Pseudotyped viral particle and use thereof

Abstract

A pseudotyped viral particle having a Maraba virus envelope glycoprotein or a variant thereof. The surface of the pseudotyped virus envelope comprises an immune cell targeting molecule and / or an immune cell activating molecule. A corresponding targeting molecule is designed according to a cell receptor to be targeted, and can target different types of cells, including immunotherapy cells such as CAR-T and CAR-NK.

Description

Pseudotyped viral particles and uses thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to and the benefit of Chinese Patent Application No. 202411232623.1, filed September 4, 2024, Chinese Patent Application No. 202411953714.4, filed December 27, 2024, Chinese Patent Application No. 202510419237.1, filed April 3, 2025, and Chinese Patent Application No. 202510569480.1, filed May 3, 2025, the contents of each of which are incorporated by reference in their entirety.

[0003] SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing, which is incorporated by reference in its entirety. TECHNICAL FIELD

[0005] The present application relates to the field of biological medicine, in particular to a novel engineered viral particle and its application. BACKGROUND

[0006] Adoptive cell therapy (ACT) is one of the immune cell therapies. In recent years, with the remarkable effect of chimeric antigen receptor (CAR) modified T cells in the treatment of hematological tumor, the demand for adoptive cell therapy is increasing. Chimeric antigen receptor is to engineer T cells to produce ligands that specifically bind to tumor cell surface antigens on their surface. This binding is transmitted to the cell through the hinge and transmembrane regions of CAR, activating the T cell. The activated T cell kills tumor cells by secreting cytokines and perforin.

[0007] Lentiviral vector is a viral vector modified from human immunodeficiency virus (HIV), which is a type of retrovirus. Its genome is RNA, which can integrate exogenous genes into the genome to achieve stable expression using reverse transcriptase. It has the characteristics of infecting both dividing and non-dividing cells. The original HIV virus carries a gp120 and gp41 complex that can recognize CD4 molecules, thereby facilitating HIV infection of cells. The modified lentiviral vector does not express gp120 and gp41, but expresses vesicular stomatitis virus envelope glycoprotein VSVG. Since VSVG can target and recognize low-density lipoprotein receptors (LDLR), and LDLR is widely expressed, lentiviral vectors carrying VSVG can infect a variety of types of cells, such as T cells, liver cells, cardiac cells, neurons, endothelial cells, and stem cells. When transducing cells in vivo or one type of cell in a mixed cell transduction, the virus vector pseudotyped with VSVG has poor specificity.

[0008] The engineered T cells are constructed by first extracting peripheral blood from a patient or a healthy person, isolating T lymphocytes, and further modifying the isolated T lymphocytes to obtain CAR-T cells. The modification is achieved by integrating a vector, such as a lentivirus vector or a retrovirus vector. The commonly used method is to add a T cell-activating monoclonal antibody, such as anti-CD3 / CD28 Dynabeads, to the cell culture medium to activate the T cells. The activated T cells have a high expression of LDLR on the surface, and LDLR is an important receptor for mediating the entry of lentivirus into cells. The lentivirus packaged with CAR enters the T cells by binding to LDLR, and the CAR gene is integrated into the T cell genome to form CAR-T cells. This traditional preparation method consumes a large amount of activated magnetic beads and cytokines, and these consumables are single-use, which is a relatively expensive part of the CAR-T cell production process. In addition, the VSVG envelope lentivirus is almost unable to transduce unactivated primary T cells.

[0009] There have been reports of developing targeted lentiviruses to address the above problems, such as CD3 antibody-based targeted lentiviruses. By expressing CD3 antibodies or other antibodies that can activate T cells with a transmembrane sequence on the surface of lentiviruses, the virus particles can be activated and infect T cells.

[0010] Currently, there is still a need to develop lentivirus vectors that can efficiently and specifically transduce unactivated T cells, thereby optimizing the preparation method of CAR-T cells.

[0011] SUMMARY

[0012] To solve the above technical problems, the present application provides a virus particle with an engineered virus envelope, which comprises a Maraba-G envelope glycoprotein derived from a Maraba virus or a variant thereof. By further expressing immune cell targeting molecules and / or immune cell activating molecules on the surface of the virus envelope, the virus vector can directly infect immune cells in vivo or in vitro without the need for prior activation of immune cells.

[0013] Accordingly, in one aspect, the present application provides a Maraba virus envelope glycoprotein (Maraba-G) variant that has reduced or completely abolished binding ability to low density lipoprotein receptor (LDLR) on the surface of T cells compared to wild-type Maraba-G comprising the amino acid sequence of SEQ ID NO: 33, while retaining the ability to mediate membrane fusion, and the Maraba-G variant comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.3%, or at least 99.5% sequence identity to SEQ ID NO: 33. In some embodiments, the Maraba-G variant comprises an amino acid substitution or deletion at one or more positions selected from H8, Q10, K47, A182, S183, H209, R354, I331, M347, and T352 relative to wild-type Maraba-G comprising the amino acid sequence of SEQ ID NO: 33. In some preferred embodiments, the Maraba-G variant comprises one or more of the following amino acid substitutions or deletions relative to wild-type Maraba-G: H8A, Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, S183P, H209A, H209E, R354A, R354G, R354S, R354E, R354W, I331E, M347E, and T352E.

[0014] The present application also provides a Maraba-G variant that comprises an amino acid substitution or deletion at one or more positions selected from H8, Q10, K47, A182, S183, H209, and R354 relative to wild-type Maraba-G comprising the amino acid sequence of SEQ ID NO: 33; the variant has significantly reduced binding ability to the LDLR receptor while retaining the ability to mediate membrane fusion. In some preferred embodiments, the Maraba-G variant comprises one or more of the following amino acid substitutions or deletions relative to wild-type Maraba-G: H8A, H8V, H8P, Q10A, K47Q, K47E, K47A, AK47, A182E, A182W, A182F, AA182, S183P, S183F, AS183, H209A, H209E, AH209, R354A, R354E, R354W, and AR354.

[0015] Therefore, the pseudotyped virus vector constructed based on the Maraba-G variant can specifically recognize the marker molecules of target cells through targeting molecules, and the targeting molecules mediate the entry of virus particles into specific cell types through a non-LDLR-dependent mechanism, thereby solving the problem of non-specific transduction of viruses.

[0016] In another aspect, the present application provides a virus particle comprising a wild-type Maraba-G envelope glycoprotein or the aforementioned Maraba-G variant.

[0017] In other aspects of the present application, nucleic acid molecules encoding the aforementioned Maraba-G variant, vectors comprising the nucleic acid molecules, vector combinations comprising the nucleic acid molecules or vectors, host cells for preparing the aforementioned virus particles, compositions and kits comprising the aforementioned virus particles, methods for preparing target cells expressing a gene of interest (e.g., CAR) in vivo using the aforementioned virus particles, methods for directly transducing immune cells in vitro without activation, methods for preventing or treating tumors, viral infections, aging or autoimmune diseases using the aforementioned virus particles, and the use of the aforementioned virus particles in the preparation of related drugs are also provided.

[0018] Applicants have found that pseudotyped viruses constructed based on Maraba virus envelope proteins have higher virus titers and higher transduction efficiency than VSV-G pseudotyped virus vectors, which are expected to reduce the production cost of lentivirus vectors.

[0019] The technical solutions of the present application have the following advantages:

[0020] Compared with VSV-G pseudotyped viruses, the pseudotyped viruses constructed by Maraba-G in the present application have higher virus titers and higher transduction efficiency, and the engineered cells after transduction have stronger amplification capacity. The Maraba-G variant provided in the present application loses the binding function to LDLR, and the pseudotyped viruses prepared therefrom can effectively avoid transfection of non-target cells, thereby improving the safety of virus vectors in transfecting target cells in vivo.

[0021] By adding immune cell targeting molecules and / or immune cell activation molecules, such as combinations of TCRa / b antibodies, TCRy / d antibodies or CD3 antibodies and CD7 antibodies, on the surface of the virus envelope, the infection efficiency of the virus in infecting primary immune cells such as T cells can be significantly improved, and the positive proportion of proteins such as CAR is significantly improved. At the same time, it helps to maintain the memory phenotype of initial T cells or NK cells and other immune cells, prolongs the survival time of engineered T or NK cells and other immune cells in vivo, and improves the killing ability of engineered immune cells on tumor cells or cells causing autoimmune diseases.

[0022] In vivo and in vitro, the immune cells are directly infected without the need for additional expensive coated antibody magnetic beads to pre-activate the immune cells, saving the cost of preparing engineered immune cells, and eliminating the culture time of pre-activating immune cells with antibody magnetic beads, greatly reducing the preparation time of engineered cells, and reducing the waiting time of the subjects.

[0023] In some specific embodiments, the present application provides an engineered lentiviral vector, the modified lentiviral envelope has Maraba-G variant, and the surface displays a polypeptide that can target activated T cells. The lentiviral vector can directly target and activate infected T cells without the need for pre-activation of T cells with anti-CD3 / CD28 antibodies, reducing the cost of expensive CAR-T cell preparation, and saving patient waiting time. At the same time, the CAR-T cells prepared by this method are superior to traditional preparation methods in terms of memory phenotype, prolonging the in vivo persistence time of CAR-T cells and improving the killing ability of CAR-T cells on tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0025] Figure 1. Schematic diagram of lentiviral envelope plasmid structure with ICAM-1 ligand or other ligands.

[0026] Figure 2. Schematic diagram of flow cytometry detection of CAR positive rate of PBMC cells infected with lentivirus with ICAM-1 ligand and other lentiviruses on day 3 and day 7.

[0027] Figure 3. Statistical diagram of CAR positive rate of PBMC cells infected with lentivirus with ICAM-1 ligand and other lentiviruses on day 3 and day 7.

