen cell populations. For example, expression may target LSECs.

[0334] As used herein, the term “suppress expression” may refer to a reduction in the expression of a transgene in a relevant cell type (may be one) in which one or more miRNA target sequences are operably ligated, compared to transgene expression under conditions where one or more miRNA target sequences are absent but otherwise substantially identical. In some embodiments, transgene expression is suppressed by at least 50%. In some embodiments, transgene expression is suppressed by at least 60%, 70%, 80%, 90%, or 95%. In some embodiments, transgene expression is substantially prevented.

[0335] Preferably, one or more miRNA target sequences suppress transgene expression in hepatic sinusoidal endothelial cells (LSECs) and / or hepatocytes.

[0336] In some embodiments, one or more miRNA target sequences suppress transgene expression in hepatocytes and / or LSECS. For example, the vector may include (i) one or more copies of a miRNA target sequence that suppresses transgene expression in LSECs, and / or (ii) one or more copies of a miRNA target sequence that suppresses transgene expression in hepatocytes.

[0337] Preferably, 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.

[0338] The miR-126 target sequence is an exemplary miRNA target sequence that suppresses transgene expression in LSECs. miR-126 is a microRNA expressed in endothelial cells (e.g., LSECs), and when it binds to its target sequence, it reduces the expression of the target gene.

[0339] In some embodiments of the present invention, the miR-126 target sequence includes or consists of a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 3. Preferably, the miR-126 target sequence includes or consists of a nucleotide sequence or fragment thereof that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 3. In some embodiments of the present invention, the miR-126 target sequence includes or consists of the nucleotide sequence or fragment thereof of SEQ ID NO: 3.

[0340] Example miRT-126 CGCATTATTACTCACGGTACGA (Sequence ID 3)

[0341] The miR-122 target sequence is an exemplary miRNA target sequence that suppresses transgene expression in hepatocytes. miR-122 is the most abundant microRNA in hepatocytes, and when it binds to its target sequence, it reduces the expression of the target gene.

[0342] In some embodiments of the present invention, the miR-122 target sequence includes or consists of a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 4. Preferably, the miR-122 target sequence includes or consists of a nucleotide sequence or fragment thereof that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 4. In some embodiments of the present invention, the miR-122 target sequence includes or consists of the nucleotide sequence or fragment thereof of SEQ ID NO: 4.

[0343] An example: miRT-122 ACAAACACCATTGTCACACTCCA (Sequence No. 4)

[0344] Further miRNA target sequences that suppress transgene expression in LSEC and / or hepatocytes can be identified by any preferred method, for example, by miRNA expression analysis as described in Oda, S., et al., 2018. The American Journal of Pathology, 188(4), pp.916-928.

[0345] 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. Preferably, the target sequences are separated by spacer sequences.

[0346] In some embodiments of the present invention, one or more miRNA target sequences include or consist of nucleotide sequences or fragments thereof that are at least 70% identical to one or more of sequence numbers 5 to 7. Preferably, one or more miRNA target sequences include or consist of nucleotide sequences or fragments thereof that are at least 80%, at least 90%, or at least 95% identical to one or more of sequence numbers 5 to 7.

[0347] In some embodiments of the present invention, one or more miRNA target sequences include or consist of one or more nucleotide sequences or fragments thereof from sequence numbers 5 to 7.

[0348] Exemplary miRT-122 4 x miRT TCTAGATAAACAAACACCATTGTCACACTCCATTCGAAACAAACACCATTGTCACACTCCAACGCGTACAAACACCATTGTCACACTCCAATGCATACAAACACCATTGTCACACTCCACCCGGGTCGAGCTCGGT ACC (Sequence ID 5)

[0349] Exemplary miRT-126 4 x miRT GGTACCAGCAAACGCATTATTACTCACGGTACGACCATCGCATTATTACTCACGGTACGAACTTCGCATTATTACTCACGGTACGAACGCATTATTACTCACGGTACGACACGTGTCGGTACC (Sequence ID 6)

[0350] Exemplary miRT-122 and miR126 4 x miRT GGTACCAGCGCTACAAACACCATTGTCACACTCCAACATACAAACACCATTGTCACACTCCAGATTACAAACACCATTGTCACACTCCACAGAACAAACACCATTGTCACACTCCAGTTTAAACGCATTATTACTCACGGTACGACCATCGCATTATTACTCACGGTACGAACTTCGCATTATTACTCACGGTACGACGAACGCATTATTACTCACGGTACGACACGTGTCGGTACC (Sequence ID 7)

[0351] In some embodiments of the present invention, one or more miRNA target sequences include or consist of nucleotide sequences or fragments thereof that are at least 70% identical to SEQ ID NO: 36. Preferably, one or more miRNA target sequences include or consist of nucleotide sequences or fragments thereof that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 36.

[0352] In some embodiments of the present invention, one or more miRNA target sequences include or consist of the nucleotide sequence of SEQ ID NO: 36 or a fragment thereof.

[0353] An example is AfeI-4xmiRT122-4xmiRT126-PmlI AGCGCTACAAACACCATTGTCACACTCCAACATACAAACACCATTGTCACACTCCAGATTACAAACACCATTGTCACACTCCACAGAACAAACACCATTGTCACACTCCAGTTTAAACGCATTATTACTCACGTACGACCATCGCATTATTACTCACGGTACGAACTTCGCATTATTACTCACGGTACGACGAACGCATTATT ACTCACGGTACGACACGTGTC (Sequence No. 36)

[0354] In some embodiments, one or more miRNA target sequences suppress transgene expression in certain liver macrophages and / or certain spleen macrophages. For example, one or more miRNA target sequences may suppress transgene expression in M2-like macrophages. For example, one or more miRNA target sequences may suppress transgene expression in Kupffer cells and / or MRC1+ spleen macrophages.

[0355] In some embodiments, one or more miRNA target sequences suppress transgene expression in splenic phagocytes (e.g., splenic macrophages).

[0356] miRNA target sequences that suppress transgene expression in some liver macrophages and / or some spleen macrophages can be identified by any preferred method, for example, by miRNA expression analysis as described in Zhang, Y., et al., 2013. International Journal of Molecular Medicine, 31(4), pp.797-802.

[0357] Other expression regulatory sequences The vector of the present invention may further include one or more regulatory sequences that can act pre-transcriptionally or post-transcriptionally. Preferably, the transgene is operably linked to one or more regulatory elements that can act pre-transcriptionally or post-transcriptionally. One or more regulatory elements can promote the expression of the transgene in phagocytic cells.

[0358] A "regulatory element" is any nucleotide sequence that promotes polypeptide expression, for example, by increasing transcript expression or enhancing mRNA stability.

[0359] Suitable regulatory sequences include, for example, promoters, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites.

[0360] Post-transfer regulatory elements The vector of the present invention may include one or more post-transcriptional regulatory elements. Preferably, the transgene is operably linked to one or more post-transcriptional regulatory elements. The post-transcriptional regulatory elements can improve gene expression.

[0361] The vector of the present invention may contain a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Preferably, the transgene is operably ligated to the WPRE.

[0362] In some embodiments of the present invention, the WPRE comprises or consists of a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 35. Preferably, the WPRE comprises or consists of a nucleotide sequence or fragment thereof 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: 35.

[0363] In some embodiments of the present invention, WPRE comprises or consists of the nucleotide sequence of SEQ ID NO: 35 or a fragment thereof.

[0364] Exemplary SalI-WPRE GTCGACCCGACAGTTTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTC CTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGAGCGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCC TCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGAC GTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACC (Sequence ID 35)

[0365] destabilized domain The vector of the present invention may include a nucleotide sequence encoding a destabilization domain. Preferably, the transgene is operably linked to the destabilization domain, i.e., in frame with the transgene product, so that when the transgene is translated, a fusion protein containing the destabilization domain fused to the transgene product is produced.

[0366] A destabilization domain (DD) is inherently unstable and represents a fusion protein component that destabilizes other proteins during uptake, leading to proteolysis. A well-known example of a DD is the Shield system, which incorporates lampamycin-binding protein (FKBP12) into a protein as an embedded destabilization domain to induce proteolysis in cells. 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).

[0367] Another exemplary destabilizing domain is dihydrofolate reductase (DHFR) or its variants. In mammalian cells, fusion proteins containing the DHFR protein are rapidly ubiquitinated and degraded by the proteasome system. The antibiotic trimethoprim (TMP) or TMP-derived small molecules can bind to the DHFR protein, preventing its degradation and thus allowing the fusion protein to escape degradation (Peng, H., et al., 2019. Molecular Therapy-Methods & Clinical Development, 15, pp.27-39).

[0368] The vector of the present invention may include a dihydrofolate reductase coding sequence, or a variant or derivative thereof. Preferably, the transgene is operably linked to the dihydrofolate reductase coding sequence (or a variant or derivative thereof), i.e., linked in-frame with the transgene product, so that when the transgene is translated, a fusion protein containing the dihydrofolate reductase coding sequence (or a variant or derivative thereof) fused to the transgene product is produced.

[0369] Polyadenylated sequence The vector of the present invention may include a polyadenylated sequence. Preferably, the transgene is operably linked to the polyadenylated sequence. To improve the expression of the transgene, the polyadenylated sequence can be inserted after the transgene.

[0370] A polyadenylated sequence typically includes 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 the cleavage site where a poly-A tail is attached to the mRNA; and the downstream element is a GT-rich region, usually located immediately downstream of the polyadenylation site, which is important for efficient processing.

[0371] Kozak Array The vector of the present invention may include a Kosak sequence. Preferably, the transgene is operably ligated to the Kosak sequence. To improve translation initiation, the Kosak sequence may be inserted before the start codon.

[0372] In some embodiments of the present invention, the Kozak sequence includes or consists of a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 33. Preferably, the Kozak sequence includes or consists of a nucleotide sequence or fragment thereof 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: 33.

[0373] In some embodiments of the present invention, the Kozak sequence comprises or consists of the nucleotide sequence of sequence number 33 or a fragment thereof.

[0374] An example of BamHI-Kosak GGATCCGCCACC (Sequence ID 33)

[0375] Transgene The vector of the present invention may contain one or more transgenes. Preferably, one or more expression regulatory sequences are operably ligated to the transgenes.

[0376] The introduced gene is not particularly limited, and any suitable introduced gene can be used.

[0377] The introduced gene may encode a naturally occurring human gene, or a variant thereof, or / or a fragment thereof.

[0378] The introduced gene may be a therapeutic introduced gene.

[0379] The transgene may encode a therapeutic polypeptide and / or an antigenic polypeptide.

[0380] In some embodiments, the transgene includes a nucleotide sequence encoding a signal peptide, preferably the signal peptide being operably linked to an encoded polypeptide (e.g., a therapeutic polypeptide and / or an antigenic polypeptide). The signal peptide may be, for example, a native signal peptide of the encoded polypeptide. In some embodiments, the transgene does not include a nucleotide sequence encoding a signal peptide.

[0381] Therapeutic polypeptides Preferably, the transgene encodes a therapeutic polypeptide.

[0382] As used herein, “therapeutic polypeptide” is any polypeptide that can be used for therapeutic purposes. For example, a therapeutic polypeptide may include a therapeutic cytokine that can activate an immune response.

[0383] In some embodiments, the transgene encodes cytokines, such as those that can activate immune responses, particularly antitumor responses.

[0384] Cytokines are molecular messengers that enable immune system cells to communicate with each other and generate a coordinated, potent, but self-limiting response to target antigens. Cytokines directly stimulate immune effector cells and stromal cells at tumor sites and enhance tumor cell recognition by cytotoxic effector cells. Cytokines can possess broad antitumor activity (Lee, S. and Margolin, K., 2011. Cancers, 3(4), pp.3856-3893).

[0385] For example, any cytokine capable of activating an immune response, particularly an antitumor response, can be used. Exemplary cytokines include IFNα, IFNβ, IFNγ, IL-2, IL-12, TNFα, CXCL9, and IL-1β. Further exemplary cytokines include IL10, IL15, or IL18. Further exemplary cytokines include GMCSF, FLT3, IL7, or IL21.

[0386] Variants of any of these cytokines can be used, provided that the variants retain 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 variants may have at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity with any of the cytokines.

[0387] Any fragment of these cytokines (or their variants) can 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 access such fragments using methods known in the art. For example, the fragment may retain residues or domains necessary to activate the immune response.

