Cytomegalovirus vectors and methods of use thereof

Abstract

The present disclosure relates to methods and materials involved in treating a mammal (e.g., a human) with cancer and / or infectious diseases. For example, the present disclosure provides a recombinant human cytomegalovirus (hCMV) vector that includes (e.g., is designed to include) a nucleic acid encoding a viral gene transfer vector genome (e.g., a heterologous viral gene transfer vector genome) and one or more nucleic acids encoding packaging polypeptides, such that a mammalian cell infected with the hCMV vector can produce and release a viral vector (e.g., an infectious lentiviral vector), which in turn can infect cells (e.g., immune cells) in vivo and, in some cases, drive expression of an exogenous polypeptide (e.g., a therapeutic polypeptide or an antigen receptor such as a chimeric antigen receptor (CAR)) in the infected immune cell in the mammal (e.g., a human) to induce an immune response in the mammal.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Application No. 63 / 297,339, filed January 7, 2022. The disclosure of the prior application is considered part of (and is incorporated by reference into) the disclosure of this application.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file titled "07039-2095WO1_SL.xml". The XML file was created on December 12, 2022 and is 10,000 bytes in size. The contents of this XML file are hereby incorporated by reference in their entirety.

[0003] Technical Field The present disclosure relates to methods and materials involved in treating a mammal (e.g., a human) with cancer and / or infectious diseases. For example, the present disclosure provides a recombinant human cytomegalovirus (hCMV) vector that includes (e.g., is designed to include) a nucleic acid encoding a viral gene transfer vector genome (e.g., a heterologous viral gene transfer vector genome) and one or more nucleic acids encoding packaging polypeptides, such that a mammalian cell infected with the hCMV vector can produce and release a viral vector (e.g., an infectious lentiviral vector, which in turn can infect a cell (e.g., an immune cell) in vivo and / or be recognized and internalized by an antigen presenting cell (APC)), and optionally drive expression of an exogenous polypeptide (e.g., a therapeutic polypeptide or an antigen receptor such as a chimeric antigen receptor (CAR)) in the infected immune cell in the mammal (e.g., a human) to induce an immune response in the mammal. In some cases, the recombinant hCMV vectors provided herein can be administered to a mammal having cancer and / or an infectious disease to induce an immune response against the pathogen causing the cancer and / or infectious disease in the mammal (e.g., to treat the mammal). [Background technology]

[0004] Background information Viral vectors are reliable tools that can introduce foreign genetic material and genome engineering components into target cells (Warnock et al., Methods Mol. Biol., 737:1-25 (2011)). For example, lentiviral vectors can integrate foreign genes into the host genome, while adeno-associated viral (AAV) vectors can be produced at high titers and exhibit feasible in vivo infection efficiencies (Maes et al., Neurosci. Lett., 707:134310 (2019)). Summary of the Invention

[0005] overview Current protocols for generating genetically modified T cells expressing a CAR (CAR T cells) include isolation of autologous T cells from the patient, cytokine activation and expansion of the isolated T cells, ex vivo transduction of the T cells with a lentivirus designed to express the CAR, thereby forming CAR T cells, and infusion of the CAR T cells back into the patient (Tyagarajan et al., Mol. Ther. Methods Clin. Dev., 16:136-144 (2019)). The present disclosure is based, at least in part, on the design of a recombinant hCMV that can be administered to a patient to achieve in vivo immune cell editing (e.g., CAR T cell generation) in a single step.

[0006] The present disclosure relates to methods and materials involved in treating a mammal (e.g., a human) with cancer and / or an infectious disease. For example, the present disclosure provides a recombinant hCMV vector that includes (e.g., is designed to include) a nucleic acid encoding a viral vector genome (e.g., a heterologous viral gene transfer vector genome) and one or more nucleic acids encoding a packaging polypeptide, and is capable of infecting cells in a mammal. In some cases, an hCMV vector-infected cell can produce and release a viral vector (e.g., a heterologous viral gene transfer vector such as a lentiviral vector), which can infect other cells (e.g., immune cells such as T cells) in vivo, and in some cases, can drive expression of an exogenous polypeptide (e.g., a therapeutic polypeptide or an antigen receptor such as a CAR) in the viral vector-infected cell in a mammal (e.g., a human) to induce an immune response in the mammal. In some cases, hCMV vector-infected cells can produce and release a viral vector (e.g., a heterologous viral gene transfer vector, such as a lentiviral vector) derived from a virus that can cause an infection and can be recognized as a pathogen by APCs in vivo, whereby the viral vector is internalized by APCs and one or more polypeptides from the viral vector are presented as antigens to one or more immune cells (e.g., T cells) to induce an immune response in a mammal. The present specification also provides methods and materials for producing and using such recombinant hCMV vectors. In some cases, a recombinant hCMV vector provided herein can be administered to a mammal having cancer to induce an immune response against the cancer in the mammal (e.g., to treat the mammal). In some cases, a recombinant hCMV vector provided herein can be administered to a mammal having an infection to induce an immune response against a pathogen causing an infection in the mammal (e.g., to treat the mammal).

[0007] As described herein, a population of recombinant hCMV designed to infect cells in a mammal can be administered to generate hCMV vector-infected cells in the mammal, which in turn can produce and release a viral vector (e.g., a heterologous viral gene transfer vector, such as a lentiviral vector) that can infect other cells (e.g., T cells) in vivo, thereby editing one or more immune cell functions of the viral vector-infected cells (e.g., lentiviral vector-infected T cells) in vivo. For example, a recombinant hCMV vector provided herein can infect a cell in a mammal (e.g., a human) and produce one or more endogenous hCMV polypeptides to recruit one or more immune cells to the infected cell (e.g., an hCMV vector-infected fibroblast, an hCMV vector-infected endothelial cell, an hCMV vector-infected mononuclear cell, or an hCMV vector-infected glial cell), such that the viral vector produced by and released from the hCMV vector-infected cell can infect the recruited immune cell. In some cases, the viral vector produced by and released from an hCMV vector-infected cell can contain a nucleic acid encoding an exogenous polypeptide (e.g., a therapeutic polypeptide and / or an antigen receptor such as a CAR) such that upon infection of a recruited immune cell by the viral vector, the viral vector-infected recruited immune cell can express the exogenous polypeptide. For example, the recombinant hCMV vectors provided herein can be designed to produce a viral vector (e.g., a heterologous viral gene transfer vector such as a lentiviral vector) that contains a nucleic acid encoding a therapeutic polypeptide such that the viral vector can infect a recruited immune cell (e.g., a T cell) and the infected recruited immune cell can express the therapeutic polypeptide.For example, the recombinant hCMV vectors provided herein can be engineered to generate a viral vector (e.g., a heterologous viral gene transfer vector such as a lentiviral vector) that contains a nucleic acid encoding a CAR, such that the viral vector is capable of infecting recruited immune cells (e.g., T cells) and the infected recruited immune cells are capable of expressing the CAR, thereby generating a CAR in vivo. + Immune cells (e.g., CAR + T cells) are generated. Also, as demonstrated herein, the recombinant hCMV provided herein can be further engineered to have altered tropism. For example, recombinant hCMV can be engineered to include a nucleic acid sequence encoding a functional UL131 polypeptide (e.g., a UL131 nucleic acid sequence from the Merlin strain of hCMV), allowing hCMV to infect non-fibroblastic cells, such as epithelial cells, endothelial cells, mononuclear cells, and glial cells.

[0008] The ability to induce an immune response in a mammal, and in some cases generate CAR T cells in a mammal, as described herein, provides a unique opportunity to use immunotherapy to target (e.g., locate and destroy) specific cells (e.g., diseased cells, such as cancer cells or infected cells, such as virally infected cells).

[0009] In general, one aspect of the present specification is directed to a recombinant hCMV vector capable of infecting a mammalian cell. The hCMV vector can comprise heterologous nucleic acid encoding (i) a heterologous viral gene transfer vector genome and (ii) one or more helper polypeptides for amplifying and packaging the heterologous viral gene transfer vector genome to obtain an infectious vector particle, and the hCMV vector-infected cell produces and releases the infectious vector particle comprising the gene transfer vector genome. The hCMV vector can be replication-competent in the cell. The hCMV vector can lack the nucleic acid encoding the UL138 polypeptide, the UL144 polypeptide, the UL146 polypeptide, the UL147 polypeptide or the miR-UL148D. The hCMV vector can lack the UL138 polypeptide, the UL144 polypeptide, the UL146 polypeptide, the UL147 polypeptide or the miR-UL148D. The hCMV vector can be derived from the hCMV AD169 strain or the hCMV Merlin strain. The hCMV vector can lack a nucleic acid encoding a UL23 polypeptide, a US1 polypeptide, a US2 polypeptide, a US3 polypeptide, a US4 polypeptide, a US5 polypeptide, a US6 polypeptide, a US7 polypeptide, a US8 polypeptide, a US9 polypeptide, a US10 polypeptide, a US11 polypeptide, a US14 polypeptide, a US15 polypeptide, a US16 polypeptide, a US17 polypeptide, a US18 polypeptide, a US19 polypeptide, a US20 polypeptide, a US21 polypeptide, a US22 polypeptide, a US30 polypeptide, a UL82 polypeptide, or a UL83 polypeptide.The hCMV vector can lack UL23 polypeptide, US1 polypeptide, US2 polypeptide, US3 polypeptide, US4 polypeptide, US5 polypeptide, US6 polypeptide, US7 polypeptide, US8 polypeptide, US9 polypeptide, US10 polypeptide, US11 polypeptide, US14 polypeptide, US15 polypeptide, US16 polypeptide, US17 polypeptide, US18 polypeptide, US19 polypeptide, US20 polypeptide, US21 polypeptide, US22 polypeptide, US30 polypeptide, UL82 polypeptide or UL83 polypeptide. The cell can be a fibroblast, an epithelial cell, an endothelial cell, a mononuclear cell or a glial cell. The viral gene transfer vector genome can be a lentivirus vector genome, a retrovirus vector genome, an AAV vector genome, a picornavirus vector genome, a rhabdovirus vector genome or a coronavirus vector genome. The viral gene transfer vector genome can include a nucleic acid sequence encoding a therapeutic polypeptide or an antigen receptor polypeptide, and the infectious vector particle including the gene transfer vector genome can infect immune cells in a mammal, and the infected immune cells can induce the expression of the therapeutic polypeptide or the antigen receptor polypeptide. In some cases, the viral gene transfer vector genome can include a nucleic acid sequence encoding a therapeutic polypeptide. The therapeutic polypeptide can be a chemokine, a cytokine, an anti-PD1 antibody, an anti-PDL-1 antibody, an anti-CTLA-4 antibody, or a CD47 polypeptide. In some cases, the viral gene transfer vector genome can include a nucleic acid sequence encoding an antigen receptor polypeptide. The antigen receptor polypeptide can be a CAR polypeptide (e.g., an anti-CD19 CAR). The immune cell can be a T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell. The viral gene transfer vector can be replication-deficient in the infected immune cell.The viral gene transfer vector genome can include one or more packaging elements selected from the group consisting of a 5' long terminal repeat (LTR), a 3' LTR, a psi (Ψ) element, a Rev response element (RRE) and a central polypurine tract / central termination sequence (cPPT / CTS). The nucleic acid encoding one or more helper polypeptides can include a nucleic acid encoding an envelope polypeptide selected from the group consisting of a vesicular stomatitis virus G (VSV G) polypeptide and a murine leukemia virus (MLV) 4070A polypeptide. The released viral gene transfer vector can include an envelope polypeptide.