[0028] Figure 4. Statistical diagram of CAR positive cell expansion of PBMC cells infected with lentivirus with ICAM-1 ligand and other lentiviruses for 7 days.

[0029] Figure 5. Schematic diagram of lentiviral envelope plasmid structure with mb-Efalizumab ligand or other ligands.

[0030] Figure 6. Schematic diagram of flow cytometry detection of CAR positive rate of PBMC cells infected with lentivirus with mb-Efalizumab ligand or other ligands on day 3 and day 7.

[0031] Figure 7. CAR positive rate statistics of PBMC cells infected with lentivirus with mb-Efalizumab ligand and other ligands on day 3 and day 7.

[0032] Figure 8. CAR positive cell expansion statistics of PBMC cells infected with lentivirus with mb-Efalizumab ligand or other ligands after 7 days.

[0033] Figure 9. Schematic diagram of lentivirus envelope plasmid structure.

[0034] Figure 10. Schematic diagram of different structure length shuttle plasmid.

[0035] Figure 11. Schematic diagram of lentivirus titer detection.

[0036] Figure 12. Schematic diagram of CAR positive rate flow cytometry detection of T cells infected with different envelope lentivirus on day 3.

[0037] Figure 13. CAR positive rate statistics of T cells infected with different envelope lentivirus on day 3.

[0038] Figure 14. Schematic diagram of CAR positive rate flow cytometry detection of T cells infected with different envelope lentivirus on day 7.

[0039] Figure 15. CAR positive rate statistics of T cells infected with different envelope lentivirus on day 7.

[0040] Figure 16. Statistics of CAR-T cell killing results of NUGC4-luc cells prepared by different envelopes.

[0041] Figure 17. Different envelope structures carrying activation molecules and adhesion molecules.

[0042] Figure 18. Lentivirus titer statistics of different envelope packaging lentivirus carrying activation molecules and adhesion molecules.

[0043] Figure 19. Schematic diagram of CAR positive rate flow cytometry detection of PBMC cells infected with lentivirus packaged by different envelopes carrying activation molecules and adhesion molecules on day 3 and day 7.

[0044] Figure 20. Structure of fusion type activation molecules and adhesion molecules.

[0045] Figure 21. Schematic diagram of CAR positive rate flow cytometry detection of PBMC cells infected with lentivirus packaged by different envelopes carrying fusion type activation molecules and adhesion molecules on day 3 and day 7.

[0046] Figure 22. CAR positive rate statistics of PBMC cells infected with lentivirus packaged by different envelopes carrying fusion type activation molecules and adhesion molecules on day 3 and day 7.

[0047] Figure 23. Maraba-G virus efficacy in Nalm6-Luc model (live imaging).

[0048] Figure 24. Maraba-G virus efficacy in Nalm6-Luc model (fluorescence statistics).

[0049] Figure 25. Trend of animal experiment mouse weight change.

[0050] Figure 26. Peripheral blood flow detection of CAR-T (D25).

[0051] Figure 27. Statistics of peripheral blood flow detection of CAR-T (D25).

[0052] Figure 28. Maraba-G envelope mutation site information.

[0053] Figure 29. Maraba-G mutant infection of Jurkat, positive rate statistics after 4 days.

[0054] Figure 30. Maraba-G-K47 site repeated verification of infection ability on Jurkat.

[0055] Figure 31. Maraba-G combined mutation verification of infection ability on Jurkat cells.

[0056] Figure 32. Maraba-G (H8A / K47E / A182E) reduces sensitivity to serum.

[0057] Figure 33. Maraba-G (H8A / K47E / A182E) mutant infection of different cell lines.

[0058] Figure 34. Maraba-G (H8A / K47E / A182E) comparison of targeting specificity.

[0059] Figure 35. Comparison of infection efficiency of different envelopes and armors on Maraba-G mutant on Jurkat cells.

[0060] Figure 36. Comparison of infection efficiency of different envelopes and armors on Maraba-G (H8A / K47E / A182E) on Jurkat and Raji cells.

[0061] Figure 37. Comparison of activation of PBMCs by different envelopes and armors.

[0062] Figure 38. Comparison of infection efficiency of T cells in PBMCs by different envelopes and armors.

[0063] Figure 39. Comparison of killing and depletion phenotypes of target cells by CAR-T cells transfected with different envelopes and armors.

[0064] Figure 40. Comparison of cytokine secretion after target cell killing by CAR-T cells transduced with different envelopes and armors.

[0065] Figure 41. Comparison of T cell activation ability of Maraba-G (H8A / K47E / A182E) containing aCD7-aCD3 armor and other molecules

[0066] Figure 42. Comparison of T cell infection ability of Maraba-G (H8A / K47E / A182E) containing aCD7-aCD3 armor and other molecules

[0067] Figure 43. Comparison of different lentivirus infection specificities.

[0068] Figure 44. Comparison of different lentivirus infection abilities of primary T cells.

[0069] Figure 45. Preparation of CAR-T targeting different antigens and detection of their killing function by aCD7-aCD3-ICAM1 and Maraba-G (H8A / K47E / A182E) envelope.

[0070] Figure 46. In vivo generation of CD19 CAR-T for treating B cell tumors by aCD7-aCD3-ICAM1 and Maraba-G (H8A / K47E / A182E) envelope prepared viruses.

[0071] Figure 47. Comparison of in vivo efficacy of viruses prepared by aCD7-aCD3-ICAM1 and Maraba-G (H8A / K47E / A182E) envelope with other combinations.

[0072] Figure 48. In vivo generation of BCM A CAR-T for treating multiple myeloma by aCD7-aCD3-ICAM1 and Maraba-G (H8A / K47E / A182E) envelope prepared viruses.

[0073] Figure 49. Proportion of BCM A CAR-T generated in vivo by aCD7-aCD3-ICAM1 and Maraba-G (H8A / K47E / A182E) envelope prepared viruses in peripheral blood.

[0074] Figure 50. In vivo CLDN18.2 CAR virus can effectively inhibit gastric cancer tumors by directly generating CLDN18.2 CAR-T in vivo.

[0075] Figure 51. In vivo CLDN18.2 CAR virus can generate CLDN18.2 CAR-T cells in vivo.

[0076] Figure 52. Transduction efficiency of lentivirus produced using different T cell activating antibodies in the aCD7-aCD3-ICAMl configuration.

[0077] Figure 53. CAR-T cells produced using lentivirus produced using different T cell activating antibodies in the aCD7-aCD3-ICAMl configuration can kill Raji tumor cells.

[0078] Figure 54. Transduction efficiency of lentivirus produced using different aCD7 antibodies in the aCD7-aCD3-ICAMl configuration.

[0079] Figure 55. CAR-T cells produced using lentivirus produced using different aCD7 antibodies in the aCD7-aCD3-ICAMl configuration can kill Raji tumor cells.

[0080] DETAILED DESCRIPTION

[0081] Reference will now be made in detail to embodiments of the present application, one or more examples of which are described herein. Each example is provided as an explanation and not as a limitation of the present application. Indeed, it will be apparent to one of ordinary skill in the art that numerous modifications and variations of the present application are possible in light of the above teachings. For example, features described or illustrated as part of one embodiment can be used with another embodiment to yield a still further embodiment.

[0082] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are provided for better understanding of the present teachings. The terms used herein are for the purpose of describing specific embodiments and are not intended to limit the present application.

[0083] The selection range of the terms "and / or", "or / and", "and / or" used in the present application includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, which includes any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0084] The terms "containing", "having", "including" and "comprising" used in the present application are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0085] As used herein, the term "antibody" refers to an immunoglobulin molecule having the ability to specifically bind a particular antigen. Antibodies typically comprise a variable region and a constant region in each of the heavy and light chains. The variable region of the heavy and light chains of an antibody contains the binding domain that interacts with the antigen. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq (the first component in the classical pathway of complement activation). Thus, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form the portion of the antibody that binds to an antigen.

[0086] A “light chain variable region” (VL) or “heavy chain variable region” (VH) is composed of “framework” regions interspersed with “complementarity determining regions” or “CDRs.” The framework regions serve to align the CDRs that are primarily responsible for specific antigenic epitope binding. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. The VL and VH domains each comprise, from amino-terminus to carboxy-terminus, the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of a VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; and the CDR1, CDR2, and CDR3 of a VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0087] The amino acid arrangement of each VL and VH domain is consistent with any conventional definition of CDRs. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); the composite of Chothia and Kabat CDRs, in which CDR-H1 is a composite of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular’s antibody modeling software; and the CONTACT definition of Martin et al. (world wide web bioinfo.org.uk / abs).

[0088] In the present application, the amino acid sequences of the CDRs are shown according to the rules of the Kabat definition. However, it is well known in the art that the CDRs of an antibody can be defined by various methods in the art. In the technical solutions of the present application, the amino acid residues in the variable domain sequence can also be determined using the Combined definition rule which contains the rules of the Kabat definition and the Chothia definition. The Combined definition rule is to combine the ranges of the Kabat definition and the Chothia definition, and a larger range is taken based thereon. It should be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementary determining region" of a given antibody or region thereof (e.g., a variable region) are understood to encompass the complementary determining regions defined by any of the above known schemes as described herein. Although the scope claimed in the present application is based on the sequences shown according to the rules of the Kabat definition, the amino acid sequences corresponding to the definitions of other CDRs should also be within the scope claimed in the present application.

[0089] The term "antibody" as used herein is to be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antigen binding fragments of antibodies, and multi-specific antibodies (e.g., bi-specific antibodies) containing at least two antigen binding regions. Antibodies can contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations of glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing an antigenic determinant of an antibody, and immunoglobulin molecules containing any other modification to the antigen recognition site, as long as the antibodies exhibit the desired biological activity.