[0388] In some embodiments, the transgene encodes a cytokine selected from IFNα, IFNβ, IFNγ, IL-2, IL-12, TNFα, CXCL9, and IL-1β, or their variants and / or fragments. In some embodiments, the transgene encodes a cytokine selected from IL10, IL15, or IL18, or their variants and / or fragments. In some embodiments, the transgene encodes a cytokine selected from GMCSF, FLT3, IL7, or IL21.

[0389] interferon There are three main types of interferon (IFN). The human type I IFN gene encodes a family of 17 different proteins (including 13 subtypes of IFNα, as well as IFNβ, IFNε, IFNκ, and IFNω). Only a single type II IFN, IFNγ, exists. Type III IFN consists of IFNλ1, IFNλ2, IFNλ3, and IFNλ4.

[0390] All IFNs have the potential to exert direct antitumor effects by acting on tumor cells, or indirect antitumor effects by acting on immune cells (Parker, BS, et al., 2016. Nature Reviews Cancer, 16(3), p.131).

[0391] In some embodiments, the transgene encodes an 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).

[0392] IFNα Interferon-alpha (IFNα), a type 1 interferon, is a pleomorphic cytokine that plays a crucial role in the defense of organisms against viral infections. It is well-established that IFNα can exert antitumor functions, including direct tumor cell death, activation of adaptive and innate immune functions, and anti-angiogenic activity. IFNα is 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, and therefore, alternative treatment options are currently preferred.

[0393] The vector of the present invention may reduce systemic toxicity associated with IFNα delivery by selectively delivering therapeutic IFNα to tumors, along with the expected target cells as phagocytic cells where physiological turnover can accelerate the natural loss of the vector.

[0394] In some embodiments, the transgene encodes IFNα. An exemplary human interferon-alpha (IFNα) for use in this invention is UniProtKB P01562.

[0395] In some embodiments of the present invention, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 8. Preferably, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 8.

[0396] In some embodiments of the present invention, the introduced gene encodes a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or a fragment thereof.

[0397] Exemplary human interferon α: MASPFALLMVLVVLSCKSSCSLGCDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE (Sequence 8)

[0398] In some embodiments of the present invention, the transgene contains or consists of a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 34. Preferably, the transgene contains or consists of a nucleotide sequence or fragment thereof 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: 34.

[0399] In some embodiments of the present invention, the introduced gene includes or consists of the nucleotide sequence of SEQ ID NO: 34 or a fragment thereof.

[0400] Exemplary human IFNA-transformed genes: ATGGCCTCGCCCTTTGCTTTACTGATGGTCCTGGTGGTGCTCAGCTGCAAGTCAAGCTGCTCTCTGGGCTGTGATCTCCCTGAGACCCACAGCCTGGATAACAGGAGGACCTTGATGCTCCTGGCACAAATGAGCAGAATCT CTCCTTCCTCCTGTCTGATGGACAGACATGACTTTGGATTTCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCCAGCCATCTCTGTCCTCCATGAGCTGATCCAGCAGATCTTCAACCTCTTTACCACAAAAGAT TCATCTGCTGCTTGGGATGAGGACCTCCTAGACAAATTCTGCACCGAACTCTACCAGCAGCTGAATGACTTGGAAGCCTGTGTGATGCAGGAGGAGAGGGTGGGAGAAACTCCCCTGATGAATGCGGACTCCATCTTGGCTG TGAAGAAATACTTCCGAAGAATCACTCTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCCCTCTCTTTATCAACAAACTTGCAAGAAAGATTAAGGAGGAAGGAATAA (Sequence ID 34)

[0401] IFNβ In some embodiments, the transgene encodes IFNβ. An exemplary human IFNβ for use in this invention is UniProtKB P01574.

[0402] In some embodiments of the present invention, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 9. Preferably, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 9.

[0403] In some embodiments of the present invention, the introduced gene encodes a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or a fragment thereof.

[0404] Exemplary human interferon β: MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN (Sequence ID 9)

[0405] IFNγ In some embodiments, the transgene encodes IFNγ. An exemplary human IFNγ for use in this invention is UniProtKB P01579.

[0406] In some embodiments of the present invention, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 10. Preferably, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 10.

[0407] In some embodiments of the present invention, the introduced gene encodes a polypeptide comprising or consisting of the polypeptide sequence or a fragment thereof of SEQ ID NO: 10.

[0408] Exemplary human interferon-γ: MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHEL IQVMAELSPAAKTGKRKRSQMLFRGRRASQ (Sequence ID 10)

[0409] Other cytokines IL-2 Interleukin-2 (IL-2), and other members of the IL-2-related family of T cell growth factors (e.g., IL-4, IL-7, IL-9, IL-15, and IL-21), utilize a common receptor signaling pathway that leads to the activation and proliferation of CD4+ and CD8+ T cells (Lee, S. and Margolin, K., 2011. Cancers, 3(4), pp.3856-3893).

[0410] 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 for use in the present invention is UniProtKB P60568.

[0411] In some embodiments of the present invention, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 11. Preferably, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 11.

[0412] In some embodiments of the present invention, the introduced gene encodes a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 11 or a fragment thereof.

[0413] Exemplary human interleukin-2: MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVE FLNRWITFCQSIISTLT (Sequence ID 11)

[0414] IL-12 In some embodiments, the transgene encodes IL-12. Exemplary human IL-12α and β subunits for use in the present invention are UniProtKB P29459 and P29460.

[0415] In some embodiments of the present invention, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 12. Preferably, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 12.

[0416] In some embodiments of the present invention, the introduced gene encodes a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 12 or a fragment thereof.

[0417] Exemplary human interleukin-12 subunit α: MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRI RAVTIDRVMSYLNAS (Sequence ID 12)

[0418] In some embodiments of the present invention, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 13. Preferably, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 13.

[0419] In some embodiments of the present invention, the introduced gene encodes a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or a fragment thereof.

[0420] Exemplary human interleukin-12 subunit β: MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSD PQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (Sequence ID 13)

[0421] In some embodiments, the transgene encodes single-chain IL12. Single-chain IL12 may include IL12 subunit β (e.g., 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 IL12 subunit α (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). Single-chain IL12 may also be a fusion protein containing IL12 subunit β and IL12 subunit α. IL12 subunit β and IL12 subunit α can be linked by a linker sequence. The linker sequence may include 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.

[0422] RRAGGGGSGGGGSGGGGSRT (Sequence ID 42)

[0423] In some embodiments, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 37 or 46. Preferably, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 37 or 46.

[0424] In some embodiments of the present invention, the introduced gene encodes a polypeptide comprising or consisting of the amino acid sequence or a fragment thereof of SEQ ID NO: 37 or 46.

[0425] Exemplary single-chain human interleukin-12 sequences: MCPQKLTISWFAIVLLVSPLMAIAGQLMWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCE AKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYF SLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSRAGGGGSGGGGSGGGGSRTRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTST VEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (Sequence ID 37)

[0426] WELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLT FSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKR EKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSRAGGGGSGGGGSGGGGSRTRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPL ELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (Sequence No. 46)

[0427] In some embodiments of the present invention, the introduced gene contains or consists of a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 40. Preferably, the introduced gene contains or consists of a nucleotide sequence or fragment thereof 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: 40.

[0428] In some embodiments of the present invention, the introduced gene includes or consists of the nucleotide sequence of SEQ ID NO: 40 or a fragment thereof.

[0429] (Sequence No. 40)

[0430] IL10 In some embodiments, the transgene encodes IL-10.

[0431] In some embodiments of the present invention, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 38. Preferably, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 38.

[0432] In some embodiments of the present invention, the introduced gene encodes a polypeptide comprising or consisting of the amino acid sequence or a fragment thereof of SEQ ID NO: 38.

[0433] Exemplary human interleukin-10: MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (Sequence No. 38)

[0434] In some embodiments of the present invention, the transgene contains or consists of a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 39. Preferably, the transgene contains or consists of a nucleotide sequence or fragment thereof 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: 39.

[0435] In some embodiments of the present invention, the introduced gene contains or consists of the nucleotide sequence of SEQ ID NO: 39 or a fragment thereof.

[0436] ATGCCAGGCTCCGCCCTGCTGTGCTGTCTGCTGCTGCTGACCGGCATGAGGATCAGCAGAGGACAGTACTCCCGGGAGGACAACAATTGCACCCACTTCCCTGTGGGACAGTCCCACATGCTGCTGGAGCTGCG CACAGCTTTTTCTCAGGTGAAGACCTTTCTTTCAGACAAAGGACCAGCTGGATAACATCCTGCTGACCGACAGCCTGATGCAGGATTTCAAGGGCTACCTGGGATGTCAGGCCCTGTCCGAGATGATCCAGTTTT ATCTGGTGGAGGTGATGCCTCAGGCTGAGAAGCACGGCCCCGAGATCAAGGAGCACCTGAATTCTCTGGGAGAGAAGCTGAAGACACTGCGGATGCGCCTGAGGAGATGCCACAGGTTCCTGCCTTGTGAGAAC AAGTCTAAGGCCGTGGAGCAGGTGAAGAGCGACTTTAATAAGCTGCAGGATCAGGGCGTGTACAAGGCCATGAACGAGTTCGATATCTTTATCAATTGCATCGAGGCTTATATGATGATCAAGATGAAGAGCTGA (Sequence ID 39)

[0437] IL15 In some embodiments, the transgene encodes IL-15.

[0438] In some embodiments of the present invention, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 44. Preferably, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 44.

[0439] In some embodiments of the present invention, the introduced gene encodes a polypeptide comprising or consisting of the amino acid sequence or a fragment thereof of SEQ ID NO: 44.

[0440] Exemplary human interleukin-15: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID 44)

[0441] IL18 In some embodiments, the transgene encodes IL-18.

[0442] In some embodiments of the present invention, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 45 or 47. Preferably, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 45 or 47.

[0443] In some embodiments of the present invention, the introduced gene encodes a polypeptide comprising or consisting of the amino acid sequence or a fragment thereof of SEQ ID NO: 45 or 47.

[0444] Exemplary human interleukin-18 sequences: YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (Sequence No. 45)

[0445] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISAYGDSRARGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (Sequence ID 47)

[0446] TNFα In some embodiments, the transgene encodes tumor necrosis factor alpha (TNFα). An exemplary human TNFα for use in the present invention is UniProtKB P01375.

[0447] In some embodiments of the present invention, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 14. Preferably, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 14.

[0448] In some embodiments of the present invention, the introduced gene encodes a polypeptide comprising or consisting of the polypeptide sequence or a fragment thereof of SEQ ID NO: 14.

[0449] Exemplary human TNFα: MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANG VELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (Sequence ID 14)

[0450] CXCL9 In some embodiments, the transgene encodes CXC motif chemokine 9 (CXCL9).

[0451] An exemplary human CXCL9 for use in this invention is UniProtKB Q07325.

[0452] In some embodiments of the present invention, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 15. Preferably, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 15.

[0453] In some embodiments of the present invention, the introduced gene encodes a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 15 or a fragment thereof.

[0454] Exemplary human CXCL9: MKKSGVLFLLGIILLVLIGVQGTPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNPDSADVKELIKKWEKQVSQKKKQKNGKKHQKKKVLKVRKSQRSRQKKTT (Sequence ID 15)

[0455] IL-1β In some embodiments, the transgene encodes interleukin-1β (IL-1β). An exemplary human IL-1β for use in the present invention is UniProtKB P01584.

[0456] In some embodiments of the present invention, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 16. Preferably, the transgene encodes a polypeptide comprising, or consisting of, an amino acid sequence or fragment thereof that is at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 16.

[0457] In some embodiments of the present invention, the introduced gene encodes a polypeptide comprising or consisting of the polypeptide sequence or a fragment thereof of SEQ ID NO: 16.

[0458] Exemplary human IL-1β: MAEVPELASEMMAYYSGNEDDLFFEADGPKQMKCSFQDLDLCPLDGGIQLRISDHHYSKGFRQAASVVVAMDKLRKMLVPCPQTFQENDLSTFFPFIFEEEPIFFDTWDNEAYVHDAPVRSLNCTLRDSQQKSL VMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSS (Sequence ID 16)

[0459] Antigenic polypeptide Preferably, the transgene encodes an antigenic polypeptide.