[0010] In another aspect, the present disclosure is directed to a method for treating a mammal having cancer. The method can comprise or consist essentially of administering a recombinant hCMV vector to a mammal having cancer, the hCMV vector being capable of infecting cells of the mammal, the hCMV comprising heterologous nucleic acid encoding (i) a heterologous viral gene transfer vector genome comprising a nucleic acid sequence encoding an antigen receptor polypeptide targeting a cancer antigen of the cancer, and (ii) one or more helper polypeptides for amplifying and packaging the heterologous viral gene transfer vector genome to obtain infectious vector particles, the hCMV-infected cells produce and release infectious vector particles comprising the gene transfer vector genome, the released infectious vector particles being capable of infecting immune cells in the mammal and inducing expression of the antigen receptor polypeptide by the infected immune cells, and the infected immune cells expressing the antigen receptor reduce the number of cancer cells in the mammal. The mammal can be a human. The cancer can be brain cancer, B-cell lymphoma, ALL, CLL, neuroblastoma, breast cancer or lung cancer. The cancer antigen may be cluster of differentiation 19 (CD19), alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, mucin 1 (MUC-1), epithelial tumor antigen (ETA), melanoma associated antigen (MAGE), human epidermal growth factor receptor 2 (HER2), biotin, EGFRvIII, CD171 or mesothelin. The hCMV vector may be replication competent in the cell. The hCMV vector may lack a nucleic acid encoding a UL138 polypeptide, a UL144 polypeptide, a UL146 polypeptide, a UL147 polypeptide or miR-UL148D. The hCMV vector may lack a UL138 polypeptide, a UL144 polypeptide, a UL146 polypeptide, a UL147 polypeptide or miR-UL148D. The hCMV vector may be derived from the hCMV AD169 strain or the hCMV Merlin strain.The hCMV vector can lack a nucleic acid encoding a UL23 polypeptide, a US1 polypeptide, a US2 polypeptide, a US3 polypeptide, a US4 polypeptide, a US5 polypeptide, a US6 polypeptide, a US7 polypeptide, a US8 polypeptide, a US9 polypeptide, a US10 polypeptide, a US11 polypeptide, a US14 polypeptide, a US15 polypeptide, a US16 polypeptide, a US17 polypeptide, a US18 polypeptide, a US19 polypeptide, a US20 polypeptide, a US21 polypeptide, a US22 polypeptide, a US30 polypeptide, a UL82 polypeptide, or a UL83 polypeptide. The hCMV vector can lack a UL23 polypeptide, a US1 polypeptide, a US2 polypeptide, a US3 polypeptide, a US4 polypeptide, a US5 polypeptide, a US6 polypeptide, a US7 polypeptide, a US8 polypeptide, a US9 polypeptide, a US10 polypeptide, a US11 polypeptide, a US14 polypeptide, a US15 polypeptide, a US16 polypeptide, a US17 polypeptide, a US18 polypeptide, a US19 polypeptide, a US20 polypeptide, a US21 polypeptide, a US22 polypeptide, a US30 polypeptide, a UL82 polypeptide, or a UL83 polypeptide. The cell may be a fibroblast, an epithelial cell, an endothelial cell, a mononuclear cell or a glial cell. The viral gene transfer vector genome may be a lentivirus vector genome, a retrovirus vector genome, an AAV vector genome, a picornavirus vector genome, a rhabdovirus vector genome or a coronavirus vector genome. The viral gene transfer vector may comprise a nucleic acid encoding one or more packaging elements selected from the group consisting of 5'LTR, 3'LTR, a psi (Ψ) element, an RRE and a cPPT / CTS. The nucleic acid encoding one or more helper polypeptides may comprise a nucleic acid encoding an envelope polypeptide selected from the group consisting of a VSV G protein or an MLV 4070A protein. The released viral gene transfer vector may comprise an envelope polypeptide.

[0011] In another aspect, the present disclosure is directed to a method for treating a mammal with an infectious disease. The method can comprise or consist essentially of administering a recombinant hCMV vector to a mammal with an infectious disease, the hCMV vector being capable of infecting cells of the mammal, the hCMV vector comprising heterologous nucleic acid encoding (i) a heterologous viral gene transfer vector genome derived from a virus causing the infectious disease, and (ii) one or more helper polypeptides for amplifying and packaging the heterologous viral gene transfer vector genome to obtain an infectious vector particle, the hCMV vector-infected cells produce and release infectious vector particles comprising the gene transfer vector genome, the infectious vector particles being recognized by APCs in the mammal, whereby the APCs present antigens from the viral gene transfer vector to T cells in the mammal, and the T cells recognize and destroy the virus causing the infectious disease in the mammal. The mammal can be a human. The infectious disease can be viral pneumonia, acquired immune deficiency syndrome, or viral hepatitis. The hCMV vector can be replication-competent in the cells. The hCMV vector can lack the nucleic acid encoding UL138 polypeptide, UL144 polypeptide, UL146 polypeptide, UL147 polypeptide or miR-UL148D. The hCMV vector can lack the nucleic acid encoding UL138 polypeptide, UL144 polypeptide, UL146 polypeptide, UL147 polypeptide or miR-UL148D. The hCMV vector can be derived from hCMV AD169 strain or hCMV Merlin strain. The hCMV vector can lack the nucleic acid encoding UL23 polypeptide, US1 polypeptide, US2 polypeptide, US3 polypeptide, US4 polypeptide, US5 polypeptide, US6 polypeptide, US7 polypeptide, US8 polypeptide, US9 polypeptide, US10 polypeptide, US11 polypeptide, US14 polypeptide, US15 polypeptide, US16 polypeptide, US17 polypeptide, US18 polypeptide, US19 polypeptide, US20 polypeptide, US21 polypeptide, US22 polypeptide, US30 polypeptide, UL82 polypeptide or UL83 polypeptide.The hCMV vector can lack UL23 polypeptide, US1 polypeptide, US2 polypeptide, US3 polypeptide, US4 polypeptide, US5 polypeptide, US6 polypeptide, US7 polypeptide, US8 polypeptide, US9 polypeptide, US10 polypeptide, US11 polypeptide, US14 polypeptide, US15 polypeptide, US16 polypeptide, US17 polypeptide, US18 polypeptide, US19 polypeptide, US20 polypeptide, US21 polypeptide, US22 polypeptide, US30 polypeptide, UL82 polypeptide or UL83 polypeptide. The cell can be a fibroblast, an epithelial cell, an endothelial cell, a mononuclear cell or a glial cell. The viral gene transfer vector genome can be a lentivirus vector genome, a retrovirus vector genome, an AAV vector genome, a picornavirus vector genome, a rhabdovirus vector genome or a coronavirus vector genome. The viral gene transfer vector genome can include a nucleic acid encoding one or more packaging elements selected from the group consisting of 5'LTR, 3'LTR, psi (Ψ) element, RRE and cPPT / CTS. The nucleic acid encoding the one or more helper polypeptides can include a nucleic acid encoding an envelope polypeptide selected from the group consisting of a VSV G protein or an MLV 4070A protein. The released viral gene transfer vector can include an envelope polypeptide.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.The present invention can be carried out using methods and materials similar or equivalent to those described herein, and suitable methods and materials are described below.All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will take precedence.In addition, the materials, methods and examples are illustrative only and are not intended to be limiting.

[0013] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will become apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0014] [Figure 1] Illustrates the construction of an exemplary hCMV-BAC. A BAC vector containing a left homology arm (hCMV short-stranded unique (US) US27-28 sequence) and a right homology arm (hCMV US29-30 sequence) was linearized and co-transfected with AD169 or Merlin linear genome into MRC-5 cells to generate recombinant hCMV-BAC virus. Green plaques of recombinant virus were purified and circular viral DNA was isolated from hCMV-BAC infected cells. Circular viral BAC DNA was transformed into E. coli DH10B cells, and colonies containing full-length hCMV genome were selected. BAC plasmids carrying the hCMV genome were then extracted and introduced into E. coli SW102 cells containing the lambda red recombination system. HCMV-BAC plasmids can be genetically manipulated in SW102 cells, and the BAC sequence can be transferred by transfecting the BAC containing plasmid into cells stably expressing Cre recombinase. [Diagram 2] 2A-2C are diagrams showing the construction of an exemplary tropism-restored hCMV. Fig. 2A: The long-chain unique (UL) UL131a-like sequence of the hCMV AD169 strain is replaced with a functional UL131 from the Merlin strain. Fig. 2B: The restored tropism is demonstrated in U251 human glioma cells. [Figure 3A]1 shows the construction of an exemplary hCMV vector that can encode and drive the synthesis of lentivirus. Two hCMV vectors were constructed. Each vector contains nucleic acid that can express the HIV-derived lentiviral vector genome and also express structural polypeptides for packaging the HIV-derived lentiviral vector genome (e.g., gag nucleic acid encoding Gag protein, pol nucleic acid encoding DNA polymerase, rev nucleic acid encoding transactivator protein, and nucleic acid encoding glycoprotein or envelope (Env) protein). One hCMV vector expresses VSV-G glycoprotein (AD169-BAC R5.3) for packaging the HIV-derived lentiviral vector genome, and the other hCMV expresses amphotropic murine leukemia virus (MLV) 4070A Env protein (AD169-BAC R5.4) for packaging the HIV-derived lentiviral vector genome. The HIV-derived lentiviral vector genome contains the HIV-LTR, HIV-1Ψ, RRE, cPPT / CTS, a transgene driven by a promoter sequence (here, dTomato is used as a reporter gene) and an enhancer sequence. [Figure 3B] 1 shows the construction of an exemplary hCMV vector capable of encoding and driving the synthesis of lentivirus. Expression of the reporter gene from AD169-BAC R5.4 was demonstrated in MRC-5 human fibroblast cells 9 days post-transfection (9 dpt). [Figure 4] Figure 1 shows that hCMV vectors can encode and drive the synthesis of infectious lentivirus. hCMV vectors (AD169-BAC R5.3 or AD169-BAC R5.4) were transfected into 293T cells. Supernatants were harvested and refreshed every day. The harvested supernatants were then added to fresh 293T cells. Red plaques of lentivirus-transduced dTomato+GFP-293T were observed and counted on day 4 post-infection. [Diagram 5]Figure 3. Optimization of lentivirus synthesis efficiency of hCMV R5.4. Mutant hCMV R5.4 vectors were constructed by deletion of US1-US11, US14-US22 or UL23 and named R5.4.1, R5.4.2 and R5.4.4, respectively. R5.4.1, R5.4.2 and R5.4.4 vectors were transfected into 293T cells. Supernatants were collected on days 5 and 8 post-transfection. The collected supernatants were then added to fresh 293T cells. The red plaques of lentivirus-transduced dTomato+GFP-293T were observed and counted on day 4 post-infection. [Figure 6] Figure 1. Optimization of lentivirus synthesis efficiency of hCMVR5.3 and R5.4. Mutant hCMVR5.4 / R5.3 vectors were constructed by deletion of US14-US22, US30, UL23, UL83(pp65) or UL83(pp71) and named R5.4.2 / R5.3.2, R5.4.3 / R5.3.3, R5.4.4 / R5.3.4, R5.4.5 / R5.3.5, and R5.4.6 / R5.3.6, respectively. These mutant vectors were transfected into 293T cells. Supernatants were collected on days 3 and 6 after transfection. The collected supernatants were then added to fresh 293T cells. The red plaques of lentivirus-transduced dTomato+GFP-293T were observed and counted on day 4 after infection. [Figure 7]1 shows the construction of an exemplary hCMV vector that can encode lentivirus and drive its synthesis in a controllable manner. The vector expresses the HIV-derived lentivirus vector genome and contains nucleic acids capable of expressing structural polypeptides for packaging the HIV-derived lentivirus vector genome (e.g., gag nucleic acid encoding Gag protein, pol nucleic acid encoding DNA polymerase, rev nucleic acid encoding transactivator protein, and nucleic acid encoding Env protein). Expression of transactivator protein and VSV-G Env protein is driven by a drug-controllable promoter such as the tetracycline (tet)-off system (pRSV-tTA-pTight). The HIV-derived lentivirus vector genome includes HIV-LTR, HIV-1 Ψ, RRE, cPPT / CTS, and a transgene (CD19CAR) driven by lymphocyte-specific CD43 promoter sequence. dTomato was used as the reporter gene.