[0090] It should be understood that the antibodies described herein encompass full-length antibodies or antigen binding fragments thereof. Examples of antigen binding fragments include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is essentially a Fab with part of the hinge region; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fd' fragment having VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment, which consists of a VH domain; (viii) an isolated complementarity determining region (CDR); (ix) a nanobody (VHH), a heavy chain variable region containing only a single variable domain; (x) a single chain Fv (scFv), using recombinant methods to link VL and VH using a synthetic linker that enables them to form a single protein chain where the VL and VH regions pair to form a monovalent molecule.

[0091] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "nucleotide sequence," and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or triple-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising, consisting of, or consisting essentially of, purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0092] The term "sequence identity" refers to the sequence similarity between two polypeptides or between two nucleic acid sequences. The percent identity can be determined by comparing a position in each sequence, which can be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of identity between sequences is dependent on the number of matching positions shared by the sequences. "Unrelated" or "non-homologous" sequences share less than 40% identity, less than 25% identity with one of the sequences of the present application. Alignment and percent sequence identity of a nucleic acid or amino acid sequence provided herein can be determined using ClustalW, available at https: / / genome.jp / tools-bin / clustalw / , by importing the nucleic acid or amino acid sequence into ClustalW.

[0093] The terms "equivalent" or "functional variant" can be used interchangeably when referring to a particular molecule, biological material, or cellular material, and refer to those that have minimal homology while still retaining the desired structure or function. Non-limiting examples of equivalent polypeptides include a polypeptide that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference polypeptide; or a polypeptide encoded by a polynucleotide that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference polynucleotide.

[0094] The term "vector" generally refers to a nucleic acid molecule capable of self- replicating in a suitable host, which transfers an inserted nucleic acid molecule into and / or between host cells. The vector can include a vector that is primarily used for inserting DNA or RNA into a cell, a vector that is primarily used for replication of DNA or RNA, and a vector that is primarily used for transcription and / or translation of expression of DNA or RNA. The vector also includes a vector having a plurality of the above-mentioned functions. The vector can be a polynucleotide that is capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0095] In the present context, the term "subject" generally refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). The term "primate" generally refers to monkey and ape species, and includes monkey species such as monkeys from the genus Macaca (such as Macaca fascicularis and / or Macaca mulatta) and Papio (Papio ursinus), as well as marmosets (species from the genus Callithrix), tamarins (species from the genus Saguinus), and squirrel monkeys (species from the genus Saimiri), and ape species such as Pan troglodytes, and also Homo sapiens.

[0096] As used herein, the term "administering" is intended to mean delivering a substance to a subject, such as an animal or human. Administration can be carried out with single doses, continuously or intermittently over the course of therapy. Methods of determining the most effective dosage and schedule of administration are known to those skilled in the art and will vary with the composition used for therapy, the purpose of the therapy, and the age, health, or gender of the subject being treated, among other factors. In some embodiments, a single or multiple administrations can be carried out with the dose level and pattern being selected by a medical practitioner, or in the case of a pet or other animal, by a veterinarian.

[0097] As used herein, “treatment” includes limiting, slowing or stopping the progression or severity of an existing symptom, condition, disease or disorder in a patient. “Treatment,” as understood in the art, is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired results can include alleviation of one or more symptoms, diminishment or delay of disease or condition progression, amelioration or palliation of the state of disease or condition (whether partial or total). The term “prevention” means prophylactic administration of a viral particle or viral vector of the application to an asymptomatic patient or a patient who does not exhibit overt symptoms to prevent the onset or progression of a disease.

[0098] “Pharmaceutically acceptable” means suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio as well as being effective for their intended use within the scope of sound medical judgment.

[0099] Maraba virus envelope glycoprotein (Maraba-G) variants

[0100] In some aspects, the present application provides Maraba virus envelope glycoprotein (Maraba-G) variants that have reduced or completely eliminated binding ability to low-density lipoprotein receptor (LDLR) on the surface of T cells compared to wild-type Maraba-G comprising the amino acid sequence of SEQ ID NO: 33, and the Maraba-G variants comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.3%, or at least 99.5% sequence identity to SEQ ID NO: 33.

[0101] The Maraba-G variants described herein comprise an amino acid substitution or deletion at one or more positions compared to wild-type Maraba-G such that the Maraba-G variants have reduced or completely eliminated binding ability to LDLR, but still retain the ability to mediate membrane fusion. In embodiments of the present application, the positions of the amino acid substitution or deletion are defined relative to the mature wild-type Maraba-G protein, such as the amino acid sequence set forth in SEQ ID NO: 33. Based on the amino acid substitution or deletion positions disclosed herein relative to the mature wild-type Maraba-G protein, one of skill in the art would be able to determine the corresponding positions on the full-length Maraba-G (comprising the N-terminal signal peptide) amino acid sequence, and variants comprising an amino acid substitution or deletion at the corresponding positions relative to the full-length Maraba-G protein (such as the amino acid sequence set forth in SEQ ID NO: 34) are also encompassed within the scope of the present application.

[0102] In some embodiments, the Maraba-G variant comprises one or more amino acid substitutions or deletions relative to the wild-type Maraba-G comprising the amino acid sequence of SEQ ID NO:33 at one or more positions selected from H8, Q10, K47, A182, S183, H209, R354, I331, M347, and T352. In some embodiments, the variant comprises one or more amino acid substitutions or deletions selected from H8A, Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, S183P, H209A, H209E, R354A, R354G, R354S, R354E, R354W, I331E, M347E, and T352E relative to the wild-type Maraba-G. In some embodiments, the variant comprises two or more amino acid substitutions or deletions selected from H8A, Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, S183P, H209A, H209E, R354A, R354G, R354S, R354E, R354W, I331E, M347E, and T352E relative to the wild-type Maraba-G. In some embodiments, the variant comprises three or more amino acid substitutions or deletions selected from H8A, Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, S183P, H209A, H209E, R354A, R354G, R354S, R354E, R354W, I331E, M347E, and T352E relative to the wild-type Maraba-G.

[0103] In some embodiments, the present application provides Maraba virus envelope glycoprotein (Maraba-G) variants comprising one or more amino acid substitutions or deletions relative to a wild-type Maraba-G comprising the amino acid sequence of SEQ ID NO:33 selected from the group consisting of: H8, Q10, K47, A182, S183, H209, and R354. In some embodiments, the substitution or deletion of H8 is selected from the group consisting of: H8A, H8V, and H8P. In some embodiments, the substitution or deletion of Q10 is Q10A. In some embodiments, the substitution or deletion of K47 is selected from the group consisting of: K47A, K47E, K47Q, or AK47. In some embodiments, the substitution or deletion of A182 is selected from the group consisting of: A182E, A182W, A182F, or AA182. In some embodiments, the substitution or deletion of S183 is selected from the group consisting of: S183P, S183F, or AS183. In some embodiments, the substitution or deletion of H209 is selected from the group consisting of: H209A, H209E, or AH209. In some embodiments, the substitution or deletion of R354 is selected from the group consisting of: R354A, R354E, R354W, or AR354.

[0104] In some embodiments, the variant comprises one or more amino acid substitutions or deletions relative to a wild-type Maraba-G selected from the group consisting of: H8A, H8V, H8P, Q10A, K47Q, K47E, K47A, AK47, A182E, A182W, A182F, AA182, S183P, S183F, AS183, H209A, H209E, AH209, R354A, R354E, R354W, and AR354. In some embodiments, the variant comprises two or more amino acid substitutions or deletions relative to a wild-type Maraba-G selected from the group consisting of: H8A, H8V, H8P, Q10A, K47Q, K47E, K47A, AK47, A182E, A182W, A182F, AA182, S183P, S183F, AS183, H209A, H209E, AH209, R354A, R354E, R354W, and AR354. In some embodiments, the variant comprises three or more amino acid substitutions or deletions relative to a wild-type Maraba-G selected from the group consisting of: H8A, H8V, H8P, Q10A, K47Q, K47E, K47A, AK47, A182E, A182W, A182F, AA182, S183P, S183F, AS183, H209A, H209E, AH209, R354A, R354E, R354W, and AR354.

[0105] In some embodiments, the variant comprises at least two mutations selected from the group consisting of H8A, Q10A, K47E / K47A / K47Q, A182E, S183P, H209A, and R354A relative to wild-type Maraba-G.

[0106] In some embodiments, the variant comprises at least two mutations selected from the group consisting of H8A, K47E / K47A / K47Q, A182E, and R354A relative to wild-type Maraba-G.

[0107] In some embodiments, the variant comprises an amino acid substitution or deletion at the K47 position. In some preferred embodiments, the variant comprises an amino acid substitution or deletion at the K47 position selected from the group consisting of K47G, K47T, K47W, K47P, K47Q, K47E, K47A, and AK47. In some more preferred embodiments, the variant comprises a K47E or K47Q amino acid substitution at the K47 position. In some embodiments, in addition to the K47 position, the variant comprises an amino acid substitution or deletion at one or more positions selected from the group consisting of H8, Q10, A182, S183, H209, and R354. In some preferred embodiments, the variant further comprises one or more amino acid substitutions selected from the group consisting of H8A, Q10A, A182E, S183P, H209A, and R354A.

[0108] In some embodiments, the variant comprises an amino acid substitution or deletion at the K47 position. In some preferred embodiments, the variant comprises an amino acid substitution or deletion at the K47 position selected from the group consisting of K47G, K47T, K47W, K47P, K47Q, K47E, K47A, and AK47. In some embodiments, in addition to the K47 position, the variant comprises an amino acid substitution or deletion at one or more positions selected from the group consisting of H8, Q10, A182, I331, S183, H209, M347, T352, and R354. In some preferred embodiments, the variant further comprises one or more amino acid substitutions selected from the group consisting of H8A, Q10A, A182E, I331E, S183P, H209A, H209E, M347E, T352E, R354A, R354A, R354G, R354S, R354E, and R354W.