[0460] As used herein, “antigenic polypeptide” is any polypeptide capable of inducing an immune response. In particular, antigenic polypeptides can be internalized and presented by antigen-presenting cells (APCs). Antigen presentation enables the specificity of adaptive immunity and can contribute to immune responses against both intracellular and extracellular pathogens. APCs also naturally play a role in fighting tumors by stimulating B cells and cytotoxic T cells, respectively, to produce antibodies against tumor-associated antigens and kill malignant cells.

[0461] Antigens can be patient-specific.

[0462] Tumor antigen In some embodiments, the transgene encodes a tumor antigen, such as a tumor-specific antigen or a tumor-associated antigen.

[0463] As used herein, “tumor antigen” refers to an antigenic substance (e.g., 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 and also on some normal cells.

[0464] Any suitable tumor antigen can be used. Suitable tumor antigens are well known to those skilled in the art, and for example, tumor antigens are recorded in the Cancer Antigenic Peptide Database.

[0465] 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.

[0466] Certain tumors are rich in specific tumor antigens. Therefore, these specific tumor antigens can be used as tumor markers and also as tumor antigen vaccines in cancer treatment.

[0467] Similar to vaccines against pathogens, oncology vaccines involve the delivery of inactivated cancer cells or tumor antigens (TAs) in combination with adjuvants. Oncology vaccines also include DCs challenged in vitro with TAs. Despite several years of experimentation, oncology vaccines have yielded mostly disappointing results, with only one approved for clinical use. Identifying novel vaccine delivery systems that circumvent barriers to effective cancer vaccines should enable their therapeutic applicability.

[0468] The vectors of the present invention may represent an effective strategy for designing tumor vaccines.

[0469] In some embodiments, the transgene encodes a tumor antigen abundant in liver metastases.

[0470] In some embodiments, the transgene encodes a tumor antigen selected from carcinoembryonic antigen (CEA), TRP2, melanoma-associated antigen (MAGE) family, cancer germline (CAGE) family, melanoma B antigen (BAGE-1), synovial sarcoma X breakpoint 20 (SSX-2), sarcoma antigen (SAGE) family, LAGE1, NY-ESO-1, HER2, EGFR, MUC-1, and GAST.

[0471] In some embodiments, the transgene encodes the OVA.

[0472] In some embodiments of the present invention, the introduced gene contains or consists of a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 41 or 43. Preferably, the introduced gene contains or consists of a nucleotide sequence or fragment thereof 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: 41 or 43.

[0473] In some embodiments of the present invention, the introduced gene contains or consists of the nucleotide sequence of SEQ ID NO: 41 or 43 or a fragment thereof.

[0474] In some embodiments, the transgene encodes TRP2.

[0475] In some embodiments of the present invention, the introduced gene contains or consists of a nucleotide sequence or fragment thereof that is at least 70% identical to SEQ ID NO: 48 or 49. Preferably, the introduced gene contains or consists of a nucleotide sequence or fragment thereof 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: 48 or 49.

[0476] In some embodiments of the present invention, the introduced gene includes or consists of the nucleotide sequence of SEQ ID NO: 48 or 49 or a fragment thereof.

[0477] This invention envisions the combined use of the cytokine gene therapy of the present invention and the tumor vaccine of the present invention.

[0478] In another embodiment, the present invention provides an product (e.g., a composition or kit) comprising 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.

[0479] In another embodiment, the present invention provides an product (e.g., a composition or kit) comprising a cell comprising 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.

[0480] In another embodiment, the present invention provides an product (e.g., a composition or kit) comprising a first vector of the present invention comprising a transgene encoding a cytokine (preferably IL12), and cells comprising a second vector of the present invention comprising a transgene encoding a tumor antigen.

[0481] In another embodiment, the present invention provides an product (e.g., a composition or kit) comprising a first cell comprising a first vector of the present invention comprising a transgene encoding a cytokine (preferably IL12), and a second cell comprising a second vector of the present invention comprising a transgene encoding a tumor antigen.

[0482] The composition may be a pharmaceutical composition disclosed herein.

[0483] In another embodiment, the present invention provides a first vector comprising a transgene encoding a cytokine (preferably IL12) for therapeutic use, the first vector being administered to a subject simultaneously, sequentially, or separately in combination with a second vector comprising a transgene encoding a tumor antigen.

[0484] In another embodiment, the present invention provides a second vector comprising a transgene encoding a tumor antigen for therapeutic use, the second vector being administered to a subject simultaneously, sequentially, or separately in combination with a first vector comprising a transgene encoding a cytokine (preferably IL12).

[0485] In another embodiment, the present invention provides the use of a first vector of the present invention comprising a transgene encoding a cytokine (preferably IL12) for the manufacture of a pharmaceutical product, wherein the first vector is administered to a subject simultaneously, sequentially, or separately in combination with a second vector of the present invention comprising a transgene encoding a tumor antigen.

[0486] In another embodiment, the present invention provides the use of a second vector of the present invention comprising a transgene encoding a tumor antigen for the manufacture of a pharmaceutical product, the second vector being administered to a subject simultaneously, sequentially, or separately in combination with a first vector of the present invention comprising a transgene encoding a cytokine (preferably IL12).

[0487] In a preferred embodiment, the therapeutic use is for the treatment or prevention of cancer.

[0488] In another embodiment, the present invention provides a method for treating or preventing cancer, comprising administering a first vector of the present invention, which includes a transgene encoding a cytokine (preferably IL-12), and a second vector of the present invention, which includes a transgene encoding a tumor antigen, to a subject requiring such treatment. The first and second vectors may be administered, for example, simultaneously, sequentially, or separately.

[0489] In some embodiments, the first vector and / or the second vector are administered by intravenous injection, portal vein injection, or hepatic artery injection.

[0490] In another embodiment, the present invention provides cells comprising a first vector of the present invention comprising a transgene encoding a cytokine (preferably IL12) and / or a second vector of the present invention comprising a transgene encoding a tumor antigen.

[0491] Exemplary vector In a preferred embodiment, the vector comprises, from 5' to 3', an MRC1 promoter, a transgene, and one or more miRNA target sequences as defined herein. In another preferred embodiment, the vector comprises, from 5' to 3', an MRC1 enhancer, an MRC1 promoter, a transgene, and one or more miRNA target sequences as defined herein.

[0492] In some embodiments, the vector comprises, from 5' to 3', an MRC1 promoter, a transgene encoding IFNα, and one or more miRNA target sequences that suppress transgene expression in hepatocytes and / or hepatic sinusoidal endothelial cells and / or splenic phagocytes.

[0493] In some embodiments, the vector comprises, from 5' to 3', an MRC1 enhancer, an MRC1 promoter, a transgene encoding IFNα, and one or more miRNA target sequences that suppress transgene expression in hepatocytes and / or hepatic sinusoidal endothelial cells and / or splenic phagocytes.

[0494] In some embodiments, the vector comprises, from 5' to 3', an MRC1 enhancer, an MRC1 promoter, a Kozak sequence, a transgene encoding IFNα, a WPRE, and one or more miRNA target sequences that suppress transgene expression in hepatocytes and / or hepatic sinusoidal endothelial cells and / or splenic phagocytes.

[0495] In some embodiments, the vector includes, from 5' to 3', 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.

[0496] In some embodiments, the vector includes, from 5' to 3', 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.

[0497] In some embodiments, the vector includes, from 5' to 3', 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.

[0498] In some embodiments, the vector comprises, from 5' to 3', an MRC1 promoter, a transgene encoding IL10, and one or more miRNA target sequences that suppress transgene expression in hepatocytes and / or hepatic sinusoidal endothelial cells and / or splenic phagocytes.

[0499] In some embodiments, the vector comprises, from 5' to 3', an MRC1 enhancer, an MRC1 promoter, a transgene encoding IL10, and one or more miRNA target sequences that suppress transgene expression in hepatocytes and / or hepatic sinusoidal endothelial cells and / or splenic phagocytes.

[0500] In some embodiments, the vector comprises, from 5' to 3', an MRC1 enhancer, an MRC1 promoter, a Kozak sequence, a transgene encoding IL10, a WPRE, and one or more miRNA target sequences that suppress transgene expression in hepatocytes and / or hepatic sinusoidal endothelial cells and / or splenic phagocytes.

[0501] In some embodiments, the vector includes, from 5' to 3', 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.

[0502] In some embodiments, the vector includes, from 5' to 3', 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.

[0503] In some embodiments, the vector includes, from 5' to 3', 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.

[0504] In some embodiments, the vector comprises, from 5' to 3', an MRC1 promoter, a transgene encoding IL12, and one or more miRNA target sequences that suppress transgene expression in hepatocytes and / or hepatic sinusoidal endothelial cells and / or splenic phagocytes.

[0505] In some embodiments, the vector comprises, from 5' to 3', an MRC1 enhancer, an MRC1 promoter, a transgene encoding IL12, and one or more miRNA target sequences that suppress transgene expression in hepatocytes and / or hepatic sinusoidal endothelial cells and / or splenic phagocytes.

[0506] In some embodiments, the vector comprises, from 5' to 3', an MRC1 enhancer, an MRC1 promoter, a Kozak sequence, a transgene encoding IL12, a WPRE, and one or more miRNA target sequences that suppress transgene expression in hepatocytes and / or hepatic sinusoidal endothelial cells and / or splenic phagocytes.

[0507] In some embodiments, the vector includes, from 5' to 3', 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.

[0508] In some embodiments, the vector includes, from 5' to 3', 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.

[0509] In some embodiments, the vector includes, from 5' to 3', 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.

[0510] Immune checkpoint inhibitors As used herein, the term "immune checkpoint inhibitor" refers to a molecule, compound, antibody, or drug that inhibits, blocks, prevents, reduces, or downmodulates the expression of an inhibitory checkpoint molecule, or is otherwise antagonistic to an inhibitory checkpoint molecule. When expressed on a cell surface, inhibitory checkpoint molecules inhibit or weaken the T cell-mediated immune response against that cell. For example, the expression of an inhibitory checkpoint molecule may prevent cells from being killed by the T cell response. This mechanism is particularly harmful when cancer cells express inhibitory checkpoint molecules, as this can allow cancer cells to evade the host T cell response. Therefore, if inhibitory checkpoint molecules on tumor cells are inhibited by an immune checkpoint inhibitor, an enhancement of the host T cell response against the tumor cells should occur.

[0511] In some embodiments, the immune checkpoint inhibitor is 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 cell death 1 receptor), PD-L1 (PD-1 ligand 1), PD-L2 (PD-1 ligand 2), TIM-3 (T cell immunoglobulin domain The inhibitory checkpoint molecules selected from the group consisting of mucin domains3), VISTA (V domain Ig inhibitor of T cell activation), B7-1 (CD80), B7-2 (CD86), TGFB (transforming growth factor β) pathway-related proteins, 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-related protein 2 (PVRL2)), LGALS3 (galectin 3), and CD47 (integrin-related protein) are also inhibited. Combinations of checkpoint inhibitors may also be used.

[0512] In some embodiments, the TGFB pathway-related proteins are selected from the group consisting of TGFB1 (transforming growth factor β1), TGFB2 (transforming growth factor β2), TGFB3 (transforming growth factor β3), LTBP1 (latent 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-containing 32).

[0513] 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 anti-CTLA4 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-PDL2 antibody, and anti-LAG-3 antibody.

[0514] In some embodiments, the immune checkpoint inhibitor is a CTLA4 inhibitor, and preferably, the CTLA4 inhibitor is an anti-CTLA4 antibody.

[0515] In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor, and preferably, the PD-1 inhibitor is an anti-PD-1 antibody.

[0516] In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor, and preferably, the PD-L1 inhibitor is an anti-PD-L1 antibody.

[0517] In some embodiments, the immune checkpoint inhibitor is a PD-L2 inhibitor, and preferably, the PD-L2 inhibitor is an anti-PD-L2 antibody.

[0518] In some embodiments, the immune checkpoint inhibitor is a LAG-3 inhibitor, and preferably, the LAG-3 inhibitor is an anti-LAG-3 antibody.

[0519] As used herein, the term “antibody” is understood to mean a polypeptide substantially encoded by an immunoglobulin gene or a plurality of immunoglobulin genes or fragments thereof that specifically binds to and recognizes an antigen (e.g., a cell surface marker). As used herein, the term “antibody” refers to a complete or intact antibody molecule (e.g., IgM, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA, IgD, or IgE) or any antigen-binding fragment thereof.