[0015] Detailed Description The present disclosure relates to methods and materials involved in the treatment of mammals (e.g., humans) with cancer and / or infectious diseases. For example, the present disclosure provides recombinant hCMV vectors that are replication-competent in infected cells and designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn produce and release viral vectors (e.g., heterologous viral gene transfer vectors such as lentiviral vectors that can infect other cells (e.g., T cells) in vivo and / or be recognized and internalized by APCs), and optionally drive the expression of exogenous polypeptides (e.g., therapeutic polypeptides or antigen receptors such as CARs) in the infected cells in the mammal (e.g., humans) to induce an immune response in the mammal. In some cases, the viral vector produced by and released from a cell infected with an hCMV vector provided herein can include a nucleic acid encoding an exogenous polypeptide (e.g., a therapeutic polypeptide or an antigen receptor such as a CAR), such that when the viral vector infects a cell (e.g., an immune cell such as a T cell recruited by an hCMV vector-infected cell), the viral vector-infected cell can express the exogenous polypeptide. For example, the recombinant hCMV vector provided herein can be designed such that the viral vector produced by and released from an hCMV vector-infected cell (e.g., a heterologous viral gene transfer vector such as a lentiviral vector) can include a nucleic acid encoding a therapeutic polypeptide, such that the viral vector can infect a cell (e.g., an immune cell such as a T cell recruited by an hCMV vector-infected cell) and the viral vector-infected cell can express the therapeutic polypeptide.For example, the recombinant hCMV vectors provided herein can be designed such that a viral vector (e.g., a heterologous viral gene transfer vector such as a lentiviral vector) produced by and released from an hCMV vector-infected cell can contain a nucleic acid encoding a CAR, such that the viral vector is capable of infecting a cell (e.g., an immune cell such as a T cell recruited by the hCMV vector-infected cell) and the viral vector-infected cell can express the CAR, thereby producing a CAR in vivo. + Immune cells (e.g., CAR + In some cases, the viral vector produced by and released from cells infected with the hCMV vectors provided herein can be derived from a pathogenic virus, so that the viral vector is internalized by APCs. For example, the recombinant hCMV vectors provided herein can be designed so that the viral vector produced by and released from cells infected with hCMV vectors (e.g., heterologous viral gene transfer vectors such as lentivirus vectors) can be recognized as pathogens by APCs, so that the viral vector is internalized by APCs and one or more polypeptides derived from the viral vector are presented as antigens to immune cells (e.g., T cells).

[0016] The recombinant hCMV vectors provided herein (e.g., recombinant hCMV vectors designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn generate and release viral vectors, such as lentiviral vectors, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) can infect any type of cell. In some cases, the recombinant hCMV vectors provided herein can infect fibroblasts, endothelial cells, mononuclear cells, and / or glial cells.

[0017] In some cases, a recombinant hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn produce and release viral vectors, such as lentiviral vectors, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) can be replication-competent (e.g., can be replication-competent in hCMV vector-infected cells).

[0018] In some cases, the recombinant hCMV vectors provided herein (e.g., recombinant hCMV vectors designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn produce and release viral vectors, such as lentiviral vectors, that are capable of infecting other cells, such as T cells, in vivo and / or that can be recognized and internalized by APCs) can be non-pathogenic (e.g., to the mammal being treated as described herein).

[0019] In some cases, the recombinant hCMV vectors provided herein (e.g., recombinant hCMV vectors designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn produce and release viral vectors, such as lentiviral vectors, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) are capable of infecting dividing cells (e.g., are capable of infecting only dividing cells).

[0020] In some cases, the recombinant hCMV vectors provided herein (e.g., recombinant hCMV vectors designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn produce and release viral vectors, such as lentiviral vectors, that are capable of infecting other cells, such as T cells, in vivo and / or that can be recognized and internalized by APCs) are capable of infecting non-dividing cells (e.g., are capable of infecting only non-dividing cells).

[0021] The recombinant hCMV vectors provided herein (e.g., recombinant hCMV vectors designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal that in turn generate and release viral vectors, such as lentiviral vectors, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) can be derived from any suitable hCMV (e.g., can include genomic elements, such as a nucleic acid encoding a polypeptide (or a fragment thereof)). Examples of hCMV include, but are not limited to, the hCMV AD169 strain, the hCMV Merlin strain, the hCMV Towne strain, the hCMV TB40 strain, and the hCMV Toledo strain.

[0022] The recombinant hCMV vectors provided herein (e.g., recombinant hCMV vectors designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn generate and release viral vectors, such as lentiviral vectors, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) can include one or more heterologous nucleotide sequences. As used herein, a heterologous nucleotide sequence can be any nucleotide sequence that is not naturally occurring in the hCMV (e.g., not naturally occurring in the hCMV prior to recombination). A nucleotide sequence that is not naturally occurring in the hCMV can be derived from any suitable source. In some cases, a nucleotide sequence that is not naturally occurring in the hCMV can be derived from a non-viral organism. In some cases, the nucleotide sequence that does not naturally occur in hCMV can be derived from a virus other than hCMV (e.g., a lentivirus, such as human immunodeficiency virus (HIV), adeno-associated virus (AAV), a retrovirus, vesicular stomatitis virus (VSV) or a murine leukemia virus (MLV), such as an amphotropic MLV). In some cases, the nucleotide sequence that does not naturally occur in hCMV can be derived from a CMV obtained from a different species. In some cases, the nucleotide sequence that does not naturally occur in hCMV can be derived from a different strain of hCMV (e.g., a serotypically distinct strain). In some cases, the nucleotide sequence that does not naturally occur in hCMV can be a synthetic nucleotide sequence.

[0023] In some cases, a recombinant hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn generate and release a viral vector, such as a lentiviral vector, capable of infecting other cells, such as T cells, in vivo and / or being recognized and internalized by APCs) can include one or more nucleic acids that enable the hCMV vector-infected cells to generate and release a viral vector (e.g., a heterologous viral gene transfer vector, such as a lentiviral vector). For example, a recombinant hCMV vector provided herein can include a nucleic acid that can encode one or more polypeptides for amplifying and / or packaging a viral vector genome (e.g., a heterologous viral gene transfer vector genome) to obtain an infectious vector particle comprising the viral vector genome. In some cases, a recombinant hCMV vector can include a viral vector genome (e.g., a viral vector genome derived from a virus other than hCMV) that includes a viral vector packaging element, and can include one or more nucleic acids encoding viral vector packaging polypeptides (e.g., a polypeptide that can package a viral vector genome to generate a viral vector).

[0024] The viral vector genome that can be encoded by the recombinant hCMV vector provided herein can be any suitable viral vector genome.In some cases, the viral vector genome is not a herpesvirus vector genome.In some cases, the viral vector genome is a viral gene transfer vector genome.The examples of the viral vector genome that can be encoded by the recombinant hCMV vector provided herein include, but are not limited to, lentivirus vector genome, retrovirus vector genome, AAV vector genome, picornavirus vector genome, rhabdovirus vector genome and coronavirus vector genome.The viral vector packaging elements that can be included in the viral vector genome that can be encoded by the recombinant hCMV vector provided herein include, but are not limited to, long terminal repeat (LTR), including 5'LTR and 3'LTR (each can include U3 region, R region and U5 region), psi (Ψ) element, Rev response element (RRE) and central polypurine tract / central termination sequence (cPPT / CTS). Nucleic acids encoding viral vector packaging polypeptides (e.g., polypeptides capable of packaging a viral vector genome to generate a viral vector) that can be included in the recombinant hCMV vectors provided herein can encode any suitable viral vector packaging polypeptide. Examples of nucleic acids encoding viral vector packaging polypeptides include, but are not limited to, gag nucleic acids (e.g., nucleic acids encoding group-specific antigen (Gag) proteins), pol nucleic acids (e.g., nucleic acids encoding DNA polymerases), rev nucleic acids (e.g., nucleic acids encoding transactivator proteins), and env nucleic acids (e.g., nucleic acids encoding envelope proteins with or without glycoproteins).