[0109] In yet other embodiments, the variant comprises an amino acid substitution or deletion at the H8 position. In some preferred embodiments, the variant comprises an H8A substitution at the H8 position. In some embodiments, in addition to the H8 position, the variant comprises an amino acid substitution or deletion at one or more positions selected from the group consisting of Q10, K47, A182, S183, H209, and R354. In some preferred embodiments, the variant further comprises one or more amino acid substitutions or deletions selected from the group consisting of Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, S183P, H209A, and R354A.

[0110] In yet other embodiments, the variant comprises an amino acid substitution or deletion at the H8 position. In some preferred embodiments, the variant comprises an H8A substitution at the H8 position. In some embodiments, in addition to the H8 position, the variant comprises an amino acid substitution or deletion at one or more positions selected from the group consisting of Q10, K47, A182, I331, S183, H209, M347, T352, and R354. In some preferred embodiments, the variant further comprises one or more amino acid substitutions or deletions selected from the group consisting of Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, I331E, S183P, H209A, H209E, M347E, T352E, R354A, R354A, R354G, R354S, R354E, and R354W.

[0111] In yet other embodiments, the variant comprises an amino acid substitution or deletion at the Q10 position. In some preferred embodiments, the variant comprises a Q10A substitution at the Q10 position. In some embodiments, in addition to the Q10 position, the variant comprises an amino acid substitution or deletion at one or more positions selected from the group consisting of H8, K47, A182, S183, H209, and R354. In some preferred embodiments, the variant further comprises one or more amino acid substitutions or deletions selected from the group consisting of H8A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, S183P, H209A, and R354A.

[0112] In yet other embodiments, the variant comprises an amino acid substitution or deletion at position Q10. In some preferred embodiments, the variant comprises a Q10A substitution at position Q10. In some embodiments, in addition to position Q10, the variant comprises an amino acid substitution or deletion at one or more positions selected from the group consisting of H8, K47, A182, I331, S183, H209, M347, T352, and R354. In some preferred embodiments, the variant further comprises one or more amino acid substitutions or deletions selected from the group consisting of H8A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, I331E, S183P, H209A, H209E, M347E, T352E, R354A, R354A, R354G, R354S, R354E, and R354W.

[0113] In yet other embodiments, the variant comprises an amino acid substitution or deletion at position A182. In some preferred embodiments, the variant comprises an A182E substitution at position A182. In some embodiments, in addition to position A182, the variant comprises an amino acid substitution or deletion at one or more positions selected from the group consisting of H8, Q10, K47, S183, H209, and R354. In some preferred embodiments, the variant further comprises one or more amino acid substitutions or deletions selected from the group consisting of H8A, Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, S183P, H209A, and R354A.

[0114] In yet other embodiments, the variant comprises an amino acid substitution or deletion at position A182. In some preferred embodiments, the variant comprises an A182E substitution at position A182. In some embodiments, in addition to position A182, the variant comprises an amino acid substitution or deletion at one or more positions selected from the group consisting of H8, Q10, K47, I331, S183, H209, M347, T352, and R354. In some preferred embodiments, the variant further comprises one or more amino acid substitutions or deletions selected from the group consisting of H8A, Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, I331E, S183P, H209A, H209E, M347E, T352E, R354A, R354A, R354G, R354S, R354E, and R354W.

[0115] In yet other embodiments, the variant comprises an amino acid substitution or deletion at position S183. In some preferred embodiments, the variant comprises a S183P substitution at position S183. In some embodiments, in addition to position S183, the variant comprises an amino acid substitution or deletion at one or more positions selected from the group consisting of H8, Q10, K47, A182, H209, and R354. In some preferred embodiments, the variant further comprises one or more amino acid substitutions or deletions selected from the group consisting of H8A, Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, H209A, and R354A.

[0116] In yet other embodiments, the variant comprises an amino acid substitution or deletion at position S183. In some preferred embodiments, the variant comprises a S183P substitution at position S183. In some embodiments, in addition to position S183, the variant comprises an amino acid substitution or deletion at one or more positions selected from the group consisting of H8, Q10, K47, I331, A182, H209, M347, T352, and R354. In some preferred embodiments, the variant further comprises one or more amino acid substitutions or deletions selected from the group consisting of H8A, Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, I331E, A182E, H209A, H209E, M347E, T352E, R354A, R354A, R354G, R354S, R354E, and R354W.

[0117] In yet other embodiments, the variant comprises an amino acid substitution or deletion at position H209. In some preferred embodiments, the variant comprises a H209A substitution at position H209. In some embodiments, in addition to position H209, the variant comprises an amino acid substitution or deletion at one or more positions selected from the group consisting of H8, Q10, K47, A182, S183, and R354. In some preferred embodiments, the variant further comprises one or more amino acid substitutions or deletions selected from the group consisting of H8A, Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, S183P, and R354A.

[0118] In yet other embodiments, the variant comprises an amino acid substitution or deletion at the H209 position. In some preferred embodiments, the variant comprises an H209A or H209E substitution at the H209 position. In some embodiments, in addition to the H209 position, the variant comprises an amino acid substitution or deletion at one or more positions selected from the group consisting of H8, Q10, K47, I331, A182, S183, M347, T352, and R354. In some preferred embodiments, the variant further comprises one or more amino acid substitutions or deletions selected from the group consisting of H8A, Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, I331E, A182E, S183P, M347E, T352E, R354A, R354A, R354G, R354S, R354E, and R354W.

[0119] In yet other embodiments, the variant comprises an amino acid substitution or deletion at the R354 position. In some preferred embodiments, the variant comprises an R354A substitution at the R354 position. In some embodiments, in addition to the R354 position, the variant comprises an amino acid substitution or deletion at one or more positions selected from the group consisting of H8, Q10, K47, A182, S183, and H209. In some preferred embodiments, the variant further comprises one or more amino acid substitutions or deletions selected from the group consisting of H8A, Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, S183P, and H209A.

[0120] In yet other embodiments, the variant comprises an amino acid substitution or deletion at the R354 position. In some preferred embodiments, the variant comprises a substitution at the R354 position selected from the group consisting of R354A, R354G, R354S, R354E, and R354W. In some embodiments, in addition to the R354 position, the variant comprises an amino acid substitution or deletion at one or more positions selected from the group consisting of H8, Q10, K47, I331, A182, S183, M347, and T352. In some preferred embodiments, the variant further comprises one or more amino acid substitutions or deletions selected from the group consisting of H8A, Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, I331E, A182E, S183P, H209A, H209E, M347E, and T352E.

[0121] In some preferred embodiments of the application, the Maraba-G variant comprises an amino acid substitution or deletion or combination of substitutions selected from the group consisting of K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, I331E, H209E, M347E, T352E, R354A, R354G, R354S, R354E, R354W, K47E / R354A, K47E / A182E, K47E / S183P, H8A / K47E / A182E, K47E / A182E / R354A, and H8A / Q10A / K47Q / A182E / H209A / R354A. In some more preferred embodiments, the Maraba-G variant comprises the combination of H8A / K47E / A182E amino acid substitutions, i.e., the variant comprises H8A, K47E, and A182E amino acid substitutions.

[0122] In some preferred embodiments, the Maraba-G variant comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.3%, at least 99.5%, or 100% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 38-55, 57-61, and 63.

[0123] In some embodiments, the N-terminus of the Maraba-G variant of the application further comprises a signal peptide. In some preferred embodiments, the signal peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% sequence identity with SEQ ID NO: 35. In some more preferred embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0124] Nucleic acid molecules, vectors, and vector combinations

[0125] In some aspects, the application provides a nucleic acid molecule comprising a nucleotide sequence encoding a wild-type Maraba-G and / or a Maraba-G variant as disclosed herein.

[0126] For example, the present application provides a nucleic acid molecule encoding any of the Maraba-G variants disclosed herein. In some embodiments, the Maraba-G variant comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.3%, at least 99.5%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 38-55, 57-61, and 63.

[0127] For example, the present application provides a nucleic acid molecule encoding any of the Maraba-G variants disclosed herein. In some embodiments, the Maraba-G variant comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.3%, at least 99.5%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 38-55, 57-61, and 63.

[0128] In some embodiments, the nucleic acid molecule is a ribonucleic acid molecule (RNA) or a deoxyribonucleic acid molecule (DNA). In some embodiments, the present application provides a ribonucleic acid molecule (RNA) comprising a nucleotide sequence encoding a Maraba-G variant disclosed herein. In some embodiments, the present application provides a deoxyribonucleic acid molecule (DNA) comprising a deoxynucleotide sequence encoding a Maraba-G variant disclosed herein.

[0129] In some embodiments, the DNA can be introduced into a cell in vivo. In some embodiments, the DNA of the present application is comprised in a vector or delivery agent. In some embodiments, the DNA of the present application is integrated into the genome of a cell.

[0130] In some embodiments, the RNA can be introduced into a cell in vivo. In some embodiments, the RNA of the present application is comprised in a vector or delivery agent.

[0131] In other aspects, the present application provides a vector comprising a nucleic acid molecule disclosed herein.

[0132] Any vector can be suitable for use in the present application. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, an adeno-associated virus (AAV) vector, a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pMD2.G, pBY, pGAR, pBABE-puro, pBABE-neo large T cDNA, pBABE-hygro-hTERT, pMKO.lGFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0133] In some preferred embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding wild-type Maraba-G and / or a Maraba-G variant is located on a lentiviral envelope plasmid vector. In some more preferred embodiments, the envelope plasmid vector is a pMD2.G vector.

[0134] In other aspects, the present application provides a vector combination comprising a vector carrying a nucleic acid molecule comprising a nucleotide sequence encoding wild-type Maraba-G and / or a Maraba-G variant, and one or more helper packaging plasmids capable of assisting viral packaging.