[0520] Antibodies can be polyclonal or monoclonal antibodies. Monoclonal antibodies are produced by identical immune cells (for example, hybridomas that can be generated from the fusion of antibody-producing B cell lines and cancerous B cell lines). Monoclonal antibodies directed at a specific antigen recognize a single specific epitope on that antigen. In contrast, polyclonal antibodies are produced from multiple non-identical cell lines and therefore recognize several different epitopes on a particular antigen.

[0521] Antibody antigen-binding fragments include, for example, single-chain antibodies, single-chain Fv fragments (scFv), Fd fragments, Fab fragments, Fab' fragments, or F(ab')2 fragments. The scFv fragment is a single polypeptide chain containing both the heavy chain variable region and the light chain variable region of the antibody from which the scFv is derived. Furthermore, intrabodies, minibodies, triabodies, and diabodies (see, e.g., Todolvska et al. (2001) J Immunol Methods 248(1):47-66; Hudson and Kortt (1999) J Immunol Methods 231(1):177-189; Poljak 25 (1994) Structure 2(12):1121-1123; Rondon and Marasco (1997) Annual Review of Microbiology 21:257-283) are also included in the definition of an antibody and are suitable for use in the methods described herein. As used herein, the term antibody also includes antibody fragments produced by modification of the entire antibody, or antibody fragments newly synthesized using recombinant methods.

[0522] Preferred methods for producing antibodies or antigen-binding fragments thereof directed towards a specific antigen are known in the art (see, for example, Greenfield (2014) Antibodies: A Laboratory Manual, Second Edition 201-221).

[0523] TR1 cell inhibitor 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 modulate resistance and can be autoantigen-specific or non-autoantigen-specific. An important natural role of Tr1 cells is to suppress tissue inflammation in autoimmunity and graft-versus-host disease.

[0524] In some embodiments, the Tr1 cell inhibitor inhibits molecules selected from the group consisting of Cd4, Eomes, Gzmk, Lag3, Pdcd1, Ahr, Maf, Prdm1, Ctla4, and Il10ra.

[0525] In some embodiments, the Tr1 cell inhibitor is an antibody.

[0526] combination As used herein, the terms “combination,” “in combination,” “used in combination,” or “combination preparation” may refer to the simultaneous, sequential, or separate combination administration of two or more drugs.

[0527] As used herein, the term “simultaneous” means that the drugs are administered concurrently, i.e., at the same time.

[0528] As used herein, the term “sequential” means that the drugs are administered one after another.

[0529] As used herein, the term “separate” means that the drugs are administered independently of each other, but within a time interval that allows the drugs to exert a combined, preferably synergistic, effect. Therefore, “separate” administration may allow one drug to be administered, for example, within 1 minute, 5 minutes, or 10 minutes of the other.

[0530] Variants, derivatives, analogues, and fragments In addition to the specific proteins and nucleotides referred to herein, the present invention also encompasses their variants, derivatives, and fragments.

[0531] In the context of the present invention, a “variant” of any given sequence is a sequence in which a specific sequence of residues (either amino acid residues or nucleic acid residues) is modified in such a manner that the polypeptide or polynucleotide in question retains at least one of its endogenous functions. Variant sequences can be obtained by adding, deleting, substituting, modifying, exchanging, and / or altering at least one residue present in the naturally occurring polypeptide or polynucleotide.

[0532] As used herein with respect to the protein or polypeptide of the present invention, the term “derivative” includes any substitution, alteration, modification, exchange, deletion, and / or addition of one (or more) amino acid residues to or from a sequence, provided that the resulting protein or polypeptide retains at least one of its endogenous functions.

[0533] Typically, amino acid substitutions may involve, for example, 1, 2, or 3 to 10 or 20 substitutions, provided that the modified sequence retains the desired activity or capability. Amino acid substitutions may include the use of analogues that do not exist in nature.

[0534] The proteins used in this invention may also undergo silent changes, resulting in deletions, insertions, or substitutions of amino acid residues that produce functionally equivalent proteins. Intentional amino acid substitutions may be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues, as long as the 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 with uncharged head groups having similar hydrophilic values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0535] Conservative substitutions can be performed, for example, according to the following table. Amino acids in the same block in the second column and amino acids in the same row in the third column can be substituted for each other:

[0536] [Table 1]

[0537] Typically, variants may have some degree of identity with the wild-type amino acid sequence or wild-type nucleotide sequence.

[0538] In the context of the present invention, a variant sequence is interpreted as containing an amino acid sequence that is at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the target sequence, and preferably at least 95%, 96%, 97%, 98%, or 99% identical. While variants may also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of the present invention, it is preferable to express them in terms of sequence identity.

[0539] In the context of the present invention, a variant sequence is interpreted as containing a nucleotide sequence that is at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the target sequence, and preferably at least 95%, 96%, 97%, 98%, or 99% identical. While variants may also be considered in terms of similarity, in the context of the present invention, they are preferably expressed in terms of sequence identity.

[0540] Preferably, a reference to a sequence having an identity percentage with any one of the sequence numbers detailed herein refers to a sequence having the described identity percentage with respect to the full length of the referenced sequence number.

[0541] Sequence identity comparison can be performed visually or, more commonly, using readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of identity between two or more sequences.

[0542] Identity percentages can be calculated for consecutive sequences; that is, one sequence is aligned with the other, and each amino acid or nucleotide in one sequence is directly compared, one residue at a time, with the corresponding amino acid or nucleotide in the other sequence. This is called a "gapless" alignment. Typically, such gapless alignments are performed only over relatively short number of residues.

[0543] While this is a very simple and consistent method, it fails to consider, for example, that in otherwise identical sequence pairs, a single insertion or deletion in an amino acid or nucleotide sequence can cause subsequent residues or codons to fall out of alignment, potentially resulting in a significant reduction in the identity percentage when a global alignment is performed. As a result, most sequence comparison methods are designed to produce an optimal alignment that takes possible insertions and deletions into account without excessively penalizing the overall identity score. This is achieved by attempting to maximize local identity by inserting "gaps" into the sequence alignment.

[0544] However, these more complex methods assign a "gap penalty" to each gap that occurs during alignment, resulting in sequence alignments with as few gaps as possible, reflecting a higher relevance between the two sequences being compared for the same number of identical amino acids or nucleotides, achieving a higher score than those with many gaps. A typical "affine gap cost" is used, which imposes a relatively high cost for the presence of gaps and a smaller penalty for each residue following a gap. This is the most commonly used gap scoring system. A high gap penalty naturally results in an optimized alignment with fewer gaps. Most alignment programs allow you to change the gap penalty. However, when using such software for sequence comparison, it is preferable to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for gaps and -4 for each extension.

[0545] Therefore, calculating the maximum identity percentage first requires creating an optimal alignment, taking gap penalties into account. A suitable computer program for performing such alignments is the GCG Wisconsin Bestfit package (University of Wisconsin, USA, Develeux et al. (1984) Nucleic Acids Research 12:387). Other software capable of performing sequence comparisons includes, but is not limited to, the BLAST package (Ausubel et al. (1999) ibid-Ch.18), FASTA (Atschul et al. (1990) J.Mol.Biol.403-410), EMBOSS Needle (Madeira, F., et al., 2019. Nucleic Acids Research, 47(W1), pp.W636-W641) and the GENEWORKS comparison toolset. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferable to use the GCG Bestfit program. Another tool, BLAST 2 Sequences, is also available for comparing protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174(2):247-50, FEMS Microbiol. Lett. (1999) 177(1):187-8).

[0546] While final 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 ​​commonly used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is ​​the BLOSUM62 matrix (the default matrix for the BLAST program suite). The GCG Wisconsin program generally uses either publicly available default values ​​or custom symbol comparison tables, if provided (see the user manual for further details). For some applications, it is preferable to use the publicly available default values ​​for the GCG package, or, in the case of other software, a default matrix such as BLOSUM62.

[0547] Once the software creates the optimal alignment, it becomes possible to calculate the sequence identity percentage. The software typically does this as part of the sequence comparison and generates a numerical result. The sequence identity percentage can be calculated as the number of identical residues as a percentage of the total residues in the sequence number being referred to.

[0548] A "fragment" is also a variant, and this term typically refers to a selected region of a polypeptide or polynucleotide that is functionally or, for example, in an assay, of interest. Therefore, a "fragment" refers to an amino acid sequence or nucleic acid sequence that is part of a full-length polypeptide or polynucleotide.

[0549] Such variants, derivatives, and fragments can be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. If an insertion is performed, synthetic DNA encoding the insertion can be prepared along with 5' and 3' flanking regions corresponding to the naturally occurring sequence on either side of the insertion site. The flanking regions contain convenient restriction sites corresponding to locations in the naturally occurring sequence, and as a result, the sequence can be cleaved with a suitable enzyme(s), and the synthetic DNA can be ligated to the cleavage. The DNA is then expressed according to the present invention, producing the encoded protein. These methods are merely examples of the many standard techniques known in the art for manipulating DNA sequences, and other known techniques may also be used.

[0550] cell In one embodiment, the present invention provides cells containing the product of the present invention.

[0551] In some embodiments of the products of the present invention, the vector(s) are contained within cells.

[0552] In one embodiment, the present invention provides cells containing the vector of the present invention. The cells may be isolated cells. The cells may be human cells, preferably isolated human cells. The cells may be any cell type known in the art.

[0553] The cells may contain the first vector and / or the second vector (and / or, optionally, a third vector) of the present invention.

[0554] Methods for producing cells The vectors of the present invention can be introduced into cells using various techniques known in the art, such as transfection, transduction, and transformation. Preferably, the vectors of the present invention are introduced into cells by transfection or transduction.

[0555] In one embodiment, the present invention provides a method for producing the cells of the present invention. This method may include, for example, introducing the vector of the present invention into cells by transfection or transduction.

[0556] Preferably, the cells may be derived from a sample isolated from the subject (e.g., peripheral blood, bone marrow, or umbilical cord blood). The cells may be further isolated from the sample by any preferred method.

[0557] The cells of the present invention can be produced by a method comprising the following steps: (i) Isolation of cell-containing samples from the subject or provision of cell-containing samples, (ii) Transfection or introduction of a cell-containing sample using the vector of the present invention is performed to provide a population of manipulated cells.

[0558] Cells can be cultured before or after the introduction of the vector of the present invention. The process can be carried out in a closed, sterile cell culture system.

[0559] Hematopoietic stem cells / hematopoietic progenitor cells and differentiated cells Preferably, the cells may be hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs) (e.g., bone marrow / monocyte-associated progenitor cells), or differentiated cells (e.g., macrophages or monocytes). Preferably, the cells may be autologous and / or allogeneic to the subject.

[0560] Hematopoietic stem cells (HSCs) are pluripotent stem cells that can be found, for example, in peripheral blood, bone marrow, and umbilical cord blood. HSCs are capable of self-renewal and differentiation into any blood cell lineage. They can re-establish erythrocyte and myeloid lineages in the entire immune system, as well as in all hematopoietic tissues (e.g., bone marrow, spleen, and thymus). They provide lifelong production of all hematopoietic cell lineages.

[0561] Hematopoietic progenitor cells (HPCs) have the ability to differentiate into specific types of cells. However, in contrast to stem cells, they are already far more specific and 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.

[0562] Differentiated cells are more specialized than stem cells or progenitor cells. Differentiated cells include hematopoietic lineage differentiated cells such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells, T cells, B cells, and NK cells. For example, hematopoietic lineage differentiated cells can be distinguished from undifferentiated cells (HSCs and HPCs) by detecting cell surface molecules that are not expressed or are expressed at lower levels on HSCs and HPCs. Examples of suitable human lineage markers include CD33, CD13, CD14, CD15 (bone marrow), CD19, CD20, CD22, CD79a (B), CD36, CD71, CD235a (erythrocytes), CD2, CD3, CD4, CD8 (T), and CD56 (NK).

[0563] The cells of the present invention may be used for adoptive cell transfer. As used herein, the term “adoptive cell transfer” refers to the administration of a cell population to a patient. The cells may be isolated from the subject, and the vector of the present invention may be introduced by the method described herein before the cells are administered to the patient.

[0564] Adoptive cell transfer can be allogeneic or autologous. “Autologous cell transfer” should be understood as the cell starting population being obtained from the same subject to which the transduced cell population is administered. Autologous transfer is advantageous because it avoids problems associated with immunological incompatibility and is available to the subject regardless of the availability of genetically compatible donors. “Allogeneic cell transfer” should be understood as the cell starting population being obtained from a different subject to which the transduced cell population is administered. By choice, the donor may be genetically compatible with the subject to which the cells are administered to minimize the risk of immunological incompatibility. Alternatively, the donor may be a mismatch and irrelevant to the patient. A suitable dose of the transduced cell population is one that is therapeutically and / or prophylactically effective. The dose administered may depend on the subject and condition being treated and can be readily determined by those skilled in the art.