[0025] In some cases, the viral vector genome (e.g., a viral gene transfer vector genome, such as a lentiviral vector genome) that can be encoded by a recombinant hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn generate and release a viral vector, such as a lentiviral vector, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) can also include a transgene (e.g., a nucleic acid encoding a therapeutic polypeptide or an antigen receptor, such as a CAR). When a recombinant hCMV vector provided herein includes a transgene, the transgene can be any suitable transgene. In some cases, the transgene can be a nucleotide sequence that encodes a detectable label. Examples of detectable labels include, but are not limited to, fluorescent dyes (e.g., green fluorescent protein (GFP), mCherry, yellow fluorescent protein (YFP), cyan fluorescent protein (CFP) and dTomato), enzymes (e.g., luciferase, CRISPR-associated protein 9 (Cas9), Cre recombinase, restriction enzymes, convertases, thymidine kinase and sodium / iodide symporter (NIS)).

[0026] In some cases, a transgene that can be included in a viral vector genome (e.g., a viral gene transfer vector genome, such as a lentiviral vector genome) that can be encoded by a recombinant hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn generate and release a viral vector, such as a lentiviral vector, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) can include a nucleotide sequence encoding a receptor (e.g., a CAR). A receptor, such as a CAR, can target any suitable antigen (e.g., a cancer antigen). Examples of antigens that may be targeted by receptors encoded by transgenes in the recombinant hCMV vectors provided herein include, but are not limited to, cluster of differentiation 19 (CD19, associated with B cell lymphoma, acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL)), B cell maturation antigen (BCMA, associated with multiple myeloma (MM)), alpha fetoprotein (AFP, associated with germ cell tumors and / or hepatocellular carcinoma), carcinoembryonic antigen (CEA , associated with intestinal, lung and / or breast cancer), CA-125 (associated with ovarian cancer), mucin 1 (MUC-1, associated with breast cancer), epithelial tumor antigen (ETA, associated with breast cancer), melanoma associated antigen (MAGE, associated with malignant melanoma), human epidermal growth factor receptor 2 (HER2, associated with breast cancer), biotin (associated with EGFRvIII positive cancers), EGFRvIII (associated with glioma), CD171 (associated with neuroblastoma) and mesothelin (associated with mesothelioma, pancreatic cancer and non-small cell lung cancer).

[0027] In some cases, a transgene that can be included in a viral vector genome (e.g., a viral gene transfer vector genome, such as a lentiviral vector genome) that can be encoded by a recombinant hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal that in turn generate and release a viral vector, such as a lentiviral vector, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) can include a nucleotide sequence that encodes a therapeutic polypeptide. Examples of therapeutic polypeptides include, but are not limited to, immune regulators, such as chemokines and cytokines, antibodies, such as antibodies that inhibit immune checkpoint molecules (e.g., PD-1, PDL-1, and CTLA-4), and CD47 polypeptides.

[0028] When a transgene that can be included in a viral vector genome (e.g., a viral gene transfer vector genome, such as a lentiviral vector genome) that can be encoded by a recombinant hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn generate and release a viral vector, such as a lentiviral vector, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) includes a nucleotide sequence that encodes a CAR, the CAR can be any suitable CAR. The CAR can include an antigen binding domain, an optional hinge, a transmembrane domain, and one or more signaling domains. The antigen binding domain of a CAR that can be expressed by a viral vector (e.g., a heterologous viral gene transfer vector, such as a lentiviral vector) generated by and released from a cell infected with a recombinant hCMV vector provided herein that can be administered to a mammal (e.g., a human) as described herein can be any suitable antigen binding domain. In some cases, the antigen binding domain can include an antibody or a fragment thereof that targets an antigen (e.g., a cancer antigen such as a CD19 polypeptide). Examples of antigen binding domains include, but are not limited to, an antigen binding fragment (Fab), a variable region of an antibody heavy (VH) chain, a variable region of a light (VL) chain, a single chain variable fragment (scFv), and a domain of a growth factor that binds to a cancer cell-specific receptor (e.g., a domain of EGF, PDGR, FGF, TGF, or derivatives thereof). In some cases, the antigen binding domain can target (e.g., target and bind to) a tumor-specific antigen. For example, the recombinant hCMV vectors provided herein can be designed to generate a viral vector (e.g., a heterologous viral gene transfer vector such as a lentiviral vector) that can express a CAR that can bind to a tumor-specific antigen (e.g., an antigen that is present on cancer cells and has minimal or no expression on non-cancer cell types).In some cases, the antigen binding domain of the CAR may be as described elsewhere (e.g., U.S. Patent Application Publication No. 2017 / 0183418, e.g., U.S. Patent Application Publication No. 2017 / 0183418, paragraph

[0015] and the Sequence Listing; U.S. Patent Application Publication No. 2017 / 0183413, e.g., U.S. Patent Application Publication No. 2017 / 0183413, paragraph

[0049] , FIG. 2, Table 9 and the Sequence Listing; U.S. Patent Application Publication No. 2018 / 0291079, e.g., U.S. Pat. See U.S. Patent Application Publication No. 2018 / 0291079, paragraphs

[0041] -

[0045] and Table 4; U.S. Patent Application Publication No. 2020 / 0289563, e.g., U.S. Patent Application Publication No. 2020 / 0289563, paragraphs

[0006] -

[0053] ,

[0186] -

[0189] and Table 1; and U.S. Patent Application Publication No. 2003 / 0211097, e.g., U.S. Patent Application Publication No. 2003 / 0211097, paragraphs

[0081] and [0211-0215] and the sequence listing).

[0029] In some cases, the CAR that can be expressed by a viral vector (e.g., a heterologous viral gene transfer vector, such as a lentiviral vector) produced by and released from a cell infected with a recombinant hCMV vector provided herein can include an optional hinge region. In some cases, the hinge region can be located between the antigen-binding domain and the transmembrane domain of the CAR. In some cases, the hinge region can provide a CAR with improved flexibility of the antigen-binding domain. For example, the hinge region can reduce the spatial restriction of the antigen-binding domain of the CAR and its target antigen (e.g., to increase the binding between the antigen-binding domain of the CAR and its target antigen). Examples of hinge regions that can be used as described herein include, but are not limited to, the membrane proximal region from IgG, the membrane proximal region from CD8, and the membrane proximal region from CD28. In some cases, the hinge region of the CAR may be as described elsewhere (see, e.g., U.S. Patent Application Publication No. 2018 / 0000914, e.g., U.S. Patent Application Publication No. 2018 / 0000914, paragraph

[0168] and Table 1; U.S. Patent Application Publication No. 2017 / 0183418, e.g., U.S. Patent Application Publication No. 2017 / 0183418, paragraphs

[0034] ,

[0037] ,

[0040] and Table 2; U.S. Patent Application Publication No. 2017 / 0183413, e.g., U.S. Patent Application Publication No. 2017 / 0183413, paragraph

[0116] ; and U.S. Patent Application Publication No. 2017 / 0145094, e.g., U.S. Patent Application Publication No. 2017 / 0145094, paragraph

[0104] ).

[0030] The transmembrane domain of the CAR that can be expressed by a viral vector (e.g., a heterologous viral gene transfer vector, such as a lentiviral vector) produced by and released from a cell infected with a recombinant hCMV vector provided herein that can be administered to a mammal (e.g., a human) as described herein can include any suitable transmembrane domain. The transmembrane domain can be located between the antigen binding domain and the signaling domain of the CAR, and / or between the hinge and the signaling domain of the CAR. In some cases, the transmembrane domain can provide structural stability to the CAR. For example, the transmembrane domain can include a structure (e.g., a hydrophobic alpha-helical structure) that can span the cell membrane and anchor the CAR to the cell membrane. Examples of transmembrane domains that can be used as described herein include, but are not limited to, CD3zeta transmembrane domain, CD4 transmembrane domain, CD8 (e.g., CD8α) transmembrane domain, CD28 transmembrane domain, CD16 transmembrane domain, and erythropoietin receptor transmembrane domain. In some cases, the transmembrane domain of the CAR may be as described elsewhere (e.g., U.S. Patent Application Publication No. 2016 / 0120906, e.g., U.S. Patent Application Publication No. 2016 / 0120906, paragraphs

[0155] ,

[0161] ,

[0269] , FIG. 4 and FIG. 11; U.S. Patent Application Publication No. 2019 / 0209616, e.g., U.S. Patent Application Publication No. 2019 / 0209616, paragraph

[0026] ; U.S. Patent Application Publication No. 2018 / 0000914, e.g., U.S. Patent Application Publication No. 2018 / 000 (See U.S. Patent Application Publication No. 2017 / 0183414, paragraphs

[0168] -

[0171] ; U.S. Patent Application Publication No. 2017 / 0183418, e.g., U.S. Patent Application Publication No. 2017 / 0183418, paragraphs

[0116] -

[0118] ; U.S. Patent Application Publication No. 2017 / 0183413, e.g., U.S. Patent Application Publication No. 2017 / 0183413, paragraphs

[0116] -

[0118] ; and U.S. Patent Application Publication No. 2017 / 0145094, e.g., U.S. Patent Application Publication No. 2017 / 0145094, paragraphs

[0104] -

[0107] ).

[0031] The one or more signaling domains of a CAR that can be expressed by a viral vector (e.g., a heterologous viral gene transfer vector, such as a lentiviral vector) produced and released by a cell infected with a recombinant hCMV vector provided herein that can be administered to a mammal (e.g., a human) as described herein can include any suitable signaling domain or combination of signaling domains (e.g., a combination of two, three, or four signaling domains). In some cases, the signaling domain of a CAR can be an intracellular signaling domain normally found in T cells or NK cells. Examples of signaling domains that can be used as described herein include, but are not limited to, CD2 signaling domain, CD3ζ signaling domain, CD28 signaling domain, Toll-like receptor (TLR) signaling domain (e.g., TLR3 or TLR4 signaling domain), CD27 intracellular signaling domain, OX40 (CD134) intracellular signaling domain, 4-1BB (CD137) intracellular signaling domain, CD278 intracellular signaling domain, DAP10 intracellular signaling domain, DAP12 intracellular signaling domain, FceRly intracellular signaling domain, CD278 intracellular signaling domain, CD122 intracellular signaling domain, CD132 intracellular signaling domain, CD70 intracellular signaling domain, cytokine receptor intracellular signaling domain, and CD40 intracellular signaling domain. In some cases, a CAR for use as described herein can be designed to be a first generation CAR with a CD3ζ intracellular signaling domain. In some cases, a CAR for use as described herein can be designed to be a second generation CAR with a CD28 intracellular signaling domain and then a CD3ζ intracellular signaling domain.In some cases, a CAR for use as described herein can be designed to be a third generation CAR having (a) a CD28 intracellular signaling domain, followed by (b) a CD27 intracellular signaling domain, an OX40 intracellular signaling domain, or a 4-1BB intracellular signaling domain, followed by (c) a CD3ζ intracellular signaling domain. In some cases, the intracellular signaling domain of the CAR can be as described elsewhere (see, e.g., U.S. Patent Application Publication No. 2018 / 0000914, e.g., U.S. Patent Application Publication No. 2018 / 0000914, paragraphs

[0164] -

[0167] , and U.S. Patent Application Publication No. 2017 / 0183413, e.g., U.S. Patent Application Publication No. 2017 / 0183413, paragraphs

[0112] -

[0115] ).