[0135] In some embodiments, the vector combination includes an envelope plasmid for lentiviral packaging, and one or more helper packaging plasmids, wherein the envelope plasmid carries a nucleic acid molecule comprising a nucleotide sequence encoding wild-type Maraba-G and / or a Maraba-G variant. In some embodiments, the vector combination includes an envelope plasmid for lentiviral packaging, one or more helper packaging plasmids, and a transfer plasmid or a shuttle plasmid carrying a gene of interest, wherein the envelope plasmid carries a nucleic acid molecule comprising a nucleotide sequence encoding wild-type Maraba-G and / or a Maraba-G variant.

[0136] In some preferred embodiments, the envelope plasmid vector is pMD2.G. In some preferred embodiments, the helper packaging plasmid is selected from one or more of: pRSV-Rev, pMDLg-pRRE, psPAX2, pCMV-dR8.91.

[0137] The vector combination disclosed herein can be used to prepare lentiviral particles, under the action of the envelope plasmid and one or more helper packaging plasmids, the transfer plasmid or shuttle plasmid carrying the target gene is packaged into lentiviral particles, so as to be used for delivering the target gene to target cells. The transfer plasmid or shuttle plasmid vector structure suitable for packaging into lentiviral particles is well known to those skilled in the art, for example, the transfer plasmid or shuttle plasmid vector comprises 5' long terminal repeat (LTR), 3' LTR, Psi packaging sequence (Ψ), polyadenylation signal and the like elements, so as to realize the packaging of lentivirus and the expression of the target gene in target cells. Those skilled in the art will be able to easily utilize known or optimized transfer plasmid or shuttle plasmid vectors to construct a transfer plasmid or shuttle plasmid expressing a target gene.

[0138] Host cell

[0139] In some aspects, the present application provides a host cell comprising a nucleic acid molecule disclosed herein or a vector or vector combination disclosed herein.

[0140] Any cell can be used as a host cell for the nucleic acid molecules or vectors or vector combinations of the present application. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobactehaceae, such as Escherichia, for example, E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium; Serratia, for example, Serratia marcescans and Shigella; Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell includes, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, HEK293FT, and HEK293FS.

[0141] In some embodiments, the host cells of the present application can be used to produce viral particles described herein. In some preferred embodiments, the host cells include HEK293 cells, for example, HEK293FT cells.

[0142] Viral particles

[0143] In some aspects, the present application provides viral particles comprising a wild-type Maraba-G envelope glycoprotein or a Maraba-G variant as disclosed herein.

[0144] In some embodiments, the viral particles are selected from a retroviral particle, a lentiviral particle, or a virus-like particle (VLP). In some preferred embodiments, the viral particles are lentiviral particles.

[0145] Virus-like particles (VLP) are a class of nanoscale particles that resemble viruses, with structures such as envelope, capsid, etc. that can be used for transduction of a gene of interest, but do not contain viral genes. VLPs can also be "pseudotyped" by replacing their envelope proteins, modifying the targeting of the VLP.

[0146] In some embodiments, the virus particles described herein comprise an expression cassette comprising a gene of interest encoding a protein or polypeptide of interest. In some embodiments, the gene of interest encodes a therapeutic protein, a therapeutic polypeptide, or a therapeutic nucleic acid. In some embodiments, the therapeutic protein, the therapeutic polypeptide, or the therapeutic nucleic acid is capable of treating and / or preventing a disease or disorder, including but not limited to, a tumor and an autoimmune disease.

[0147] In some embodiments, the gene of interest encodes a molecule selected from any one or a combination of the following: siRNA, shRNA, non-coding RNA (e.g., guide RNA of CRISPR system), polypeptide, and protein.

[0148] In some embodiments of the present application, the gene of interest encodes a chimeric antigen receptor (CAR) or a TCR. In some embodiments, the gene of interest encodes a CAR comprising: an extracellular domain comprising a binding domain that specifically binds to a target molecule; an intracellular signaling domain, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM) and an optional costimulatory signaling domain; and a transmembrane domain linking the extracellular domain and the intracellular signaling domain.

[0149] In some embodiments, the viral envelope surface of the virus particles disclosed herein further comprises one or more cell targeting molecules. In some embodiments, the targeting molecule is a membrane-bound protein. In some embodiments, the viral envelope surface of the virus particles disclosed herein further comprises at least one heterologous membrane-bound protein; preferably, the viral envelope surface further comprises at least two heterologous membrane-bound proteins. When the viral envelope surface further comprises two or more heterologous membrane-bound proteins, the titer and transduction efficiency of Maraba-G pseudotyped viruses are improved more significantly than VSVG pseudotyped viruses.

[0150] In some embodiments, the cell targeting molecule is capable of binding to one or more cells selected from the group consisting of: normal tissue cells, immune cells, tumor cells, and stem cells. In some embodiments, the immune cells are selected from one or more cells selected from the group consisting of: T cells, B cells, NK cells, dendritic cells, macrophages, NKT cells, and peripheral blood mononuclear cells (PBMCs); preferably, the immune cells are T cells.

[0151] The viral particles disclosed herein are capable of directly transducing immune cells, such as directly transfecting immune cells in PBMCs, without the need to sort and activate the immune cells, saving on manufacturing costs. In some preferred embodiments, the immune cells comprise T cells, NK cells, or B cells. In some more preferred embodiments, the immune cells comprise T cells.

[0152] In some embodiments of the application, the viral envelope surface of the viral particle comprises a wild-type Maraba-G envelope glycoprotein or a Maraba-G variant, and further comprises one or more T cell targeting molecules selected from a ligand, a receptor, or an antibody that binds to a molecule specifically expressed on the surface of a T cell.

[0153] In some embodiments, the molecule specifically expressed on the surface of a T cell is selected from one or more of CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD30, CD39, CD40, CD44, CD45, CD45RA, CD45RO, CD62L, CD70, CD73, OX40, 4-1BB, ICOS, IL2RB, IL7R, IL12R, IL18R, IL21R, LFA-1, VLA-4, PSGL-1, CXCR2, CXCR3, CXCR4, CCR5, CCR7, CCR4, CCR8, and TCR. In some preferred embodiments, the molecule specifically expressed on the surface of a T cell is selected from one or more of CD3, TCR, CD7, LFA-1, and CD28. In some preferred embodiments, the T cell targeting molecule is selected from one or more of a CD3 antibody, a TCR alpha / beta antibody, a TCR gamma / delta antibody, a CD7 antibody, a LFA-1 antibody, a LFA-1 ligand (such as ICAM1), and a CD28 ligand (such as CD80 and / or CD86).

[0154] In some embodiments of the application, the viral envelope surface of the viral particle comprises a wild-type Maraba-G envelope glycoprotein or a Maraba-G variant and further comprises one or more immune cell activating molecules. In some embodiments, the immune cell activating molecule is a T cell activating molecule. In some embodiments, the T cell activating molecule is selected from the group consisting of: CD3 antibody, CD28 antibody, LFA-1 antibody, CD80, CD86, CD58, HHLA2, ICAM-1, OX40L, 4-lBBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and B7-H6. In some embodiments, the T cell activating molecule is selected from the group consisting of: any protein or polypeptide that specifically binds CD2, CD3, CD28H, LFA-1, DNAM-1, CD27, ICOS, LIGHT, GITR, CD30, SLAM, Ly-9, CD84, Ly108, NKG2D, NKp46, NKp44, NKp30, CD244, TCR alpha chain, TCR beta chain, TCR zeta chain, TCR gamma chain, TCR delta chain, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, or NKp80 and is capable of activating or stimulating a T cell. In some preferred embodiments, the T cell activating molecule is selected from the group consisting of: CD3 antibody, CD28 antibody, LFA-1 antibody, CD80, CD86.

[0155] In some embodiments, the viral envelope further comprises one or more specific binding molecules for an immune cell surface adhesion molecule. In some embodiments, the specific binding molecule for an immune cell surface adhesion molecule is selected from the group consisting of: CD80, CD86, CD58, HHLA2, ICAM-1, OX40L, 4-lBBL, CD40, CD155, CD70, HVEM, GITRL, ICOSL, CD30L, SLAM, Ly-9, CD84, Ly108, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or B7-H6. In some preferred embodiments, the specific binding molecule for an immune cell surface adhesion molecule is selected from the group consisting of: CD58, ICAM-1, CD80, CD86.

[0156] In some embodiments, the viral envelope further comprises a fusion protein consisting of a plurality of immune cell activating molecules and immune cell surface adhesion molecules specific binding molecules. The immune cell activating molecules and immune cell surface adhesion molecules are as defined above.

[0157] In some embodiments of the application, the viral envelope surface of the viral particle comprises a wild-type Maraba-G envelope glycoprotein or a Maraba-G variant, and further comprises an immune cell targeting molecule and / or an immune cell activating molecule.

[0158] In some embodiments, the immune cell targeting molecule comprises a ligand, receptor or antibody that specifically binds an immune cell marker comprising:

[0159] a) a T cell marker selected from the group consisting of: CD3, CD28, 4-1BB, AhR, CD2, CD7, CD4, CD8, CD25, CD44, CD45RA, CD47, CD62L, CD69, CD94, CD95, CD127, CD161, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5), CD193 (CCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, PD-1, TCRa / b, CD5, CD27, CD45RO, CD45RB, CD57, CD103, CD122, P2RX7, LAG-3, TIM-3, IL6ST, TCRy / d, Vd1, Vd2, TCR (Va24-Ja18), CD185 (CXCR5), CXCR6, IL-21R, Va7.2, Ja33, CXCR6, IL-18R, and KLRB1 (CD161); or

[0160] b) an NK cell marker selected from the group consisting of: CD56, CD57, NKp30, NKp44, NKp46, CD16, KIR(s), NKG2 proteins, KLRB1 (CD161), KLRD1 (cd94), IL2Rb (CD122), IL-21R, SLAMF6 (CD352), SLAMF7 (CD319), and IL-18R; or

[0161] c) a B cell marker selected from the group consisting of: CD19, CD20, CD21, CD22, CD24, CD38, CD40, CD72, CD32b, CD268, CD269, CD267, CD86, CD80, CD52, CD138, CD27, CD28, CD23, CD84, CD257, CD270, CD37, CD74, and CD269.