[0565] Producer cells and packaging cells Preferably, the cells may be producer cells. The term "producer cells" includes cells that produce viral particles after transient transfection, stable transfection, or vector transduction of all elements necessary for producing viral particles, or any cells that have been manipulated to stably contain the elements necessary for producing viral particles. Suitable producer cells are well known to those skilled in the art. Suitable producer cell lines include HEK293 (e.g., HEK293T), HeLa, and A549 cell lines.

[0566] Preferably, the cells may be packaging cells. The term “packaging cells” includes cells that contain some or all of the elements necessary for packaging infectious recombinant viruses. Packaging cells may lack a 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 necessary for the production of enveloped viral particles are useful as intermediate reagents in the generation of viral particle producer cell lines through subsequent steps of transient transfection, transduction, or stable incorporation of each additional required element. These intermediate reagents are encompassed within the term “packaging cells.” Suitable packaging cells are well known to those skilled in the art.

[0567] In some embodiments, cells are genetically engineered to reduce the expression of CD47 and / or HLA on the cell surface. In some embodiments, cells include genetically engineered disruptions of the gene encoding CD47 and / or the gene encoding β2-microglobulin and / or one or more genes encoding the MHC-Iα chain. Cells may include genetically engineered disruptions of all copies of the gene encoding CD47. The expression of CD47 and / or HLA on the cell surface may be reduced such that the cell substantially lacks CD47 and / or HLA molecules exposed on its surface. In some embodiments, cells do not contain any CD47 and / or HLA molecules exposed on their surface.

[0568] In one embodiment, the present invention provides a method for producing viral vector particles of the present invention. The method may include the step of culturing viral particle producer cells or packaging cells containing the vector of the present invention under conditions suitable for the production of viral particles. The method may include (a) introducing the vector of the present invention into viral particle producer cells or packaging cells, for example, by transfection or transduction, and (b) culturing the cells under conditions suitable for the production of viral particles. Such conditions are well known to those skilled in the art.

[0569] Pharmaceutical composition A pharmaceutical composition is a composition containing or comprising a therapeutically effective amount of a pharmaceutically active agent. It preferably contains a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof).

[0570] In some embodiments, the pharmaceutical composition is a cancer vaccine. A "cancer vaccine" is a vaccine that treats existing cancer or prevents the development of cancer.

[0571] "Pharmacologically acceptable" includes the fact that the formulation is sterile and free of pyrogens. The carrier, diluent, and / or excipient must be "acceptable" in the sense that they are compatible with the vector and not harmful to its recipient. Typically, the carrier, diluent, and excipient are sterile, pyrogen-free saline or infusion medium, but other acceptable carriers, diluents, and excipients may be used.

[0572] Acceptable carriers, diluents, and excipients for therapeutic use are well known in the field of pharmacy. The choice of pharmaceutical carrier, excipient, or diluent can be made in relation to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may contain, or in addition to, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), or solubilizer(s) as carriers, excipients, or diluents.

[0573] Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, wax, petrolatum, vegetable oil, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, fragrance oils, fatty acid monoglycerides and fatty acid diglycerides, petroleum ether fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0574] Products, vectors, inhibitors, cells, or pharmaceutical compositions according to the present invention may be administered in an appropriate manner to treat and / or prevent the diseases described herein. The dosage and frequency of administration will be determined by factors such as the condition of the subject and the type and severity of the disease, but the appropriate dosage may be determined by clinical trials. Pharmaceutical compositions may be formulated accordingly.

[0575] The products, vectors, inhibitors, cells, or pharmaceutical compositions according to the present invention may be administered parenterally, for example, intravenously or by infusion techniques. The products, vectors, inhibitors, cells, or pharmaceutical compositions may be administered in the form of a sterile aqueous solution that may contain other substances, such as salts or glucose sufficient to make the solution isotonic with blood. The aqueous solution may be preferably buffered (preferably to a pH of 3-9). The pharmaceutical compositions may be formulated accordingly. Preparation of suitable parenteral formulations under sterile conditions is readily achieved by standard pharmaceutical techniques well known to those skilled in the art.

[0576] The products, vectors, inhibitors, cells, or pharmaceutical compositions according to the present invention can be administered systemically, for example, by intravenous injection.

[0577] The products, vectors, inhibitors, cells, or pharmaceutical compositions according to the present invention may be administered topically, for example, by targeted administration to the liver. Preferably, the products, vectors, inhibitors, cells, or pharmaceutical compositions may be administered by intra-portal injection or intra-hepatic artery injection.

[0578] The pharmaceutical composition may contain the product of the present invention, a vector, an inhibitor, or cells in an injection medium, such as a sterile isotonic solution. The pharmaceutical composition may be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0579] Products, vectors, inhibitors, cells, or pharmaceutical compositions may be administered in single or multiple doses. In particular, products, vectors, inhibitors, cells, or pharmaceutical compositions may be administered in a single dose. Pharmaceutical compositions may be formulated accordingly.

[0580] Products, vectors, inhibitors, cells, or pharmaceutical compositions may be administered in various doses (e.g., measured in vector genome (vg) per kg). In any case, the physician will determine the actual dose best suited to the individual subject, which will vary depending on the age, weight, and response of the particular subject.

[0581] The pharmaceutical composition may further comprise one or more other therapeutic agents. The product, vector, inhibitor, cell, or pharmaceutical composition may be administered in combination with one or more other therapeutic agents.

[0582] The present invention further includes the use of a kit comprising the products, vectors, inhibitors, cells, and / or pharmaceutical compositions of the present invention. Preferably, the kit is for use in the methods described herein, for example, the therapeutic methods described herein, and is used as described herein. Preferably, the kit includes instructions for the use of the kit components.

[0583] Methods for treating and / or preventing diseases In one embodiment, the present invention provides a product, vector, inhibitor, cell, or pharmaceutical composition for use as a pharmaceutical.

[0584] In related embodiments, the present invention provides the use of the products, vectors, inhibitors, cells, or pharmaceutical compositions according to the present invention in the manufacture of pharmaceuticals.

[0585] In related embodiments, the present invention provides a method for administering a product, vector, inhibitor, cell, or pharmaceutical composition according to the present invention to a subject requiring such administration. Preferably, the subject is a human subject.

[0586] cancer The products, vectors, inhibitors, cells, or pharmaceutical compositions according to the present invention may be used to prevent or treat cancer in a subject. Preferably, the subject is a human subject.

[0587] In one embodiment, the present invention provides a product, vector, inhibitor, cell, or pharmaceutical composition according to the present invention for use in the prevention or treatment of cancer.

[0588] In related embodiments, the present invention provides the use of the products, vectors, inhibitors, cells, or pharmaceutical compositions according to the present invention for the manufacture of pharmaceuticals for the prevention or treatment of cancer.

[0589] In related embodiments, the present invention provides a method for preventing or treating cancer, comprising administering a product, vector, inhibitor, cell, or pharmaceutical composition according to the present invention to a subject in need thereof.

[0590] The subjects may already have cancer, or they may be at risk of developing cancer.

[0591] Participants may have previously been determined to be at risk of developing cancer. Increased risk may be determined by genetic screening and / or a review of the participant's family history. Participants may have been determined to express one or more genetic markers indicating an increased risk of developing cancer.

[0592] Preferably, a person skilled in the art is aware of genetic risk factors (e.g., genetic markers) associated with an increased risk of developing cancer. A person skilled in the art may use any suitable method or technique known in the art to determine whether a subject is at high risk of developing cancer.

[0593] Participants may have previously received cancer treatment. Participants may be in remission from cancer. Participants may be resistant to chemotherapy.

[0594] Liver metastasis In some embodiments, the cancer is liver cancer, for example, secondary liver cancer (e.g., liver metastasis).

[0595] In some embodiments, subjects have or are at risk of developing secondary liver cancer (e.g., liver metastasis), and the products, vectors, inhibitors, cells, or pharmaceutical compositions are used to prevent or treat secondary liver cancer.

[0596] In some embodiments, the subject has primary cancer (e.g., of colorectal, pancreatic, or breast origin), and the product, vector, inhibitor, cell, or pharmaceutical composition is used to prevent or treat secondary liver cancer (e.g., liver metastasis).

[0597] Metastasis is the development of secondary malignant growth away from the primary site of cancer. Metastasis most commonly occurs when cancer cells detach from the primary tumor and enter the bloodstream or lymphatic system.

[0598] 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 diseases can be explained by different hypotheses of metastatic spread. The dual blood supply to the liver by the portal vein and hepatic artery facilitates the capture of circulating cancer cells, according to the "mechanical or hemodynamic hypothesis," which explains the high incidence of liver metastases in patients with gastrointestinal cancers. On the other hand, some primary tumors selectively target the liver as a metastatic site, according to the "seed and soil" hypothesis. Examples include patients with uveal melanoma with a deletion of chromosome 3, as well as patients with breast cancer that is positive for human growth factor receptor 2 (HER-2) combined with estrogen receptor (ER) and progesterone receptor (PR) positivity (de Ridder, J., et al., 2016. Oncotarget, 7(34), p.55368).

[0599] The majority of liver metastases are carcinomas, particularly adenocarcinomas. The primary tumor may be any primary tumor, and the primary tumor may be unknown. However, the most common primary tumors in patients with adenocarcinoma are those originating from the colorectal, pancreatic, or breast (de Ridder, J., et al., 2016. Oncotarget, 7(34), p.55368).

[0600] The subject may be diagnosed with liver metastasis by any suitable method known to those skilled in the art. For example, the subject may be diagnosed by a liver protocol, colonoscopy, and CT imaging with EGD.

[0601] The products, vectors, inhibitors, cells, or pharmaceutical compositions of the present invention may be used in combination with any other suitable therapy to treat or prevent liver metastases, for example, in combination with surgical resection and / or chemotherapy of liver metastases.

[0602] Those skilled in the art will understand that all features of the invention disclosed herein can be combined without departing from the scope of the invention as disclosed.

[0603] Preferred features and embodiments of the present invention are described herein as non-limiting examples.

[0604] The implementation of this invention will utilize conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology, which are within the scope of the skills of those skilled in the art unless otherwise specified. Such techniques are described in the literature. For example, Sambrook, J., Fritsch, EFand Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Ausubel, FM et al. Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley&Sons, Polak, JMand 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, DMand. Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part See A:Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference. [Examples]

[0605] Example 1 result Development of an LV platform enabling in vivo liver macrophage gene manipulation The inventors first generated a LV containing a putative 1.8Kb promoter sequence obtained from the mouse mannose receptor C-1 type (Mrc1) gene (Figure 8A). MRC1 is expressed by most macrophage subsets, including KCs, and is upregulated by alternatively activated macrophages such as tumor-associated macrophages (TAMs). Next, the inventors cloned a GFP coding sequence downstream of the Mrc1 promoter sequence (originating from Mrc1.GFP LV) to generate a VSV G pseudotype Mrc1.GFP LV stock (Figure 1A). Mrc1.GFP LV induced robust transgene expression in IL4-exposed (M2-like) bone marrow-derived macrophages (BMDMs), but not in LPS / IFNγ-exposed (M1-like) BMDMs (Figures 1B-1E). Intravenous injection (iv) of Mrc1.GFP LV into immunodeficient mice selectively resulted in GFP expression in hepatocytes (KCs and hepatic sinusoidal endothelial cells, LSECs) and some spleen cells (Mrc1-positive macrophages). We did not observe GFP expression or integrated LV copies in blood cells, bone marrow, or other organs such as the lungs, lymph nodes, small intestine, and brain (Figures 1B-1D). To further fine-tune gene expression in KCs, we utilized microRNA (miRNA) regulation. We first investigated the suppression of transgene expression in off-target cell types using a bidirectional LV containing a selected miRNA target sequence (miRT) downstream of GFP. Four tandem copies of mirT-122-5p completely prevented GFP expression in hepatocytes while preserving it in KC cells (Figures 8F-9I), while four copies of miRT-126-3p prevented GFP expression in LSEC cells but not in Kupffer cells (Figures 8J and 8K). Based on these results, the inventors incorporated four copies each of miRT-122-5p and miRT-126-3p downstream of GFP in Mrc1.GFP.LV to generate Mrc1.GFP.miRT LV (Figure 1A).To investigate the in vivo expression of the novel Mrc1.GFP.miRT LV in the presence of liver tumors, we induced experimental liver metastases by transplanting either mCherry (red fluorescent protein)-expressing MC38 colorectal cancer cells or colorectal epithelial cancer cells derived from APCΔ716;KrasG12D;Tgfbr2- / -;Trp53R270H;Fbxw7- / - mice, which we refer to herein as AKTPF cancer cells. We intravenously injected Mrc1.GFP or Mrc1.GFP.miRT LV into the liver metastases-challenged mice. Consistent with our findings in tumor-free mice, Mrc1.GFP selectively induced GFP expression in KC, LSEC, and spleen Mrc1-positive macrophages, but in the presence of miRNA regulation (Mrc1.GFP.miRT LV), GFP expression in LSEC was virtually completely reduced (Figure 1E). In both MC38-derived and AKTPF-derived metastatic lesions, we found enhanced GFP expression in the perimetastatic region of the liver. This indicates enrichment of transduced KCs and / or upregulation of Mrc1 promoter activity in these regions, including tumor-associated macrophages derived from euna monocytes (Figure 1F and Figure 8L). GFP expression was not observed in other organs such as the brain, small intestine, lungs, and lymph nodes (Figure 8M). In summary, the selective in vivo distribution and expression of the newly developed Mrc1.GFP.miRT LV in KCs, along with its abundant expression in the region surrounding tumor lesions, supports the feasibility of in vivo genetic manipulation of KCs (including liver metastasis-associated macrophages) for the delivery of therapeutic molecules to liver metastatic lesions.