[0032] Examples of CARs that may be expressed by viral vectors (e.g., heterologous viral gene transfer vectors such as lentiviral vectors) produced and released from cells infected with a recombinant hCMV vector provided herein as described herein include, but are not limited to, EGFRvIII CARs, GD2 CARs, IL-13RA CARs, CD19 CARs, BCMA CARs, CD138 CARs, NKG2-D CARs, HER2 CARs, CD137 CARs, and B7-H3 CARs.

[0033] In addition to a transgene (e.g., a nucleic acid encoding a therapeutic polypeptide or an antigen receptor such as a CAR), a viral vector genome that may be encoded by a recombinant hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn generate and release a viral vector, such as a lentiviral vector, capable of infecting other cells, such as T cells, in vivo and / or being recognized and internalized by APCs) may also include one or more regulatory elements operably linked to the transgene. Such regulatory elements may include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements that regulate the expression (e.g., transcription or translation) of the nucleic acid. The choice of regulatory elements that may be included in the viral vector genome depends on several factors, including, but not limited to, inducibility, targeting, and the desired expression level. For example, a promoter may be included in the viral vector genome to facilitate transcription of the transgene. The promoter may be a naturally occurring promoter or a recombinant promoter. Promoters can be ubiquitous or inducible (e.g., in the presence of tetracycline) and can affect expression of nucleic acids encoding polypeptides in a general or tissue-specific manner (e.g., T cell-specific promoters, NK cell-specific promoters, B cell-specific promoters and NKT cell-specific promoters).Examples of promoters that can be used to drive expression of an importin polypeptide in a cell include, but are not limited to, the cytomegalovirus / chicken β-actin (CBA) promoter, the cytomegalovirus (CMV) promoter, the ubiquitin C (UbC) promoter, the EF1a promoter, the Rous sarcoma virus long terminal repeat (RSV) promoter, the human T-lymphotropic virus type I (HTLV-I) promoter, the CD43 promoter, the B29 promoter, the CD14 promoter, and the CD3D promoter. As used herein, "operably linked" refers to the positioning of a regulatory element in the viral vector genome relative to a transgene so as to permit or promote expression of the encoded polypeptide. For example, the viral vector genome can include a promoter and a transgene. In this case, the promoter is operably linked to the transgene and drives expression of the transgene in the cell.

[0034] In some cases, the viral vector genome that can be encoded by the recombinant hCMV vectors provided herein can include the nucleic acid sequence set forth in SEQ ID NO:1 (see, e.g., Example 2).

[0035] In some cases, the viral vector genome that may be encoded by the recombinant hCMV vectors provided herein may be as described elsewhere (see, e.g., FIG. 2 of U.S. Patent Application Publication No. 2004 / 014702).

[0036] In some cases, the genome of a recombinant hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal that in turn generate and release a viral vector, such as a lentiviral vector, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) can include a functional UL132 sequence. For example, the genome of a recombinant hCMV vector provided herein can include a UL132 sequence that can encode a functional UL132 polypeptide that enables the recombinant hCMV vector to be replication-competent (e.g., to maintain infectious virus production) in infected cells in a mammal.

[0037] In some cases, the genome of a recombinant hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn generate and release a viral vector, such as a lentiviral vector, capable of infecting other cells, such as T cells, in vivo and / or being recognized and internalized by APCs) can include one or more modifications to one or more nucleic acids encoding a polypeptide and / or one or more viral elements of the hCMV genome. The one or more modifications can be any suitable modification. In some cases, the one or more modifications in the hCMV genome can alter the tropism of the recombinant hCMV vector. In some cases, the one or more modifications in the hCMV genome can promote viral latency. In some cases, the one or more modifications in the hCMV genome can recruit bone marrow cells. In some cases, the one or more modifications in the hCMV genome can minimize or inhibit an immune response to the recombinant hCMV vector. Examples of modifications that can be made to the nucleic acid or viral element encoding polypeptide include, but are not limited to, deletion, insertion and substitution.For example, the recombinant hCMV vector provided herein can comprise one or more deletions in the hCMV genome.In some cases, the recombinant hCMV vector provided herein can have at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) deletion (e.g., complete or partial deletion) of UL23, US1-US11, US30, US14-22, US30, UL83, UL82, UL138, UL144, UL146, UL147 and miR-UL148D.For example, the recombinant hCMV vectors provided herein can have a deletion of at least one of UL23, US1-US11, US30, US14-22, US30, UL83, UL82, UL138, UL144, UL146, UL147, and miR-UL148D, such that the recombinant hCMV vector has deleted a nucleic acid encoding a UL23 polypeptide, a nucleic acid encoding a US1-US11 polypeptide, a nucleic acid encoding a US30 polypeptide, a nucleic acid encoding a US14-22 polypeptide, a nucleic acid encoding a US30 polypeptide, a nucleic acid encoding a UL83 polypeptide, a nucleic acid encoding a UL82 polypeptide, a nucleic acid encoding a UL138 polypeptide, a nucleic acid encoding a UL144 polypeptide, a nucleic acid encoding a UL146, and / or a nucleic acid encoding a UL147 polypeptide. For example, the recombinant hCMV vectors provided herein can have a deletion (e.g., a complete or partial deletion) of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) of UL23, US1-US11, US30, US14-22, US30, UL83, UL82, UL138, UL144, UL146 and UL147, such that the recombinant hCMV vector is deleted from a UL23 polypeptide, a US1-US11 polypeptide, a US30 polypeptide, a US14-22 polypeptide, a US30 polypeptide, a UL83 polypeptide, a UL82 polypeptide, a UL138 polypeptide, a UL144 polypeptide, a UL146 and / or a UL147 polypeptide. In some cases, the recombinant hCMV vectors provided herein can include one or more substitutions within the hCMV genome. For example, the recombinant hCMV vectors provided herein can have (e.g., can be engineered to have) a replacement of the UL131 sequence with a heterologous UL131 sequence (e.g., replacement of the defective AD169 UL131a sequence with a functional UL131 sequence from an hCMV Merlin strain) or can have a repaired UL131 sequence (e.g., deletion of a single A nucleotide from the defective AD169 UL131a sequence at position 77).For example, a recombinant hCMV vector provided herein can have a UL131 sequence having the nucleotide sequence set forth in SEQ ID NO:2 (see, eg, Example 3).

[0038] When a recombinant hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn generate and release viral vectors, such as lentiviral vectors, capable of infecting other cells, such as T cells, in vivo and / or being recognized and internalized by APCs) contains one or more modifications to the hCMV genome, the one or more modifications can be effective to increase the production of viral vectors (e.g., heterologous viral gene transfer vectors, such as lentiviral vectors) from the recombinant hCMV vector (e.g., compared to a recombinant hCMV vector lacking the mutation). For example, a recombinant hCMV vector having a deletion of UL23, US1-US11, US30, US14-22, US30, UL83 and / or UL82 can exhibit increased production of viral vectors from the recombinant hCMV vector. In some cases, recombinant hCMV vectors having deletions of UL23, US1-US11, US30, US14-22, US30, UL83 and / or UL82 are 3 Transduction units (TU) / mL supernatant ~ approx. 10 6 TU / mL supernatant (e.g., approximately 10 3 TU / mL supernatant ~ approx. 10 5 TU / mL supernatant, approximately 10 3 TU / mL supernatant ~ approx. 10 4 TU / mL supernatant, approximately 10 4 TU / mL supernatant ~ approx. 10 6 TU / mL supernatant, approximately 10 5 TU / mL supernatant ~ approx. 10 6 TU / mL supernatant, or approximately 10 4 TU / mL supernatant ~ approx. 10 5For example, recombinant hCMV vectors with deletions of UL23, US1-US11, US30, US14-22, US30, UL83 and / or UL82 can produce approximately 10 4 TU / mL supernatant can be generated.

[0039] When a recombinant hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal that in turn generate and release a viral vector, such as a lentiviral vector, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) contains one or more modifications to the hCMV genome, the one or more modifications can be effective to alter the tropism of the recombinant hCMV vector (e.g., compared to a recombinant hCMV vector lacking the mutation). For example, a recombinant hCMV vector having a UL131 sequence from the hCMV Merlin, hCMV Towne, hCMV TB40, or hCMV Toledo strains can infect cell types that a recombinant hCMV vector lacking the mutation cannot infect. In some cases, recombinant hCMV vectors carrying the UL131 sequence from the hCMV Merlin, Towne, TB40 or Toledo strains can infect epithelial, endothelial, mononuclear and glial cells in addition to fibroblasts.

[0040] In some cases, a recombinant hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect a cell in a mammal, thereby generating hCMV vector-infected cells in the mammal that in turn generate and release a viral vector, such as a lentiviral vector, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) can include (e.g., can be designed to include) a lentiviral (e.g., HIV) gag nucleic acid, a lentiviral (e.g., HIV) pol nucleic acid, a lentiviral (e.g., HIV) rev nucleic acid, and a VSV-G env nucleic acid, as well as nucleic acids encoding a lentiviral genome including a lentiviral (e.g., HIV) LTR, a lentiviral (e.g., HIV) psi (Ψ) element, a lentiviral (e.g., HIV) RRE, and a lentiviral (e.g., HIV) cPPT / CTS.

[0041] In some cases, a recombinant hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect a cell in a mammal, thereby generating hCMV vector-infected cells in the mammal that in turn generate and release a viral vector, such as a lentiviral vector, that is capable of infecting other cells, such as T cells, in vivo and / or being recognized and internalized by APCs) can include (e.g., can be designed to include) a lentiviral (e.g., HIV) gag nucleic acid, a lentiviral (e.g., HIV) pol nucleic acid, a lentiviral (e.g., HIV) rev nucleic acid, and an MLV env nucleic acid, as well as nucleic acids encoding a lentiviral genome including a lentiviral (e.g., HIV) LTR, a lentiviral (e.g., HIV) psi (Ψ) element, a lentiviral (e.g., HIV) RRE, and a lentiviral (e.g., HIV) cPPT / CTS.

[0042] In some cases, a recombinant hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn produce and release viral vectors, such as lentiviral vectors, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) may be as shown in FIG. 3A.

[0043] The viral vectors produced by and released from cells infected with the hCMV vectors provided herein (e.g., recombinant hCMV vectors designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn produce and release viral vectors, such as lentiviral vectors, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) can infect any type of cell. In some cases, the viral vectors produced by and released from cells infected with the hCMV vectors provided herein can infect immune cells. Examples of immune cells that can be infected with the viral vectors produced by and released from cells infected with the hCMV vectors provided herein include, but are not limited to, T cells, NK cells, and NKT cells.