[0162] In some embodiments, the immune cell activating molecule comprises:

[0163] a) a T cell activating molecule selected from the group consisting of: a CD3 antibody, a CD4 antibody, a CD8 antibody, and a TCR antibody; or

[0164] b) an NK cell activating molecule comprising a ligand or antibody for an NK cell activating receptor selected from the group consisting of: CD16, VLA-4, CD27, CD69, NKG2C, NKG2D, NKG2E, CD223, CD226, CD244, CD319, NKp30, NKp44, NKp46, Ly-49D, Ly-49H, NKR-P1C, NKR-P1A, NKR-P1F, and PILR-beta; or

[0165] c) a B cell activating molecule comprising a ligand or antibody for a B cell activating receptor selected from the group consisting of: CD79, CD19, CD21, CD24, CD81.

[0166] In some embodiments, the viral envelope surface further comprises a specific binding molecule for an immune cell surface adhesion molecule. In some embodiments, the specific binding molecule for an immune cell surface adhesion molecule comprises a ligand, receptor, or antibody for an immune cell surface adhesion molecule; preferably, the immune cell surface adhesion molecule comprises:

[0167] a) a T cell surface adhesion molecule selected from the group consisting of: LFA-1, CD2, VLA-4, CD43, and CD45; or

[0168] b) an NK cell surface adhesion molecule selected from the group consisting of: LFA-1, CD2, pTA1, Mac-1, p150, p95, CD31, CD44, CD162R, and VLA1-6; or

[0169] c) a B cell surface adhesion molecule selected from the group consisting of: ICAM-1 and CD22.

[0170] In some embodiments, the viral envelope surface further comprises an immune cell costimulatory molecule, which provides a costimulatory signal for immune cell activation. In some embodiments, the costimulatory molecule comprises:

[0171] a) a T cell costimulatory molecule selected from the group consisting of: CD80, CD86, 4-1BBL, B7-H2, CD40, IL2, CD28 antibody, 4-1BB antibody, ICOS antibody, and CD40L antibody; or

[0172] b) an NK cell costimulatory molecule selected from the group consisting of: CD160, 4-1BBL, 4-1BB antibody, IL15, IL21, and FLT3L; or

[0173] c) a B cell costimulatory molecule selected from the group consisting of: CD28, CD40L, CD40 antibody.

[0174] In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell marker is a T cell marker selected from the group consisting of: TCRa / b, TCRy / d, CD3, and CD7.

[0175] In some embodiments, the viral envelope surface comprises: 1) a TCRa / b antibody, a TCRy / d antibody, or a CD3 antibody; and 2) a CD7 antibody. Further, the viral envelope surface further comprises: 3) a specific binding molecule for at least one T cell surface adhesion molecule, wherein the T cell surface adhesion molecule is selected from the group consisting of: LFA-1, CD2, VLA-4, CD43, and CD45.

[0176] In some embodiments, the specific binding molecule for the T cell surface adhesion molecule is selected from the group consisting of: ICAM-1, LFA-1 antibody, CD58, CD2 antibody, VCAM-1, Fibronectin, VLA-4 antibody, E-selectin, SIGLEC1, ERM protein, CD43 antibody, Galectin-3, PP14 / PAEP, CD45 antibody.

[0177] In some preferred embodiments, the viral envelope surface comprises a combination of molecules selected from the group consisting of:

[0178] (1) a CD3 antibody, a CD7 antibody, and ICAM-1;

[0179] (2) a CD3 antibody, a CD7 antibody, and a LFA-1 antibody;

[0180] (3) a TCRa / b antibody, a CD7 antibody, and ICAM-1;

[0181] (4) TCR alpha / beta antibody, CD7 antibody, and LFA-1 antibody;

[0182] (5) TCR gamma / delta antibody, CD7 antibody, and ICAM-1;

[0183] (6) TCR gamma / delta antibody, CD7 antibody, and LFA-1 antibody;

[0184] (7) CD3 antibody, CD7 antibody, and CD58;

[0185] (8) TCR alpha / beta antibody, CD7 antibody, and CD58;

[0186] (9) TCR gamma / delta antibody, CD7 antibody, and CD58;

[0187] (10) CD3 antibody, CD7 antibody, and CD2 antibody;

[0188] (11) TCR alpha / beta antibody, CD7 antibody, and CD2 antibody; and

[0189] (12) TCR gamma / delta antibody, CD7 antibody, and CD2 antibody.

[0190] In yet other embodiments, the viral envelope surface comprises: 1) a TCR alpha / beta antibody, a TCR gamma / delta antibody, or a CD3 antibody; and / or 2) a CD7 antibody. Further, the viral envelope surface further comprises: 3) a specific binding molecule for at least one T cell surface adhesion molecule, wherein the T cell surface adhesion molecule is selected from the group consisting of: LFA-1, CD2, VLA-4, CD43, and CD45.

[0191] In some embodiments, the specific binding molecule for the T cell surface adhesion molecule is selected from the group consisting of: ICAM-1, LFA-1 antibody, CD58, CD2 antibody, VCAM-1, Fibronectin, VLA-4 antibody, E-selectin, SIGLEC1, ERM protein, CD43 antibody, Galectin-3, PP14 / PAEP, CD45 antibody.

[0192] In some embodiments, the viral envelope surface further comprises an immune cell costimulatory molecule, e.g., a T cell costimulatory molecule. In some embodiments, the immune cell costimulatory molecule is a T cell costimulatory molecule selected from the group consisting of: CD80, CD86, and CD28 antibody.

[0193] In some preferred embodiments, the viral envelope surface comprises a combination of molecules selected from the group consisting of:

[0194] (1) CD3 antibody, ICAM-1, and CD80;

[0195] (2) CD3 antibody, LFA-1 antibody, and CD80;

[0196] (3) TCR α / β antibody, ICAM-1, and CD80;

[0197] (4) TCR α / β antibody, LFA-1 antibody, and CD80;

[0198] (5) TCR γ / δ antibody, ICAM-1, and CD80;

[0199] (6) TCR γ / δ antibody, LFA-1 antibody, and CD80;

[0200] (7) CD3 antibody, CD7 antibody, ICAM-1, and CD80;

[0201] (8) CD3 antibody, CD7 antibody, LFA-1 antibody, and CD80;

[0202] (9) TCR α / β antibody, CD7 antibody, ICAM-1, and CD80;

[0203] (10) TCR α / β antibody, CD7 antibody, LFA-1 antibody, and CD80;

[0204] (11) TCR γ / δ antibody, CD7 antibody, ICAM-1, and CD80;

[0205] (12) TCR γ / δ antibody, CD7 antibody, LFA-1 antibody, and CD80;

[0206] (13) CD3 antibody, CD7 antibody, CD58, and CD80;

[0207] (14) TCR α / β antibody, CD7 antibody, CD58, and CD80;

[0208] (15) TCR γ / δ antibody, CD7 antibody, CD58, and CD80;

[0209] (16) CD3 antibody, CD7 antibody, CD2 antibody, and CD80;

[0210] (17) TCR α / β antibody, CD7 antibody, CD2 antibody, and CD80;

[0211] (18) TCR γ / δ antibody, CD7 antibody, CD2 antibody, and CD80; and

[0212] the above combinations in which CD80 is replaced with CD86 or a CD28 antibody.

[0213] In embodiments of the application, the virus envelope surface displays a specific binding molecule of the aforementioned immune cell targeting molecule, immune cell activation molecule, immune cell costimulatory molecule, and / or immune cell surface adhesion molecule, such as CD3 antibody, CD80, ICAM-1, which can facilitate the adhesion effect of the virus to the primary T cells, enhance the activation of the virus to the T cells, while improving the virus titer and transduction efficiency, the positive proportion of CAR expression is significantly improved, and it is helpful to maintain the memory phenotype with the initial T cells.

[0214] For the aforementioned specific binding molecules of immune cell targeting molecules, immune cell activation molecules, immune cell costimulatory molecules, and immune cell surface adhesion molecules, wherein the antibody includes a full-length antibody, an antigen-binding fragment thereof, or a combination of antigen-binding fragments. For example, the antigen-binding fragment can include Fab, Fab', F(ab')2, scFv, Diabody, VHH, dAb, etc. The antibody described herein can be any combination of these antigen-binding fragments, such as a combination of scFv and scFv, VHH and VHH, scFv and VHH, etc. A person skilled in the art can select a suitable linker, such as a GS linker known in the art, to combine the antigen-binding fragments together to achieve the desired function.

[0215] In embodiments of the application, in the combination of the aforementioned specific binding molecules of immune cell targeting molecules, immune cell activation molecules, immune cell costimulatory molecules, and / or immune cell surface adhesion molecules, each molecule can be included in the virus envelope described herein in the form of a fusion protein, or can be included in the virus envelope described herein in the form of a single protein. A person skilled in the art can select a suitable linker, such as a GS linker known in the art, to combine the molecules together to achieve the desired function.

[0216] In some embodiments of the application, the CD3 antibody comprises the following combination of CDRs:

[0217] (1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and HCDR3 as shown in SEQ ID NO: 5-10, respectively; or

[0218] (2) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and HCDR3 as shown in SEQ ID NO: 68-73, respectively.

[0219] In some embodiments, the CD3 antibody comprises a VH and a VL, wherein:

[0220] (1) the VH and VL comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 11 and 12, respectively; or

[0221] (2) the VH and VL comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 74 and 75, respectively.