[0606] In vivo LV-modified KC enables rapid, sustained, and well-tolerated IFNα production. Next, the inventors utilized engineered KC to deliver IFNα to liver metastases. For this purpose, the inventors substituted GFP with the IFNα DNA coding sequence in Mrc1.GFP.miRT LV to create what is referred to herein as IFNαLV. To efficiently transduce KC, the inventors generated LVs based on a manufacturing process aimed at obtaining high-titer LV stocks containing plasmids and low levels of contaminants such as endotoxins that may induce bystander innate immune activation or adverse systemic effects. Subsequently, the inventors engineered KC in vivo by intravenously injecting either IFNαLV or LVs having the same regulatory elements but lacking the transgene (control LV, as referred to herein) into immune-responsive mice at dose ranges previously reported to target hepatocytes with high efficiency (Figure 2A). In mice with IFNαLV-modified KCs, we observed rapid transgene output, reflected by the detection of increased plasma IFNα concentrations, peaking at 700–1,000 pg / mL after 3 weeks and then stabilizing between 200–700 pg / mL. These IFNα levels remained stable until day 240 compared to the control LV cohort, and eventually decreased to virtually undetectable levels by day 360 (Figure 2B). Integrated LV copies in the liver of IFNαLV-treated mice were lower than those found in control LV-treated mice, suggesting long-term counterselection of IFNαLV-transduced hepatocytes, including KCs (Figure 2C). IFNα expression by KCs reduced the number of circulating B cells and eosinophils over time compared to control LV-treated mice. Levels of CD4 and CD8 T lymphocytes, inflammatory and resident monocytes, neutrophils, platelets, erythrocytes, and hemoglobin were unchanged compared to control LV-treated mice (Figure 2D and Figure 9A). To investigate whether the decrease in B cells could be related to B cell activation and autoantibody production, the inventors measured the presence of autoantibodies in the plasma of either saline (PBS), control LV, or IFNαLV-treated mice on days 52 and 366. The inventors found no difference in autoantibody levels among all groups analyzed (Figure 9B).Furthermore, the inventors did not observe any changes in the levels of indicators of liver or tissue damage (i.e., alanine aminotransferase, ALT, and aspartate aminotransferase, AST), suggesting the absence of hepatotoxicity in IFNαLV-treated mice (Figure 2E). To further investigate whether KC-driven exogenous IFNα expression induced inflammation, tissue damage, or other changes, the inventors performed histopathological analysis of the most relevant organs at the end of the experiment. No abnormalities related to the treatment were observed in any of the analyzed compartments (Figures 2F and 9C). In summary, these results indicate that KC-driven IFNα expression results in robust and long-lasting levels of plasma IFNα that are safe and well-tolerated, at least in mice.

[0607] Gene-based forced IFNα expression by KC inactivates T cell activation and reduces liver metastasis proliferation. The inventors have developed two different doses (1.5 × 10) to manipulate KC in mice previously challenged with experimental liver metastasis based on MC38. 9 or 1.5 × 10 10IFNαLV was delivered systemically at a dose of TU / kg (Figure 3A). Consistent with previous results, we found dose-dependent sustained levels of IFNα in plasma (Figure 3B) and dose-dependent LV copies incorporated into the liver (Figure 10A), as well as a negative correlation between plasma IFNα levels and the number of circulating B cells in IFNαLV-treated mice (Figure 10B). We monitored liver metastatic growth by magnetic resonance and found that both IFNαLV doses delayed tumor progression and enabled complete response (CR) and long-term survival in three mice (one at low dose and two at high dose) (Figures 3C and 3D). CR mice rechallenged with MC38 tumors showed proliferative impairment, suggesting induction of adaptive immunological memory against tumor-associated antigens (Figure 10C). To better investigate the induction of tumor-responsive T cells by KC manipulation, we systemically delivered control LV or IFNαLV to syngeneic immunocompetent mice previously challenged with experimental liver metastases of MC38 cells expressing chicken ovalbumin (OVA, used as a surrogate tumor antigen). Consistent with previous results, KC-mediated IFNα expression delayed liver metastasis proliferation (Figures 3E and 10D). We found that tumor-specific T cells, identified by staining with pentameric MHCI complexed with OVA immunogenic peptide (SIINFEKL), were enriched in tumors from IFNαLV-treated mice compared to control LV-treated mice (Figure 3F). Furthermore, TAMs from liver metastases exposed to KC-derived IFNα showed an increased percentage of cells expressing CD11c, a marker associated with activation or inflammatory phenotypes, and a decreased percentage of TAMs expressing a putative M2-like phenotype (Figure 3G). These data suggest that manipulated KC-derived IFNα may delay tumor progression by biasing TAM toward an inflammatory phenotype and enabling T cell activation and proliferation of tumor-responsive T cell clones.

[0608] To further investigate the effect of forced IFNα expression on the tumor microenvironment, we induced experimental liver metastases using AKTPF CRC cells. AKTPF liver metastases reproduce some of the histopathological features of human CRC liver metastases, including epithelial glandular structures, dirty necrotic zones, fibrosis, angiogenesis, and immune infiltration formed by CRC cells (Figures 10E and 10F). IFNα-expressing KCs delayed tumor progression and resulted in complete response (CR) in 5 out of 10 treated mice in two independent experiments (Figures 3I, 3J, and 10G-10I). Based on our previous findings using the MC38 experimental metastasis model, we observed that KC-derived IFNα biased TAM toward an M1-like phenotype and increased the number of tumor-infiltrating CD8 T lymphocytes (Figures 3K-3M). To further investigate the effect of exogenous IFNα expression from KC on liver metastases from a different origin than CRC, we challenged syngeneic mice by intrahepatic injection of KrasG12DTrp53R172H pancreatic ductal adenocarcinoma (PDAC) cells (K8484) and treated them with either a control or IFNαLV. We found a strong inhibition of tumor growth in the IFNαLV group compared to the control group, including CR in 6 out of 8 treated mice (Figure 3N and Figure 10J). In summary, these experiments demonstrate that KC manipulation via systemic delivery of IFNαLV leads to IFNα expression from KC, which at least partially inhibits tumor growth by biasing the TAM phenotype and promoting CD8 T cell recruitment.

[0609] IFNαLV-mediated manipulation of KCs enables preferential IFNα signaling in the peripheral region of metastasis. To investigate the underlying mechanisms of the observed tumor response, the inventors performed a comprehensive transcriptome analysis of AKTPF liver metastases in control LV or IFNαLV-treated mice. The inventors observed increased expression of interferon-stimulated genes exhibiting IFNα activity in metastatic lesions in the IFNαLV cohort (Figure 11A). Next, the inventors used spatial transcriptome analysis to investigate whether there were regions of preferential IFNα signaling within the metastatic liver. For this purpose, the inventors assigned mice to three different cohorts: (1) control: control LV-treated mice, (2) responder: IFNαLV-treated mice with reduced metastatic volume compared to controls, and (3) resistant: IFNαLV-treated mice with a similar metastatic volume to controls (Figure 11B). Note that mice that achieved a complete response could not be analyzed because they did not have tumors at the time of analysis. Next, 36 mm of the liver containing metastatic lesions was analyzed. 2Spatial transcriptome analysis was performed on the sections. Subsequently, unsupervised clustering analysis was performed to cluster spatial spots based on similar transcriptome analysis profiles (Figure 11C). Spatial spots belonging to clusters 1 and 6 showed high expression of genes associated with adenocarcinoma and were presumed to be assigned to liver metastasis areas. In contrast, 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). Next, the inventors estimated the estimated distance of each spatial spot from the metastatic lesion interface using an unsupervised method that weighted the properties of the surrounding spatial spots (i.e., liver or metastatic lesions). The inventors grouped the spatial spots into separate spatial compartments, including internal metastasis (spatial compartments A-C), anterior metastasis (spatial compartment D), perimetastasis (spatial compartments E-G), and intact liver region (spatial compartment H), according to their relative distance from the metastasis / liver parenchymal boundary (Figures 4A and 11E). As expected, the inventors found that in all cohorts, genes belonging to cancer-related biological processes or pathways (e.g., angiogenesis, p53 pathway, epithelial-mesenchymal transition) were enriched in the metastatic region (internal and anterior regions) compared to the perimetastatic region (perimetastatic and intact liver). Consistent with this observation, epithelial cell-related genes such as epithelial cell adhesion molecule (Epcam), cadherin 1 (Cdh1), and virin 1 (Vil1) were highly expressed in the internal and anterior metastatic regions. In contrast, hepatocyte-related genes (e.g., albumin, Alb; apolipoprotein 2, Apoa2; and cytochrome p450 family 27a1, Cyp27a1), as well as gene sets belonging to liver-related pathways (e.g., adipogenesis or bile acid metabolism), were upregulated in the intact liver region.Consistent with the enhancement of transgene expression from engineered KCs in the region surrounding liver metastases upon systemic Mrc1.GFP.miRT LV delivery, we found that genes associated with the response to type I interferon (e.g., cytokine signaling repressor1, Socs1; transcriptional signaling and activator1, Stat1; and NLR family CARD domain-containing5, Nlrc5) were enriched in the liver metastases and perimetastatic regions of IFNα LV cohorts (responders and resistances), consistent with the LV platform's ability to preferentially manipulate KCs near liver metastases. Furthermore, upregulation of genes belonging to the gene ontology (GO) category, such as those related to type I interferon activity, response to interferon-γ, response to viruses, positive regulation of cytokine production, and T cell activation, was associated with the region of type I interferon signaling. Furthermore, compared to the resistance or control cohort, genes belonging to the adaptive immune activation GO category, such as those involved in the modulation 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 responders in the internal, anterior, and perimetastatic regions corresponding to sites of enhanced IFNα activity. Interestingly, genes related to antigen presentation were also highly expressed in metastatic lesions in both the responder and resistance cohorts compared to controls. Importantly, increased IL10 signaling was found in the anterior and perimetastatic regions in the resistance cohort, but not in the control or responders, suggesting that IL10 may play a role in counteracting the IFNα effect in resistance mice. Notably, markers associated with T cell exhaustion and tolerogenic phenotypes, such as transforming growth factor β1 (Tgfb1), eomesoderm (Eomes), and granzyme k (Gzmk), were also upregulated in the internal, anterior, and perimetastatic regions in the resistance group (Figures 4B and 4C). In short, KC-mediated IFNα expression was associated with selective immune activation in the liver metastases and perimetastatic regions of 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 metastasis / hepatic parenchymal boundary region.