[0044] In some cases, the viral vector produced by and released from a cell infected with an hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect a cell in a mammal, thereby generating an hCMV vector-infected cell in the mammal, which in turn produces and releases a viral vector, such as a lentiviral vector, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) can be replication-deficient (e.g., can be replication-deficient in a viral vector-infected cell, such as a viral vector-infected immune cell).

[0045] In some cases, the viral vector produced by and released from a cell infected with an hCMV vector provided herein (e.g., a recombinant hCMV vector designed to infect a cell in a mammal, thereby generating an hCMV vector-infected cell in the mammal, which in turn produces and releases a viral vector, such as a lentiviral vector, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs) can be integrated into the genome of the infected cell (e.g., can be integrated into the genome of a viral vector-infected cell, such as a viral vector-infected immune cell).

[0046] The present specification also provides methods and materials for using the recombinant hCMV vectors provided herein (e.g., one or more recombinant hCMV vectors designed to infect cells in a mammal, thereby generating hCMV vector-infected cells in the mammal, which in turn generate and release viral vectors, such as lentiviral vectors, that can infect other cells, such as T cells, in vivo and / or be recognized and internalized by APCs). In some cases, the recombinant hCMV vectors provided herein can be used to treat a mammal having or at risk of having cancer and / or an infectious disease. For example, a method of treating a mammal having cancer and / or an infectious disease can include administering one or more recombinant hCMV vectors provided herein to the mammal. When treating a mammal (e.g., a human) having cancer, one or more recombinant hCMV vectors provided herein can be administered to the mammal to reduce the number of cancer cells in the mammal (e.g., inhibit and / or slow tumor growth) and / or increase the survival of the mammal. When treating a mammal (e.g., a human) having an infectious disease, one or more recombinant hCMV vectors provided herein can be administered to the mammal to reduce the number of infected cells in the mammal and / or to reduce or eliminate the number of pathogens (e.g., pathogens that cause an infectious disease) present within the mammal.

[0047] Any suitable mammal (e.g., a mammal having cancer and / or an infectious disease) can be treated as described herein (e.g., by administration of one or more recombinant hCMV vectors provided herein). Examples of mammals that can be treated as described herein include, but are not limited to, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice, and rats. In some cases, one or more recombinant hCMVs provided herein (e.g., one or more recombinant hCMVs designed to generate and release viral vectors capable of infecting cells in vivo and / or being recognized and internalized by APCs) or nucleic acids (e.g., expression vectors) encoding the recombinant hCMV vectors provided herein can be administered to a human.

[0048] When the methods and materials provided herein are used to treat a mammal (e.g., a human) with cancer, the mammal can have any type of cancer. The cancer treatable as described herein can be a primary cancer or a metastatic cancer. In some cases, the cancer treated as described herein can include one or more solid tumors. In some cases, the cancer treated as described herein can be a blood cancer. Examples of cancer treatable as described herein include, but are not limited to, brain tumors (e.g., glioblastoma), B-cell lymphoma, ALL, CLL, neuroblastoma, breast cancer, and lung cancer.

[0049] When the methods and materials provided herein are used to treat a mammal (e.g., a human) with cancer, the method can also include a step of diagnosing the mammal as having cancer. Examples of methods for diagnosing a mammal as having cancer include, but are not limited to, physical examination, laboratory tests (e.g., blood and / or urine), biopsy, imaging tests (e.g., X-ray, PET / CT, MRI and / or ultrasound), nuclear medicine scans (e.g., bone scans), endoscopy and / or genetic testing. Once diagnosed with cancer, the mammal can be administered or instructed to self-administer one or more recombinant hCMVs described herein (e.g., one or more recombinant hCMVs designed to generate and release viral vectors capable of infecting cells in vivo and / or being recognized and internalized by APCs) or a nucleic acid (e.g., expression vector) encoding a recombinant hCMV vector provided herein.

[0050] When the methods and materials provided herein are used to treat a mammal (e.g., a human) with an infectious disease, the mammal may have or be at risk of developing any type of infectious disease. For example, one or more recombinant hCMVs described herein (e.g., one or more recombinant hCMVs designed to produce and release viral vectors capable of infecting cells in vivo and / or being recognized and internalized by APCs) or nucleic acids (e.g., expression vectors) encoding recombinant hCMV vectors provided herein may be administered to or directed to self-administer a mammal with an infectious disease to reduce the number of pathogens causing the infectious disease in the mammal. For example, one or more symptoms of the infectious disease may be reduced or eliminated by administering or directed to self-administer a nucleic acid (e.g., expression vectors) encoding one or more recombinant hCMVs described herein (e.g., one or more recombinant hCMVs designed to produce and release viral vectors capable of infecting cells in vivo and / or being recognized and internalized by APCs) or recombinant hCMV vectors provided herein to a mammal with an infectious disease. For example, the risk of developing an infectious disease can be reduced by administering or instructing a mammal at risk of developing an infectious disease to self-administer one or more recombinant hCMVs described herein (e.g., one or more recombinant hCMVs engineered to generate and release viral vectors capable of infecting cells in vivo and / or being recognized and internalized by APCs) or a nucleic acid (e.g., expression vector) encoding a recombinant hCMV vector provided herein prior to exposure to a pathogen that may cause the infectious disease. In some cases, the infectious disease may be caused by a virus. Examples of infectious diseases treatable as described herein include, but are not limited to, viral pneumonia, acquired immune deficiency syndrome (AIDS), and viral hepatitis.

[0051] When the methods and materials provided herein are used to treat a mammal (e.g., a human) having an infectious disease, the method can also include a step of diagnosing the mammal as having an infectious disease. Examples of methods for diagnosing a mammal as having an infectious disease include, but are not limited to, laboratory tests to check samples for specific microorganisms (e.g., laboratory tests of blood samples, urine samples, throat swabs, stool samples, and / or spinal tap samples) and / or imaging scans (e.g., X-rays, computed tomography (CT) and magnetic resonance imaging (MRI)). Once diagnosed with cancer, the mammal can be administered or instructed to self-administer one or more recombinant hCMVs provided herein (e.g., one or more recombinant hCMVs designed to generate and release viral vectors capable of infecting cells in vivo and / or being recognized and internalized by APCs) or a nucleic acid (e.g., an expression vector) encoding a recombinant hCMV vector provided herein.

[0052] A nucleic acid (e.g., expression vector) encoding one or more recombinant hCMV provided herein (e.g., one or more recombinant hCMVs designed to generate and release a viral vector capable of infecting cells in vivo and / or being recognized and internalized by APCs) or a recombinant hCMV vector provided herein can be administered to a mammal by any suitable route, such as intravenous, intramuscular, subcutaneous, oral, intranasal, inhalation, transdermal, and parenteral. In some cases, a nucleic acid (e.g., expression vector) encoding one or more recombinant hCMVs provided herein or a recombinant hCMV vector provided herein can be administered to a mammal (e.g., a human) intravenously.

[0053] Nucleic acids (e.g., expression vectors) encoding one or more recombinant hCMVs provided herein (e.g., one or more recombinant hCMVs designed to generate and release viral vectors capable of infecting cells in vivo and / or being recognized and internalized by APCs) or recombinant hCMV vectors provided herein can be formulated into compositions (e.g., pharmaceutical compositions) for administration to a mammal (e.g., a mammal having cancer and / or an infectious disease). For example, one or more recombinant hCMVs provided herein or nucleic acids (e.g., expression vectors) encoding recombinant hCMV vectors provided herein can be formulated into pharma- ceutically acceptable compositions for administration to a mammal having cancer and / or an infectious disease. In some cases, one or more recombinant hCMVs provided herein or nucleic acids (e.g., expression vectors) encoding recombinant hCMV vectors provided herein can be formulated with one or more pharma- ceutically acceptable carriers (additives) and / or diluents. Pharmaceutical compositions can be formulated for administration in solid or liquid forms, including, but not limited to, sterile solutions, suspensions, sustained release formulations, tablets, capsules, pills, powders, and granules. Pharmaceutically acceptable carriers, fillers and vehicles that can be used in the pharmaceutical compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool fat.

[0054] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0055] Example 1: Recombinant human cytomegalovirus vectors that synthesize non-herpes virus particles and their uses In this example, we describe the design of hCMV vectors that can be used for in vivo gene editing of immune cells.

[0056] hCMV BAC reconstruction system Because the DNA genomes of herpesviruses are too large to be cloned into a single plasmid or cosmid, bacterial artificial chromosome (BAC) technology has proven to be a valuable tool for herpesvirus reconstruction. BAC vectors can carry up to 300 kb of DNA sequence, and hCMV BACs can be generated by integration of the BAC vector into circular hCMV DNA via recombination. When combined with the lambda Red recombination system, various types of genetic manipulations can be introduced into the hCMV genome, including insertions, deletions, point mutations and fusions. All recombinant viruses and vectors were constructed using BAC technology and the lambda Red recombination system (Figure 1).

[0057] Tropism repair hCMV hCMV enters cells via the endothelial tropism pathway (via gH / gL / UL128-UL131) and the non-endothelial tropism pathway (via gH / gL / (gO)). The integrity of these two pathways affects the cell tropism of hCMV. Restoring the function of the gH / gL / UL128-UL131 complex improves cell fusion of hCMV-infected cells and confers tropism to hCMV to infect epithelial, endothelial and mononuclear cells (Figure 2), thereby improving the infectivity of hCMV vectors.

[0058] Construction of lentivirus-synthesizing hCMV vector, AD169-BAC gag-pol-rev-VSVG / 4070A-LV-EF1a-dtomato (AD169-BAC R5.3 / R5.4) Based on the HIV-derived lentiviral vector genome, hCMV AD169-BAC, HIV helper functions (gag, pol and rev) for packaging the vector genome and one type of envelope protein (VSV-G glycoprotein or amphotropic murine leukemia virus 4070A Env envelope protein) were inserted into different genomic sites of AD169-BAC to construct two types of hCMV vectors, AD169-BAC R5.3 (expressing VSVG) and AD169-BAC R5.4 (expressing 4070A) (Figure 3). The HIV-derived lentiviral gene transfer vector genome to be inserted consists of HIV-LTR, HIV-1Ψ, RRE, cPPT / CTS, a transgene driven by a promoter sequence, and an enhancer sequence.

[0059] When the lentivirus production efficiency of two types of hCMV vectors was tested in 293T cells, the AD169-BAC R5.3 vector showed higher lentivirus production (Figure 4).