[0222] In some embodiments, the CD3 antibody comprises one or more scFv comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 67.

[0223] In some embodiments, the CD7 antibody comprises a combination of CDRs:

[0224] (1) HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 85-87, respectively;

[0225] (2) HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 90-92, respectively;

[0226] (3) HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 115-117, respectively;

[0227] (4) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and HCDR3 as set forth in SEQ ID NOs: 95-100, respectively; or

[0228] (5) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and HCDR3 as set forth in SEQ ID NOs: 105-110, respectively.

[0229] In some embodiments, the CD7 antibody comprises one or more VHH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 84, 89, or 114.

[0230] In some embodiments, the CD7 antibody comprises a VH and a VL, wherein:

[0231] (1) the VH and VL comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 101 and 102, respectively; or

[0232] (2) the VH and VL comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 111 and 112, respectively.

[0233] In some embodiments, the CD7 antibody comprises one or more scFv comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 94 or SEQ ID NO: 104.

[0234] In some embodiments, the TCR a / b antibody comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 78-80, respectively.

[0235] In some embodiments, the TCR a / b antibody comprises one or more VHH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 81.

[0236] In some embodiments, the LFA-1 antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and HCDR3 as set forth in SEQ ID NOs: 25-30, respectively.

[0237] In some embodiments, the LFA-1 antibody comprises VH and VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 31 and 32, respectively.

[0238] In some embodiments, the LFA-1 antibody comprises one or more scFv comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24.

[0239] In embodiments of the application, the antibodies, e.g., CD3 antibodies, CD7 antibodies, TCRa / b antibodies, and LFA-1 antibodies, are each a membrane-bound protein, and the antibodies comprise a membrane-binding domain. In some preferred embodiments, the membrane-binding domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13, 82, or 118.

[0240] Those skilled in the art will appreciate that some of the specific binding molecules for immune cell targeting molecules, immune cell activating molecules, immune cell costimulatory molecules, and / or immune cell surface adhesion molecules involved in the present application are themselves naturally membrane-bound proteins, and do not require additional signal peptides or membrane-binding domains to achieve anchoring to the viral envelope surface, but rather utilize the naturally occurring signal peptide and membrane-binding domain already contained therein.

[0241] Furthermore, those skilled in the art will appreciate that the signal peptide of a membrane-bound protein is typically removed from the protein sequence after anchoring to the viral envelope surface. Thus, the viral envelope surface membrane-bound proteins of the viral particles provided herein typically do not comprise a signal peptide sequence.

[0242] In some preferred embodiments, the membrane-bound CD3 antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 23-335 of SEQ ID NO: 2.

[0243] In some preferred embodiments, the membrane-bound CD3 antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 23-336 of SEQ ID NO: 66.

[0244] In some preferred embodiments, the membrane-bound CD7 antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22-214 of SEQ ID NO: 83.

[0245] In some preferred embodiments, the membrane-bound CD7 antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22-215 of SEQ ID NO: 88.

[0246] In some preferred embodiments, the membrane-bound CD7 antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22-330 of SEQ ID NO: 93.

[0247] In some preferred embodiments, the membrane-bound CD7 antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 23-340 of SEQ ID NO: 103.

[0248] In some preferred embodiments, the membrane-bound CD7 antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22-213 of SEQ ID NO: 113.

[0249] In some preferred embodiments, the membrane-bound TCRa / b antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22-201 of SEQ ID NO: 76.

[0250] In some preferred embodiments, the membrane-bound LFA-1 antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22-334 of SEQ ID NO: 22.

[0251] In some preferred embodiments, the ICAM-1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 28-532 of SEQ ID NO: 18.

[0252] In some preferred embodiments, the CD80 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 35-288 of SEQ ID NO: 14.

[0253] In some preferred embodiments, CD58 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 29-250 of SEQ ID NO: 16.

[0254] In some preferred embodiments, CD86 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 24-329 of SEQ ID NO: 64.

[0255] In some preferred embodiments, VCAM-1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 25-739 of SEQ ID NO: 19.

[0256] Compositions and kits

[0257] In some aspects, the present application provides compositions comprising the viral particles disclosed herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the compositions of the present application are pharmaceutical compositions.

[0258] Exemplary carriers for use in the compositions of the present application include saline, buffered saline, dextrose, and water. Exemplary excipients for use in the compositions of the present application include fillers, binders, disintegrants, coating agents, adsorbents, antiadherents, glidants, preservatives, antioxidants, flavoring agents, coloring agents, sweetening agents, solvents, cosolvents, buffers, chelating agents, viscosity conferring agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers, and tonicity adjusting agents. The selection of a suitable excipient to prepare the compositions of the present application is known to those skilled in the art. In general, the selection of a suitable excipient depends, inter alia, on the active agent used, the disease to be treated, and the desired dosage form of the composition.

[0259] Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. These materials are well known in the art for use in the delivery of active agents. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0260] The pharmaceutical compositions of the present application can be formulated to be compatible with their intended route of administration. Preferably, the pharmaceutical compositions of the present application are administered parenterally, by injection, or orally. Preferably, the injection includes intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous, intranodal, and the like. The pharmaceutical compositions are in a form that is suitable for administration, preferably in solid, semi-solid, or liquid form, i.e., in water, saline, or other aqueous solution, non-aqueous solution, or suspension. The pharmaceutical compositions of the present application can be in unit dosage form suitable for administration, and can be administered in a form suitable for injection or other therapeutic administration. The pharmaceutical compositions of the present application can be administered in a form suitable for injection or other therapeutic administration.

[0261] The dosage levels of the pharmaceutical compositions of the present application can be adjusted according to the amount of the composition required to achieve the desired diagnostic or therapeutic result. The administration regimen can also be a single injection or multiple injections, or adjusted. The dosage level and regimen selected will depend on a variety of factors including the activity and stability (i.e., half-life) of the pharmaceutical composition, the formulation, the route of administration, combination with other drugs or therapies, the disease or condition to be detected and / or treated, and the health and prior medical history of the subject to be treated.

[0262] In some aspects, the present application provides kits comprising a viral particle and / or a pharmaceutically acceptable carrier as disclosed herein, and instructions for transducing immune cells in vitro or in vivo. Containers for use in such kits will typically include at least one vial, test tube, flask, bottle, syringe, or other container, as appropriate for the formulation being administered.

[0263] In some embodiments, the kit further comprises an administration device. For example, the administration device can be a syringe (e.g., a pre-filled syringe with a viral particle or composition of the present application, such as an auto-injector), which can include a barrel for containing the fluid to be injected and a needle (which can be used to pierce the skin and / or a blood vessel). The mode of administration can vary. The route of administration can include intraperitoneal injection, subcutaneous injection, intravenous injection, or intranodal injection, among others. In some preferred embodiments, the mode of administration is intranodal injection, such as intralymph node. In some more preferred embodiments, the lymph node is a groin lymph node.

[0264] Methods and uses

[0265] In some aspects, the present application provides the use of a wild-type Maraba-G envelope glycoprotein or a Maraba-G variant disclosed herein in the manufacture of a pseudotyped virus.

[0266] In some embodiments, the pseudotyped virus is selected from a retrovirus, a lentivirus, or a viroid. In some preferred embodiments, the pseudotyped virus is a pseudotyped lentivirus.

[0267] In some embodiments, the pseudotyped virus is capable of specifically transducing a target cell in a subject. In some preferred embodiments, the target cell is selected from one or more of the following: a T cell, a B cell, a NK cell, a dendritic cell, a macrophage, an NKT cell, and a peripheral blood mononuclear cell (PBMC). In some more preferred embodiments, the target cell is a T cell.

[0268] In some embodiments, the pseudotyped virus further comprises one or more cell targeting molecules. In some preferred embodiments, the cell targeting molecule is selected from a specific binding molecule for an immune cell targeting molecule, an immune cell activating molecule, an immune cell costimulatory molecule, and / or an immune cell surface adhesion molecule, as defined hereinbefore.

[0269] In another aspect, the present application provides a method of transducing an immune cell in a subject, the method comprising administering to the subject a Maraba-G envelope protein pseudotyped viral particle or a composition comprising a Maraba-G envelope protein pseudotyped viral particle as disclosed herein.

[0270] The present application also provides a method of producing a target cell expressing a gene of interest in a subject, the method comprising administering to the subject a Maraba-G envelope protein pseudotyped viral particle or a composition comprising a Maraba-G envelope protein pseudotyped viral particle as disclosed herein.

[0271] In some embodiments, the gene of interest encodes a therapeutic nucleic acid, a therapeutic polypeptide, or a therapeutic protein. In some preferred embodiments, the gene of interest encodes a CAR.

[0272] In some embodiments, the target cell is selected from one or more of the following: a T cell, a B cell, a NK cell, a dendritic cell, a macrophage, an NKT cell, and a peripheral blood mononuclear cell (PBMC). In some preferred embodiments, the target cell is a T cell.

[0273] The present application also provides a method of producing a chimeric antigen receptor T cell (CAR-T) in a subject, the method comprising administering to the subject a Maraba-G envelope protein pseudotyped viral particle or a composition comprising a Maraba-G envelope protein pseudotyped viral particle as disclosed herein, wherein the viral particle carries a CAR expression cassette.

[0274] The present application also provides methods of treating and / or preventing a disease in a subject, comprising administering to the subject a Maraba-G envelope protein pseudotyped viral particle disclosed herein or a composition comprising a Maraba-G envelope protein pseudotyped viral particle disclosed herein.

[0275] In some embodiments, the disease is a tumor or cancer, a viral infection, aging, or an autoimmune disease.