[0610] IFNα promotes APC immune activation and enhanced MHCII-restricted antigen presentation in responder mice. The inventors performed single-cell transcriptome analysis on viable cells from the same metastatic lesions assayed by spatial transcriptome analysis (Figure 11A). Using unsupervised clustering, the inventors identified the following distinct cell types: (1) APCs, (2) T cells and NK cells, (3) B cells, (4) neutrophils, (5) endothelial cells, (6) hepatocytes, and (7) cancer cells, which were manually annotated based on the transcriptome analysis profiles (Figures 12A-12D). The inventors then focused on cells belonging to the APC cluster. The inventors found that genes belonging to the GO category related to IFNα, IFNγ, or LPS signaling were relatively enriched in all IFNα-LV treated cohorts. On the other hand, genes related to IL10, PGE2, and IL4 signaling were upregulated in the resistance cohort compared to the responder cohort, suggesting that these genes may play a role in inducing resistance to gene-based IFNα therapeutic activity. Genes related to antigen presentation, namely the MHC protein complex and antigen processing and presentation, were upregulated in partial responders compared to the resistant or control cohort, showing the lowest expression in the control cohort (Figure 5A). Next, we performed subclustering analysis to better define cell populations and differentially expressed genes within APC clusters. Within the APC, we identified overlapping cell clusters in all three experimental cohorts, with the exception of TAM clusters reformulated by IFNα treatment, indicating that gene expression is reprogrammed upon exposure to gene-based IFNα delivery. Based on this observation, and considering the dominant effect of IFNα on the TAM genetic program, we named all TAMs belonging to clusters present in IFNα-treated LV tumors IFNα-TAM, while those present in the control LV cohort were named TAM. All other cell clusters were manually annotated based on their gene expression profiles (Figures 5B and 12E).By using differential gene expression analysis between TAM subsets, we found that in all three cohorts, genes upregulated in IFNα-TAM compared to TAM were enriched in IFNα / IFNγ responses such as Stat1, Socs1, and Nlrc5; TNFα signaling; LPS activation; and biological processes related to 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), which is consistent with the role of IFNα-TAM in positively regulating immune activation. Notably, Il10 and Tgfb1 (both genes associated with immunosuppression) were upregulated in the IFNαLV cohort, suggesting important roles of these genes in resistant mice. On the other hand, tumor promoter genes commonly associated with TAM tumorigenic activity, such as matrix metallopeptidase 8 (Mmp8), transmembrane protein 176B (Tmem176B), myeloid cell 2 (Trem2), and fibronectin 1 (Fn1) receptors, were upregulated in TAM versus IFNα-TAM (Figures 5C and 5D). Notably, professional APCs, i.e., classical dendritic cells (cDCs) and monocyte-derived DCs (MoDCs), were enriched in the responder cohort compared to the resistance and control cohorts (Figure 5E). Consistent with this observation, the inventors found that in APCs derived from responder mice, genes related to MHCII-restrictive antigen presentation, such as genes encoding MHCII subunits (H2-Aa, H2-Ab1, H2-Eb1, H2-DMb1, and H2-Oa), the MHCII transactivator (Ciita), Cd74, and Cd40, were upregulated compared to resistant or control mice.Notably, MHCI-restricted genes, such as those encoding MHCI subunits (H2-T22, H2-T23, H2-D1, and B2m), Tap1, Tap2, Tap-binding protein (Tapbp), and proteasome S20 subunits beta 8 and 9 (Psmb8 and Psmb9), were upregulated in all IFNαLV-treated (resistance and responder) cohorts. Consistent with IL10, which plays a putative 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 resistance APCs compared to the responder and control cohorts (Figure 5F). Among all APC populations, Ccr7-expressing DCs, cDCs, KCs, and Mo DCs expressed the highest levels of genes associated with MHCII-restricted antigen presentation in all cohorts. Therefore, differences in the expression levels of genes associated with MHCII-restricted antigen presentation in APCs may be at least partially attributable to the enhanced infiltration of professional APCs such as Mo DCs and cDCs in the responder cohort. On the other hand, upregulation of genes associated with MHCII-restricted antigen presentation may be upregulated due to the direct effect of IFNα on cells (Figure 5G). In summary, IFNα released from KCs promoted the reformation of APCs into an immunostimulatory phenotype by boosting antigen-presenting function. However, MHCII-restricted function and DC infiltration appeared to be reduced in resistant mice compared to responder mice. Simultaneously, IL10 signaling was enhanced in APCs derived from resistant mice, supporting a link between the lack of response and IL10 upregulation and impaired MHCII-restricted antigen presentation.

[0611] The therapeutic response to IFNα is associated with T cell activation and is offset by Eomes CD4 T cell infiltration. Next, the inventors performed differential expression analyses in the T cell and NK cell compartments across the three experimental cohorts. Similar to TAM, genes belonging to IFNα and IFNγ signaling were enriched in all IFNα-LV treated cohorts. Conversely, genes belonging to immunoactivation (i.e., regulation of T cell-mediated cytotoxicity, natural killer cell activation, or cell death) were exclusively upregulated in the partial responder cohort (Figure 6A). Next, the inventors performed unsupervised subclustering analysis to identify distinct cell populations within the T cell and NK cell compartments and manually annotated the resulting clusters. The inventors found overlapping cell clusters in all three experimental cohorts (Figures 6B and 6A). In resistant mice, the inventors selectively observed a population of regulatory CD4 T cells transcriptionally similar to previously described Tr1 cells (referred to herein as Eomes CD4 T cells) (Bonnal et al. (2021) Nature immunology 22:735-745; Roncarolo et al. (2018) Immunity 49:1004-1019). These cells expressed markers of CD4 T cell exhaustion such as Ctla4, granzyme k (Gzmk), Lag3, and PD1 (Pdcd1), as well as immunosuppressive genes such as IL10 receptor (Il10ra), Il10, and the transcription factor Eomes, and lacked expression of the transcription factor Foxp3 (Figure 6C and Figure 13B). On the other hand, selectively enriched in the responder cohort, the inventors observed a population of CD8 T effector 1 cells (Figure 6D). The latter exhibited a transcriptome signature similar to that of tissue-resident effector memory T cells, which had previously been associated with a response to immunotherapy (Figure 6E and Figure 13B) (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 in the entire CD8 T cell population.Notably, genes associated with T cell exhaustion, such as Pdcd1, Lag3, TIM3 (Havcr2), Ctla4, Eomes, thymocyte selection-associated high-mobility group box protein (Tox), Ccl3, Ccl4, and caspase 3 (Casp3), were downregulated in responders compared to control or resistant mice. In contrast, genes related to adaptive immune responses and T cell-mediated immunity and cytotoxicity (e.g., 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 more upregulated in responders than in resistant mice (Figures 6E and 13C). In summary, these data indicate that IFNα released by manipulated KCs promoted adaptive immunity in responder mice by reforming T cell infiltrates that enriched effector phenotypes associated with immunotherapy responses, while suppressing T cell exhaustion. Conversely, in resistant mice, enhanced exhaustion of infiltrating Eomes CD4 and CD8 T cells may prevent the antitumor effect.

[0612] Modified KC-derived IFNα combined with functional inhibition of regulatory T cells eradicates liver metastases. Next, the inventors investigated whether IFNα signaling is positively associated with the presence of Eomes CD4 T cells in the tumor microenvironment in human CRC liver metastases, similar to the case in mice. To this end, the inventors used bulk RNA sequencing data from human CRC liver metastases collected from their institution and found that patients with high IFNα signaling scores exhibited higher levels of Eomes CD4 signature scores (Figures 7A, 7B, and 14A). Next, the inventors performed immunohistochemical staining 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. The inventors found that most CD4 T cells expressed detectable levels of LAG3 in the high IFNα group, while CD4 T cells in the low IFNα group did not show detectable LAG3 expression (Figures 7C and 14B). Notably, LAG3 has been previously reported as a marker of T cell exhaustion and as a marker of human Tr1 cells. This observation suggests that Eomes CD4 T cells are positively associated with endogenous IFNα signaling and can, at least partially, counteract immune activation in the tumor microenvironment.

[0613] In IFNαLV-resistant mice, we simultaneously observed increased IL10 signaling, impaired MHCII-restricted antigen presentation, enhanced Eomes CD4 T cell infiltration, and enhanced CD8 T cell exhaustion. 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, having been described as suppressing antigen presentation function through the direct death of perforin-mediated DCs and inhibiting T cell activity in CRC liver metastases via IL10 secretion. Based on these observations, we inhibited IL10 signaling by using a monoclonal antibody (α-IL10R) that blocks the IL10 receptor. Mice challenged with AKTPF liver metastases and treated with IFNα or control LV were treated with either αIL10R or an unrelated IgG. Anti-IL10R blocked IFNα-inducible accumulation in EOMES CD4 T cells (Figure 7D), demonstrating that IL10 signaling is required for IFNα-inducible accumulation in these cells in liver metastases. However, the combination of IFNα and α-IL10R achieved a lower therapeutic effect than either IFNαLV or α-IL10R alone (Figure 7E), suggesting that IL10 signaling may also be required for the development of IFNα therapeutic activity. In fact, we found that the combination of α-IL10R and IFNαLV induced the highest increase in PD1 expression on CD4 and CD8 T cells circulating in peripheral blood (Figures 14C and 14D), a result consistent with the role of IL10 in T cell reactivation and prevention of their exhaustion, which may be particularly necessary in the case of IFNα T cell exposure.

[0614] The inventors observed that Ctla4 was expressed in Eomes CD4 T cells, exhausted CD4 and CD8 T cells, and Foxp3-regulating T (Treg) cells (Figure 13B). Furthermore, Ctla4 was strongly upregulated in CD8 T cells of resistant mice. Notably, CTLA4 in Tr1 cells may play a crucial role in suppressing T cell function and attenuating antigen presentation by sequestering the costimulatory molecule CD80 / CD86 during APC. Based on this observation, the inventors combined KC-based IFNα delivery with an anti-CTLA4 blocking monoclonal antibody (α-CTLA4, Figure 7F). The combination of KC-mediated IFNα and α-CTLA4 strongly inhibited liver metastatic growth compared to either treatment alone in two different experimental models of CRC liver metastases, MC38 (Figure 7G and Figure 14E) and AKTPF (Figure 7H and Figure 14F). Notably, in mice with AKTPF liver metastases, we observed up to 70% of mice showing a complete response to the combination of IFNα and CTLA4. This result indicates that enhancing antigen presentation function in APCs through inhibition of CTLA4 function in regulatory Eomes CD4 T cells, exhausted CD4 / CD8 T cells, and CD4 Treg cells strongly enhances the therapeutic activity of IFNαLV and reveals the major contribution of CTLA4 to the development of treatment resistance.

[0615] Overall, these findings demonstrate a potent synergy between our strategy of gene-based IFNα delivery via KC from within the tumor bed and checkpoint blockade targeting regulatory T cell function.

[0616] Consideration The inventors developed a novel LV platform for manipulating KCs in close proximity to liver metastases and utilized this strategy to deliver IFNα to CRC and PDAC liver metastasis models. KC-released IFNα resulted in i) reprogramming of TAMs and infiltrating DCs toward immune activation and antigen presentation, ii) increased recruitment, activation, and reduced exhaustion of CD8 T cells, and iii) enrichment of a CD8 T cell subpopulation with tissue-resident effector memory cell characteristics previously associated with positive responses to immunotherapy. This immune cell regeneration resulted in inhibition of metastasis in most mice. Detailed analysis of resistant mice revealed the emergence of an Eomes-expressing CD4 T cell population transcriptionally similar to Tr1 cells, associated with immunosuppressive and tolerogenic functions. Furthermore, APCs from resistant mice showed increased IL10 signaling and decreased MHCII-restricted antigen presentation, while CD8 T cells showed increased markers of exhaustion. The co-administration of CTLA4 blockers and IFNαLV overcame these resistance mechanisms, enabling a nearly complete therapeutic response and demonstrating the principle of a novel therapeutic strategy with potential interpretation in cancer patients with significant unmet medical needs.

[0617] Efficient KC manipulation was achieved through the preferential in vivo distribution of intravenously administered LV into the liver, as well as through vector-integrated Mrc1 promoters and miRNA target sites that enable selective transgene expression in KC, particularly in regions proximal to metastatic lesions. LV-based KC manipulation was abundant in the perimetastatic region, likely due to tumor-driven changes in the local vascular system and KC phagocytic activity undergoing remodeling. Furthermore, previous reports have shown that macrophage-mediated MRC1 expression increases in the presence of tumor.