[0060] Optimization of lentiviral synthesis efficiency of hCMV vector AD169-BAC R5.4 To further optimize the lentivirus synthesis efficiency of the hCMV vector, three regions of the hCMV genome were deleted from the AD169-BAC R5.4 backbone. Deletion of either US14-US22 or UL23 improved the generation of infectious lentivirus from undetectable to several hundred transduction units (TU) / mL supernatant (Figure 5). These results demonstrate that lentivirus production efficiency can be increased by mutating non-essential genes of hCMV.

[0061] Optimization of lentiviral synthesis efficiency of hCMV vectors AD169-BAC R5.3 and R5.4 To further optimize lentivirus synthesis efficiency of hCMV vectors, five segments of the hCMV genome were deleted from the AD169-BAC R5.3 and R5.4 backbones. Deletion of US14-US22, US30, UL23, UL83, or UL82 reduced infectious lentivirus production from undetectable to 10%. 2 ~10 4 The transduction units (TU) / mL supernatant was improved (Figure 6). These results demonstrate that lentivirus production efficiency can be further increased by mutating non-essential genes of hCMV.

[0062] Example 2: Genomic sequences of exemplary lentiviral gene transfer vectors Genomic sequence of the LV-EF1a-hCD19CAR-T2A-dTomato lentiviral gene transfer vector, which may be encoded by the recombinant hCMV vector provided herein.

[0063] SEQ ID NO:1 TIFF2025501656000001.tif36162TIFF2025501656000002.tif217162TIFF20255016560 00003.tif217162TIFF2025501656000004.tif217162TIFF2025501656000005.tif39162

[0064] Example 3: Exemplary AD169 UL131 Sequences TIFF2025501656000006.tif64162

[0065] Example 4: Generation of CAR T cells in vivo A recombinant hCMV vector capable of producing and releasing a viral vector (e.g., a heterologous viral gene transfer vector such as a lentiviral vector), the viral vector capable of infecting T cells in vivo and driving expression of a CAR in the infected T cells in a mammal (e.g., a human) to produce a CAR in the mammal. +A recombinant hCMV vector capable of generating T cells is engineered by replacing the promoter with a T cell specific promoter. For example, the nucleic acid encoding the dTomato polypeptide shown in Figure 3A is replaced with a transgene encoding a CD19 CAR, and the EF1a promoter shown in Figure 3A is replaced with a T cell specific promoter (e.g., the CD3D promoter).

[0066] Example 5: Construction of an inducible hCMV vector capable of lentivirus synthesis Based on hCMVAD169-BAC, we constructed an hCMV vector that can encode lentivirus and drive its synthesis in a drug-controllable manner. The following steps were used to generate an hCMV vector that can induce lentivirus synthesis.

[0067] The nucleic acid containing pRSV-tTA-pTight-rev was inserted between IRS1 and US1 of hCMV by homologous recombination.

[0068] The nucleic acid containing pTight-VSV-F-SV40pA-ZeoR-pEM7 was inserted into the hCMV vector in front of the pRSV-tTA-pTight-rev cassette.

[0069] The nucleic acid containing pCMV-gag-pol-β-globin polyA was inserted between UL25 and UL26 of hCMV.

[0070] The nucleic acid containing pRSV-truncated 5'UTR(HIV)-HIV-1 Ψ-RRE-cPPT / CTS-pCD43-CD19CAR-dTomato-WPRE-5'UTR(ΔU3)-SV40pA-pAmp-AmpR was inserted between US32 and US33A of hCMV.

[0071] A schematic demonstration of the construction of an hCMV vector encoding a lentivirus and capable of driving its synthesis in a drug-controllable manner is shown in FIG.

[0072] Example 6: Generation of an inducible hCMV vector capable of lentiviral synthesis A high-titer recombinant hCMV vector capable of producing and releasing a viral vector (e.g., a heterologous viral gene transfer vector such as a lentiviral vector) is produced in vitro. Fibroblast cells (e.g., human fibroblast cells such as MRC-5 cells or WI-38 cells) are infected with a recombinant hCMV vector capable of producing and releasing a viral vector (e.g., a heterologous viral gene transfer vector such as a lentiviral vector) in the absence of a specific drug (e.g., in the absence of tetracycline or doxycycline), and the synthesis of the viral vector is suppressed by administering the specific drug (e.g., tetracycline or doxycycline). For example, fibroblast cells (e.g., human fibroblast cells such as MRC-5 cells or WI-38 cells) are infected with an hCMV vector as shown in FIG. 7, and tetracycline is administered to suppress the synthesis of the viral vector. The recombinant hCMV vector is replicated by the infected cells, and a high-titer recombinant hCMV is produced. In some cases, the recombinant hCMV vector is purified from the cell culture.

[0073] Example 7: Generation of CAR T cells in vivo A recombinant hCMV vector capable of producing and releasing a viral vector (e.g., a heterologous viral gene transfer vector such as a lentiviral vector) in the absence of a particular drug (e.g., in the absence of tetracycline or doxycycline) is administered to a mammal to infect cells within the mammal. For example, an hCMV vector as shown in FIG. 7 is administered to a mammal. The hCMV vector-infected cells produce and release a viral vector (e.g., a heterologous viral gene transfer vector such as a lentiviral vector) capable of infecting T cells in vivo, and express a CAR to infect the CAR in the mammal. + T cells can be generated.

[0074] Example 8: Treatment of Cancer One or more recombinant hCMV vectors designed to produce and release a viral vector (e.g., a heterologous viral gene transfer vector, such as a lentiviral vector) capable of expressing a CD19 CAR and infecting cells in vivo are administered to a human diagnosed with a CD19-positive cancer. The recombinant hCMV vectors are capable of infecting cells in the human, such that the viral vectors produced by and released from the recombinant hCMV vectors can infect T cells in the human and express the CD19 CAR to generate CD19-CAR T cells in the human. The CD19-CAR T cells generated in vivo can target and destroy CD19-positive cancer cells in the human.

[0075] Example 9: Treatment of AIDS One or more recombinant hCMV vectors designed to produce and release HIV-derived lentiviral vectors are administered to a human diagnosed with AIDS. The recombinant hCMV vectors can infect cells in the human, so that the HIV-derived lentiviral vectors produced by and released from the recombinant hCMV vectors are internalized by APCs in vivo, so that one or more polypeptides from the HIV-derived lentiviral vectors are presented as antigens to T cells (e.g., naive T cells) in the human, inducing an immune response against HIV particles present in the human. The immune response against HIV particles present in the human reduces or eliminates one or more symptoms of AIDS.

[0076] Example 10: Prevention of AIDS One or more recombinant hCMV vectors designed to produce and release HIV-derived lentiviral vectors are administered to a human diagnosed as HIV-positive or at risk of exposure to HIV. The recombinant hCMV vectors can infect cells in the human, so that the HIV-derived lentiviral vectors produced by and released from the recombinant hCMV vectors are internalized by APCs in vivo, so that one or more polypeptides from the HIV-derived lentiviral vectors are presented as antigens to T cells (e.g., naive T cells) in the human, priming the T cells. In the presence of HIV particles, the primed T cells induce an immune response against the HIV particles present in the human. The immune response against the HIV particles present in the human prevents the onset of AIDS.

[0077] Other embodiments Although the invention has been described in conjunction with its detailed description, it is to be understood that the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention, which is defined by the appended claims, other aspects, advantages, and modifications of which are within the scope of the following claims.

Claims

1. A recombinant human cytomegalovirus (hCMV) vector capable of infecting a mammalian cell, said hCMV vector comprising a heterologous nucleic acid encoding (i) a heterologous viral gene transfer vector genome and (ii) one or more helper polypeptides for amplifying and packaging said heterologous viral gene transfer vector genome to obtain infectious vector particles, wherein said hCMV vector-infected cells produce and release said infectious vector particles comprising said gene transfer vector genome.

2. The hCMV vector of claim 1, wherein the hCMV vector is replication competent in the cell.

3. 3. The hCMV vector of claim 1 or claim 2, wherein the hCMV vector lacks a nucleic acid encoding a UL138 polypeptide, a UL144 polypeptide, a UL146 polypeptide, a UL147 polypeptide or miR-UL148D.

4. 3. The hCMV vector of claim 1 or claim 2, wherein the hCMV vector lacks a UL138 polypeptide, a UL144 polypeptide, a UL146 polypeptide, a UL147 polypeptide or miR-UL148D.

5. 5. The hCMV vector according to any one of claims 1 to 4, wherein the hCMV vector is derived from the hCMV AD169 strain or the hCMV Merlin strain.

6. 6. The hCMV vector of any one of claims 1 to 5, wherein the hCMV vector lacks a nucleic acid encoding a UL23 polypeptide, a US1 polypeptide, a US2 polypeptide, a US3 polypeptide, a US4 polypeptide, a US5 polypeptide, a US6 polypeptide, a US7 polypeptide, a US8 polypeptide, a US9 polypeptide, a US10 polypeptide, a US11 polypeptide, a US14 polypeptide, a US15 polypeptide, a US16 polypeptide, a US17 polypeptide, a US18 polypeptide, a US19 polypeptide, a US20 polypeptide, a US21 polypeptide, a US22 polypeptide, a US30 polypeptide, a UL82 polypeptide or a UL83 polypeptide.

7. 6. The hCMV vector of any one of claims 1 to 5, wherein the hCMV vector lacks a UL23 polypeptide, a US1 polypeptide, a US2 polypeptide, a US3 polypeptide, a US4 polypeptide, a US5 polypeptide, a US6 polypeptide, a US7 polypeptide, a US8 polypeptide, a US9 polypeptide, a US10 polypeptide, a US11 polypeptide, a US14 polypeptide, a US15 polypeptide, a US16 polypeptide, a US17 polypeptide, a US18 polypeptide, a US19 polypeptide, a US20 polypeptide, a US21 polypeptide, a US22 polypeptide, a US30 polypeptide, a UL82 polypeptide or a UL83 polypeptide.

8. 8. The hCMV vector of any one of claims 1 to 7, wherein the cell is a fibroblast, an epithelial cell, an endothelial cell, a mononuclear cell or a glial cell.

9. 9. The hCMV vector of any one of claims 1 to 8, wherein the viral gene transfer vector genome is a lentivirus vector genome, a retrovirus vector genome, an adeno-associated virus (AAV) vector genome, a picornavirus vector genome, a rhabdovirus vector genome or a coronavirus vector genome.

10. 10. The hCMV vector of any one of claims 1 to 9, wherein the viral gene transfer vector genome comprises a nucleic acid sequence encoding a therapeutic polypeptide or an antigen receptor polypeptide, and the infectious vector particle comprising the gene transfer vector genome is capable of infecting immune cells within the mammal, and the infected immune cells are capable of infecting immune cells for expression of the therapeutic polypeptide or the antigen receptor polypeptide.

11. The hCMV vector of claim 10, wherein the viral gene transfer vector genome comprises a nucleic acid sequence encoding the therapeutic polypeptide.