[0618] Selective exogenous expression of cytokines in KCs may limit hepatotoxicity from direct expression in hepatocytes or LSECs. Furthermore, macrophages, including KCs, have been proposed to reconfigure their genetic programs to promote tumor growth and immune evasion in the presence of tumors. Therefore, direct expression of IFNα in these cells may reconfigure their tumor-promoting genetic programs, resulting in greater therapeutic benefits. On the other hand, IFNα derived from KCs also reached systemic circulation and established sustained plasma levels. While this systemic exposure may contribute to the therapeutic benefits observed here, previous studies have only reported the prophylactic activity against metastatic dissemination of recombinant type I interferon administered via an implanted mini-osmolar pump. Expression from tissues via KC manipulation is likely to bypass the in vivo distribution and vascular barriers of systemic administration and achieve more effective concentrations for target cells within the TME. Although systemic IFNα administration has been associated with significant toxicity in preclinical models and in humans, we did not collect evidence of tissue damage or autoimmunity in our studies. This is due to the following reasons: (i) improved therapeutic index of locally produced IFNα in the hepatic interstitium and preferential IFNα signaling in hepatic areas with hepatic metastases; (ii) stable expression of IFNα compared to the peak and trough dynamics in plasma from systemically delivered cytokines (which is often associated with desensitization and toxicity); and (iii) plasma levels of IFNα within the physiological range and similar to those observed during viral infection.

[0619] Notably, LV-based cell manipulation leads to vector integration and maintenance of transgene expression. Importantly, our strategy ultimately came to a virtual demise within a year.

[0620] Termination of expression is likely due to the turnover of the manipulated KC, which occurs earlier in cells expressing exogenous IFNα than in those transduced with control LV, suggesting several counter-selections of the former. Alternatively, it may be possible to use integrase-deficient (ID) LV, which persist in the nucleus as an episomal form driving lower transient transgene expression. Therefore, the choice between using LV or IDLV may depend on the desired transgene production level and duration.

[0621] The inventors observed therapeutic benefits of IFNαLV in all mouse models of liver metastases tested, with a superior response in AKPTF CRC, which better replicates the human disease in terms of genetic variation and histopathological features. In the AKPTF model, the presence of multifocal glandular structures, which allows for closer interaction between engineered macrophages and TMEs, and slower tumor growth, which extends the therapeutic window, may have enhanced the therapeutic activity of IFNαLV.

[0622] Despite the therapeutic activity observed with IFNαLV-mediated KC manipulation as a single-dose treatment, a population of Eomes CD4 T cells exhibiting a Tr1-like gene signature neutralized IFNα activity in some resistant mice. Consistent with previous reports, we demonstrated that Eomes CD4 T cell development is dependent on stimulation with type I IFN and IL10. This observation highlights the complex and sometimes contradictory effects of IFNα, which can promote tumor growth and immune evasion in several situations. For example, in a mouse model of chronic viral infection, IFNα exposure promoted myeloid-derived suppressor cell differentiation, which in turn inhibited the CD8 T cell response or promoted the cancer stem cell phenotype in a mouse model of fibrosarcoma. Conversely, forced expression of IFNαR1 in CD8 T cells enhanced cytotoxic activity in subcutaneous MC38 mouse tumors, or restoration of IFNα signaling in cancer cells resulted in CD8-dependent therapeutic activity in separate syngeneic and xenograft tumor mouse models. Similarly, depending on the presence of its targets and other stimuli, IL10 can promote or inhibit tumor immunity. For example, by acting on DCs, IL10 impairs activation and antigen presentation, and simultaneously, by impairing DC function, IL10 prevents DC-induced CD8 T cell apoptosis. By acting on CD8 T cells, IL10 prevents T cell exhaustion and promotes T cell activation in renal cell carcinoma patients and tumor mouse models. Consistent with these observations, we found that only the blockade of either IFNαLV or IL10 promotes tumor immunity and delays liver metastasis growth. Conversely, the combination of IFNαLV and IL10 blockade had no effect on liver metastasis growth.

[0623] The inventors have found that exogenous IFNα released by KC strongly upregulates genes involved in MHCI-restricted antigen presentation in different populations of liver metastatic infiltrating APCs, including TAMs and DCs, from both responder and resistant mice, suggesting that IFNα may exert its therapeutic activity, at least partially, through the activation of antigen presentation.

[0624] Interestingly, MHCII-restricted antigen presentation was inhibited in liver metastases from resistant mice, which is partly due to lower infiltration in different populations of DCs and downregulation of genes associated with MHCII-restricted antigen presentation. Consistent with this observation, MHCII-restricted antigen presentation may be necessary for maintaining functional T cells in tumors and enabling responses to immunotherapy. Further investigation is needed to determine whether the reduction in MHCII-restricted antigen presentation in resistant mice is upstream or downstream of IL10 signaling and enhancement of Eomes CD4 T cell differentiation.

[0625] Tr1 cells, like Treg cells, have been described as suppressing immunosuppression through the expression of IL10 and CTLA4. CTLA4 may play a crucial role in reducing T cell priming via antigen presentation and CD80 / CD86 segregation, which in turn leads to defective T cell activation. Notably, CTLA4 is highly upregulated in CD8 T cells and Eomes CD4 T cells in resistant mice, suggesting it may play a vital role in preventing immune activation in the presence of high IFNα signaling. Consistent with this concept, dual intervention with IFNαLV delivery and CTLA4 blockade yielded a strong therapeutic effect, achieving complete regression of liver metastases in most mice.

[0626] The inventors' findings have clinical correlations that support their relevance. These include differential prognostic values ​​of gene signatures associated with specific subpopulations of innate or adaptive immune cells described in the inventors' studies, such as tumor-promoting macrophages, CD8 tissue-resident effector memory cells, exhausted T cells, and Eomes CD4 T cells. The inventors present evidence of a correlation between the degree of IFNα signaling and the Tr1 signature score in clinical samples of hepatic metastatic CRCs. Furthermore, the inventors found expression of the Tr1 cell marker LAG3 in CD4 T cells infiltrating human CRC liver metastases with high IFNα signaling scores. Notably, Eomes CD4 T cells are associated with a subgroup of patients exhibiting high IFNα signaling in liver metastases and may be present at low levels in patients exhibiting low IFNα signaling. Therefore, while previous studies have investigated and highlighted the complexity of CD4 T cells in CRC liver metastases, only a small number of studies have detected Tr1-like Eomes CD4 T cells infiltrating liver metastases (Bonnal et al. (2021) Nature immunology 22:735-745).

[0627] Overall, the inventors have developed a novel, off-the-shelf gene therapy tool that manipulates KC with a single, well-tolerated systemic dose, which then rapidly delivers IFNα from within the liver to liver metastases, eliciting tumor immunity against liver metastases in the relevant mouse model.

[0628] method Plasmid design To construct the Mrc1.GFP lentiviral vector (LV), the inventors inserted a putative Mrc1 promoter sequence (MM39 assembly:CHR2:14232425-14234307) containing an 1883bp DNA sequence into the previously described PGK.GFP LV by replacing the PGK promoter sequence using restriction enzyme sites XhoI and AgeI. Bidirectional miRT LVs were generated by inserting four tandem copies having complete complementarity to miR-122-5p (miRT-122-5p:5'-ACAAACACCATTGTCACACTCCA-3') or miR-126-3p (miRT-126-3p:5'-CGCATTATTACTCACGGTACGA-3') using random 4bp DNA linker sequences that separate the miRT sites. Next, four copies of the miRT sequence were inserted downstream of the bidirectional WPRE sequence of the LV, located in opposite directions and containing cleaved low-affinity nerve growth factor receptor (dlNGFR) and GFP expression-driving minimal cytomegalovirus (mCMV) and human phosphoglycerate kinase 1 (PGK) promoters, respectively. The miRT was inserted using restriction enzyme site KpnI. The Mrc1.GFP.miRT LV was constructed by inserting four copies of miRT-122-5p and four copies of miRT-126-3p downstream of the WPRE sequence of the Mrc1.GFP LV transfer vector plasmid using restriction enzyme site KpnI. The IFNαLV transfer vector plasmid was constructed by replacing the GFP sequence of the Mrc1.GFP.miRT LV transfer vector plasmid with cDNA encoding the mouse IFNα1 protein using restriction enzyme sites SalI and ScaI. Control LVs were prepared by depleting the GFP sequence of the Mrc1.GFP LV transfer vector plasmid by digestion with restriction enzymes AgeI and SalI, followed by the insertion of four copies of miRT-122-5p and four copies of miRT-126-3p downstream of WPRE using restriction enzyme site KpnI.

[0629] cell culture HEK293T cells, MC38 cells, and K8484 cells were cultured in adherent cell culture plates in Iscove's Modification of Dulbecco's Modified Eagle Medium (IMDM, Corning) supplemented with 10% fetal bovine serum (FBS, HyClone®), penicillin (100 IU / mL), and streptomycin (100 μg / mL). To generate MC38-mCherry cells expressing mCherry in virtually all cells (99.97% of all cells), MC38 cells were transduced with LV, which drives the expression of a chimeric protein formed by mCherry fused to the C-terminus of the CD81 transmembrane domain from a constitutively expressed human phosphoglycerate kinase 1 (PGK) promoter. To generate MC38-OVA cells, MC38 cells were transduced with LV driving the expression of full-length chicken oocyte albumin (OVA) from the hPGK ...

Claims

1. An organism comprising (a) a vector for phagocytic cell-specific expression of the liver and / or spleen, wherein the vector comprises a transgene operably linked to one or more expression regulatory sequences, and (b) an immune checkpoint inhibitor or a Tr1 cell inhibitor.

2. A vector for therapeutic use, wherein the vector is for phagocytic expression of the liver and / or spleen, the vector comprises a transgene operably linked to one or more expression regulatory sequences, and 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 therapeutic purposes, wherein the immune checkpoint inhibitor or Tr1 cell inhibitor is used in combination with a vector for phagocytic cell-specific expression in the liver and / or spleen, the vector comprising a transgene operably linked to one or more regulatory sequences.

4. The one or more expression regulatory sequences comprises (a) a phagocytic cell-specific promoter and / or enhancer, and / or (b) one or more miRNA target sequences, wherein optionally, the one or more miRNA target sequences suppress expression in cells other than hepatic phagocytic cells, the product or vector of an immune checkpoint inhibitor or Tr1 cell inhibitor for use according to any one of claims 1 to 3.

5. The phagocytic cells are Kupffer cells, the product or vector of an immune checkpoint inhibitor or Tr1 cell inhibitor for use according to any one of claims 1 to 4.

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

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

8. An immunotherapy product or vector for use according to any one of claims 1 to 7, wherein the transgene encodes a cytokine, and optionally 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. The transgene encodes a tumor antigen, and optionally the tumor antigen is carcinoembryonic antigen (CEA), TRP2, melanoma-associated antigen (MAGE) family, carcinogenline (CAGE) family, melanoma B antigen (BAGE-1), synovial sarcoma X breakpoint 20 (SSX-2), sarcoma antigen (SAGE) family, LAGE1, NY-ESO-1, HER2, EGFR, MUC-1, or GAST, an product or vector of an immune checkpoint inhibitor or Tr1 cell inhibitor for use according to any one of claims 1 to 8.

10. The vector is a viral vector, and optionally, the vector is a lentiviral vector, a retroviral vector, an adenovirus vector, an adeno-associated virus vector, or a herpes simplex virus vector, an product or vector of an immune checkpoint inhibitor or Tr1 cell inhibitor for use according to any one of claims 1 to 9.

11. The aforementioned immune checkpoint inhibitors include 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 activator gene-3), PD-1 (programmed cell 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), and VISTA (V domain Ig inhibitor of T cell activation). An immunotherapy checkpoint inhibitor or Tr1 cell inhibitor for use according to any one of claims 1 to 10, or a vector, which inhibits an inhibitory checkpoint molecule selected from the group consisting of B7-1 (CD80), B7-2 (CD86), TGFB (transforming growth factor β) pathway-related protein, Il13 (interleukin-13), IL4 (interleukin-4), FGL (fibrinogen-like 1), TIGIT (T cell immune receptor having 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-related protein).

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

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

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

15. An organism comprising (a) a vector for phagocytic cell-specific expression of the liver and / or spleen, wherein the vector comprises a transgene operably linked to one or more expression regulatory sequences; and (b) a second vector for phagocytic cell-specific expression of the liver and / or spleen, wherein the second vector comprises a second transgene operably linked to one or more expression regulatory sequences, wherein the transgene is different from the second transgene.

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