12. The hCMV vector of claim 11, wherein the therapeutic polypeptide is a chemokine, a cytokine, an anti-PD1 antibody, an anti-PDL-1 antibody, an anti-CTLA-4 antibody or a CD47 polypeptide.

13. The hCMV vector of claim 10, wherein the viral gene transfer vector genome comprises a nucleic acid sequence encoding the antigen receptor polypeptide.

14. The hCMV vector of claim 13, wherein the antigen receptor polypeptide is a chimeric antigen receptor (CAR) polypeptide.

15. The hCMV vector of claim 14, wherein the CAR polypeptide is an anti-CD19 CAR.

16. 16. The hCMV vector of any one of claims 10 to 15, wherein the immune cell is a T cell, a natural killer (NK) cell or a natural killer T (NKT) cell.

17. 17. The hCMV vector of any one of claims 10 to 16, wherein the viral gene transfer vector is replication-deficient in the infected immune cell.

18. 18. The hCMV vector of any one of claims 1 to 17, wherein the viral gene transfer vector genome comprises one or more packaging elements selected from the group consisting of a 5' long terminal repeat (LTR), a 3' LTR, a psi (Ψ) element, a Rev response element (RRE) and a central polypurine tract / central termination sequence (cPPT / CTS).

19. 19. The hCMV vector of any one of claims 1 to 18, wherein the nucleic acid encoding the one or more helper polypeptides comprises a nucleic acid encoding an envelope polypeptide selected from the group consisting of a vesicular stomatitis virus G (VSV G) polypeptide and a murine leukemia virus (MLV) 4070A polypeptide.

20. 20. The hCMV vector of claim 19, wherein the released viral gene transfer vector comprises the envelope polypeptide.

21. 1. A method of treating a mammal having cancer, the method comprising administering to the mammal a recombinant hCMV vector, the hCMV vector capable of infecting cells of the mammal, the hCMV comprising heterologous nucleic acid encoding (i) a heterologous viral gene transfer vector genome comprising a nucleic acid sequence encoding an antigen receptor polypeptide that targets a cancer antigen of the cancer, and (ii) one or more helper polypeptides for amplifying and packaging the heterologous viral gene transfer vector genome to obtain infectious vector particles, the hCMV infected cells producing and releasing the infectious vector particles comprising the gene transfer vector genome, the released infectious vector particles capable of infecting immune cells in the mammal and inducing expression of the antigen receptor polypeptide by the infected immune cells, and the infected immune cells expressing the antigen receptor reducing the number of cancer cells in the mammal.

22. 22. The method of claim 21, wherein the mammal is a human.

23. 23. The method of claim 21 or claim 22, wherein the cancer is selected from the group consisting of brain cancer, B-cell lymphoma, ALL, CLL, neuroblastoma, breast cancer and lung cancer.

24. 24. The method of any one of claims 21 to 23, wherein the cancer antigen is selected from the group consisting of cluster of differentiation 19 (CD19), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, mucin 1 (MUC-1), epithelial tumor antigen (ETA), melanoma associated antigen (MAGE), human epidermal growth factor receptor 2 (HER2), biotin, EGFRvIII, CD171 and mesothelin.

25. 25. The method of any one of claims 21 to 24, wherein the hCMV vector is replication competent in the cell.

26. 26. The method of any one of claims 21 to 25, wherein the hCMV vector lacks a nucleic acid encoding a UL138 polypeptide, a UL144 polypeptide, a UL146 polypeptide, a UL147 polypeptide or miR-UL148D.

27. 26. The hCMV vector of any one of claims 21 to 25, wherein the hCMV vector lacks a UL138 polypeptide, a UL144 polypeptide, a UL146 polypeptide, a UL147 polypeptide or miR-UL148D.

28. 28. The method of any one of claims 21 to 27, wherein the hCMV vector is derived from the hCMV AD169 or hCMV Merlin strains.

29. 29. The method of any one of claims 21 to 28, wherein the hCMV vector lacks a nucleic acid encoding a UL23 polypeptide, a US1 polypeptide, a US2 polypeptide, a US3 polypeptide, a US4 polypeptide, a US5 polypeptide, a US6 polypeptide, a US7 polypeptide, a US8 polypeptide, a US9 polypeptide, a US10 polypeptide, a US11 polypeptide, a US14 polypeptide, a US15 polypeptide, a US16 polypeptide, a US17 polypeptide, a US18 polypeptide, a US19 polypeptide, a US20 polypeptide, a US21 polypeptide, a US22 polypeptide, a US30 polypeptide, a UL82 polypeptide or a UL83 polypeptide.

30. 29. The method of any one of claims 21 to 28, wherein the hCMV vector lacks a UL23 polypeptide, a US1 polypeptide, a US2 polypeptide, a US3 polypeptide, a US4 polypeptide, a US5 polypeptide, a US6 polypeptide, a US7 polypeptide, a US8 polypeptide, a US9 polypeptide, a US10 polypeptide, a US11 polypeptide, a US14 polypeptide, a US15 polypeptide, a US16 polypeptide, a US17 polypeptide, a US18 polypeptide, a US19 polypeptide, a US20 polypeptide, a US21 polypeptide, a US22 polypeptide, a US30 polypeptide, a UL82 polypeptide or a UL83 polypeptide.

31. 31. The method of any one of claims 21 to 30, wherein the cell is a fibroblast, an epithelial cell, an endothelial cell, a mononuclear cell or a glial cell.

32. 32. The method of any one of claims 21 to 31, wherein the viral gene transfer vector genome is a lentivirus vector genome, a retrovirus vector genome, an AAV vector genome, a picornavirus vector genome, a rhabdovirus vector genome or a coronavirus vector genome.

33. 33. The method of any one of claims 21 to 32, wherein the viral gene transfer vector comprises a nucleic acid encoding one or more packaging elements selected from the group consisting of a 5'LTR, a 3'LTR, a psi (Ψ) element, an RRE and a cPPT / CTS.

34. 34. The method of any one of claims 21 to 33, wherein the nucleic acid encoding the one or more helper polypeptides comprises a nucleic acid encoding an envelope polypeptide selected from the group consisting of a VSV G protein or an MLV 4070A protein.

35. 35. The method of claim 34, wherein the released viral gene transfer vector comprises the envelope polypeptide.

36. 1. A method of treating a mammal having an infectious disease, the method comprising administering to the mammal a recombinant hCMV vector, the hCMV vector capable of infecting cells of the mammal, the hCMV vector comprising heterologous nucleic acid encoding (i) a heterologous viral gene transfer vector genome derived from a virus that causes an infectious disease, and (ii) one or more helper polypeptides for amplifying and packaging the heterologous viral gene transfer vector genome to obtain infectious vector particles, the hCMV vector-infected cells producing and releasing the infectious vector particles comprising the gene transfer vector genome, the infectious vector particles being recognized by antigen presenting cells (APCs) within the mammal, whereby the APCs present antigens from the viral gene transfer vector to T cells within the mammal, and the T cells recognize and destroy the virus causing the infectious disease in the mammal.

37. 37. The method of claim 36, wherein the mammal is a human.

38. 38. The method of claim 36 or claim 37, wherein the infectious disease is selected from the group consisting of viral pneumonia, acquired immune deficiency syndrome and viral hepatitis.

39. 39. The method of any one of claims 36 to 38, wherein the hCMV vector is replication competent in the cell.

40. 40. The method of any one of claims 36 to 39, wherein the hCMV vector lacks a nucleic acid encoding a UL138 polypeptide, a UL144 polypeptide, a UL146 polypeptide, a UL147 polypeptide or miR-UL148D.

41. 40. The hCMV vector of any one of claims 36 to 39, wherein the hCMV vector lacks a UL138 polypeptide, a UL144 polypeptide, a UL146 polypeptide, a UL147 polypeptide or miR-UL148D.

42. 42. The method of any one of claims 36 to 41, wherein the hCMV vector is derived from the hCMV AD169 or hCMV Merlin strain.

43. 43. The method of any one of claims 36 to 42, wherein the hCMV vector lacks a nucleic acid encoding a UL23 polypeptide, a US1 polypeptide, a US2 polypeptide, a US3 polypeptide, a US4 polypeptide, a US5 polypeptide, a US6 polypeptide, a US7 polypeptide, a US8 polypeptide, a US9 polypeptide, a US10 polypeptide, a US11 polypeptide, a US14 polypeptide, a US15 polypeptide, a US16 polypeptide, a US17 polypeptide, a US18 polypeptide, a US19 polypeptide, a US20 polypeptide, a US21 polypeptide, a US22 polypeptide, a US30 polypeptide, a UL82 polypeptide or a UL83 polypeptide.

44. 43. The method of any one of claims 36 to 42, wherein the hCMV vector lacks a UL23 polypeptide, a US1 polypeptide, a US2 polypeptide, a US3 polypeptide, a US4 polypeptide, a US5 polypeptide, a US6 polypeptide, a US7 polypeptide, a US8 polypeptide, a US9 polypeptide, a US10 polypeptide, a US11 polypeptide, a US14 polypeptide, a US15 polypeptide, a US16 polypeptide, a US17 polypeptide, a US18 polypeptide, a US19 polypeptide, a US20 polypeptide, a US21 polypeptide, a US22 polypeptide, a US30 polypeptide, a UL82 polypeptide or a UL83 polypeptide.

45. 45. The method of any one of claims 36 to 44, wherein the cell is a fibroblast, an epithelial cell, an endothelial cell, a mononuclear cell or a glial cell.

46. 46. ​​The method of any one of claims 36 to 45, wherein the viral gene transfer vector genome is a lentivirus vector genome, a retrovirus vector genome, an AAV vector genome, a picornavirus vector genome, a rhabdovirus vector genome or a coronavirus vector genome.

47. 47. The method of any one of claims 36 to 46, wherein the viral gene transfer vector genome comprises nucleic acid encoding one or more packaging elements selected from the group consisting of 5'LTR, 3'LTR, psi (Ψ) element, RRE and cPPT / CTS.

48. 48. The method of any one of claims 36 to 47, wherein the nucleic acid encoding the one or more helper polypeptides comprises a nucleic acid encoding an envelope polypeptide selected from the group consisting of a VSV G protein or an MLV 4070A protein.

49. 49. The method of claim 48, wherein the released viral gene transfer vector comprises the envelope polypeptide.

50. 21. Use of a composition comprising a recombinant hCMV according to any one of claims 1 to 20 for the treatment of cancer or an infectious disease.

51. 21. A recombinant hCMV according to any one of claims 1 to 20 for use as a medicament for treating cancer or an infectious disease.

52. 21. A recombinant hCMV according to any one of claims 1 to 20 for use in the treatment of cancer or an infectious disease.

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