[0276] In some embodiments, the tumor includes various hematological tumors and solid tumors. Solid tumors include, but are not limited to, tumors arising in any of bone, bone joints, muscle, lung, trachea, heart, spleen, arteries, veins, capillaries, lymph nodes, lymphatic vessels, lymphatic fluid, mouth, pharynx, esophagus, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gall, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testicle, vas deferens, penis, eye, ear, nose, tongue, skin, brain, brain stem, medulla, spinal cord, cerebrospinal fluid, nerve, thyroid, parathyroid, adrenal gland, pituitary, pineal, pancreatic islet, thymus, gonad, sublingual gland, and parotid gland. Hematological tumors are selected from at least one of acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, and the like in blood cells and hematopoietic system.

[0277] In some embodiments, the autoimmune disease is selected from Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes mellitus, systemic lupus erythematosus, vasculitis, Addison's disease, polymyositis, Sjogren's syndrome, progressive systemic sclerosis, glomerular inflammation.

[0278] In some embodiments, the disease is a cancer. In some embodiments, the cancer is selected from immune system cancer, breast cancer, lung cancer, melanoma, bladder cancer, stomach cancer, esophageal cancer, colorectal cancer, cervical cancer, ovarian cancer, endometrial cancer, kidney cancer, brain cancer, small intestine cancer, liver cancer, pancreatic cancer, hepatobiliary tumor, germ cell cancer, prostate cancer, Merkel cell carcinoma, head and neck cancer, thyroid cancer, and sarcoma.

[0279] In some embodiments, the immune system cancer is selected from the group consisting of lymphoma, leukemia, and myeloma, for example, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma, primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, B-lymphoblastic leukemia (B-ALL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies.

[0280] In some embodiments, the viral particle is administered by intraperitoneal injection, subcutaneous injection, intravenous injection, or intranodal injection. In some embodiments, the intranodal injection is into a lymph node; preferably, the lymph node is a groin lymph node.

[0281] In some embodiments, the subject is a mammal, including but not limited to humans, monkeys, pigs, and other farm animals, sport animals, pets, primates, horses, dogs, cats, giant pandas, rodents (including mice, rats, guinea pigs), and the like.

[0282] In other aspects, the present application provides use of a Maraba-G envelope protein pseudotyped viral particle disclosed herein or a composition comprising a Maraba-G envelope protein pseudotyped viral particle disclosed herein in the manufacture of a medicament for transducing an immune cell in a subject.

[0283] The present application also provides use of a Maraba-G envelope protein pseudotyped viral particle disclosed herein or a composition comprising a Maraba-G envelope protein pseudotyped viral particle disclosed herein in the manufacture of a medicament for producing a target cell expressing a gene of interest in a subject.

[0284] In some embodiments, the gene of interest encodes a therapeutic nucleic acid, a therapeutic polypeptide, or a therapeutic protein. In some preferred embodiments, the gene of interest encodes a CAR.

[0285] In some embodiments, the target cell is selected from one or more of the following: a T cell, a B cell, an NK cell, a dendritic cell, a macrophage, an NKT cell, and a peripheral blood mononuclear cell (PBMC). In some preferred embodiments, the target cell is a T cell.

[0286] The present application also provides use of a Maraba-G envelope protein pseudotyped viral particle disclosed herein or a composition comprising a Maraba-G envelope protein pseudotyped viral particle disclosed herein in the manufacture of a medicament for preparing a chimeric antigen receptor T cell (CAR-T) expressing cell in a subject, wherein the viral particle carries a CAR expression cassette.

[0287] The present application also provides use of a Maraba-G envelope protein pseudotyped viral particle disclosed herein or a composition comprising a Maraba-G envelope protein pseudotyped viral particle disclosed herein in the manufacture of a medicament for treating and / or preventing a disease in a subject.

[0288] In some embodiments, the disease is a tumor or cancer, a viral infection, an aging, or an autoimmune disease.

[0289] In some embodiments, the tumor includes various hematological tumors and solid tumors. The solid tumors include, but are not limited to, tumors generated in any of bone, bone joint, muscle, lung, trachea, heart, spleen, artery, vein, capillary, lymph node, lymph vessel, lymph fluid, oral cavity, pharynx, esophagus, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gall, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, urinary bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testis, vas deferens, penis, eye, ear, nose, tongue, skin, brain, brain stem, medulla, spinal cord, cerebrospinal fluid, nerve, thyroid, parathyroid, adrenal gland, pituitary, pineal gland, pancreatic islet, thymus, gonad, sublingual gland, and parotid gland. The hematological tumors are selected from at least one of acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, and the like in blood cells and hematopoietic system.

[0290] In some embodiments, the autoimmune disease is selected from Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes mellitus, systemic lupus erythematosus, vasculitis, Addison's disease, polymyositis, Sjogren's syndrome, progressive systemic sclerosis, glomerular inflammation.

[0291] In some embodiments, the disease is a cancer. In some embodiments, the cancer is selected from immune system cancer, breast cancer, lung cancer, melanoma, bladder cancer, stomach cancer, esophageal cancer, colorectal cancer, cervical cancer, ovarian cancer, endometrial cancer, kidney cancer, brain cancer, small intestine cancer, liver cancer, pancreatic cancer, hepatobiliary tumor, germ cell cancer, prostate cancer, Merkel cell carcinoma, head and neck cancer, thyroid cancer, and sarcoma.

[0292] In some embodiments, the immune system cancer is selected from the group consisting of lymphoma, leukemia, and myeloma, such as multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma, primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, B-lymphoblastic leukemia (B-ALL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies.

[0293] In some embodiments, the viral particle is administered by intraperitoneal injection, subcutaneous injection, intravenous injection, or intranodal injection. In some embodiments, the intranodal injection is into a lymph node; preferably, the lymph node is a groin lymph node.

[0294] In some embodiments, the subject is a mammal, including but not limited to humans, monkeys, pigs, and other farm animals, sport animals, pets, primates, horses, dogs, cats, giant pandas, rodents (including mice, rats, guinea pigs), and the like.

[0295] The present application also relates to the following embodiments:

[0296] 1. A novel viral vector, wherein the viral envelope surface of the viral vector comprises:

[0297] at least one heterologous immune cell targeting molecule; and / or

[0298] at least one heterologous immune cell activating molecule; and

[0299] at least one specific binding molecule for a heterologous immune cell surface adhesion molecule; and

[0300] a heterologous viral envelope glycoprotein that generates a pseudotyped viral vector.

[0301] 2. The viral vector of embodiment 1, wherein the immune cell targeting molecule comprises a ligand, receptor, or antibody that specifically binds to an immune cell marker, wherein the immune cell marker comprises:

[0302] a) a T cell marker selected from the group consisting of: CD3, CD28, 4-1BB, AhR, CD2, CD7, CD4, CD8, CD25, CD44, CD45RA, CD47, CD62L, CD69, CD94, CD95, CD127, CD161, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5), CD193 (CCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, PD-1, TCRa / b, CD5, CD27, CD45RO, CD45RB, CD57, CD103, CD122, P2RX7, LAG-3, TIM-3, IL6ST, gdTCR, Vd1, Vd2, TCR (Va24-Ja18), CD185 (CXCR5), CXCR6, IL-21R, Va7.2, Ja33, CXCR6, IL-18R, and KLRB1 (CD161); or

[0303] b) an NK cell marker selected from the group consisting of: CD56, CD57, NKp30, NKp44, NKp46, CD16, KIR(s), NKG2 proteins, KLRB1 (CD161), KLRD1 (cd94), IL2Rb (CD122), IL-21R, SLAMF6 (CD352), SLAMF7 (CD319), and IL-18R; or

[0304] c) a B cell marker selected from the group consisting of: CD19, CD20, CD21, CD22, CD24, CD38, CD40, CD72, CD32b, CD268, CD269, CD267, CD86, CD80, CD52, CD138, CD27, CD28, CD23, CD84, CD257, CD270, CD37, CD74, and CD269.

[0305] 3. The viral vector according to embodiment 1, characterized in that the immune cell activating molecule comprises:

[0306] a) a T cell activating molecule selected from the group consisting of CD3, CD4, and CD8 antibodies; or

[0307] b) an NK cell activating molecule comprising a ligand or antibody for an NK cell activating receptor selected from the group consisting of: CD16, VLA-4, CD27, CD69, NKG2C, NKG2D, NKG2E, CD223, CD226, CD244, CD319, NKp30, NKp44, NKp46, Ly-49D, Ly-49H, NKR-P1C, NKR-P1A, NKR-P1F, and PILR-beta; or

[0308] c) a B cell activating molecule comprising a ligand or antibody for a B cell activating receptor selected from the group consisting of: CD79, CD19, CD21, CD24, CD81.

[0309] 4. The viral vector of embodiment 1, wherein the specific binding molecule for an immune cell surface adhesion molecule comprises a ligand, receptor, or antibody for an immune cell surface adhesion molecule; and the immune cell surface adhesion molecule comprises:

[0310] a) a T cell surface adhesion molecule selected from the group consisting of: LFA-1, CD2, VLA-4, CD43, and CD45; or

[0311] b) an NK cell surface adhesion molecule selected from the group consisting of: LFA-1, CD2, pTA1, Mac-1, p150, p95, CD31, CD44, CD162R, and VLA1-6; or

[0312] c) a B cell surface adhesion molecule selected from the group consisting of: ICAM-1 and CD22.

[0313] 5. The viral vector of embodiment 1, wherein the viral membrane surface further comprises an immune cell costimulatory molecule that provides a costimulatory signal for immune cell activation.

[0314] 6. The viral vector of embodiment 5, wherein the costimulatory molecule comprises:

[0315] a) a T cell costimulatory molecule selected from at least one of the group consisting of: CD80, CD86, 4-1BBL, B7-H2, CD40, IL2, CD28 antibody, 4-1BB antibody, ICOS antibody, and CD40L antibody; or

[0316] b) an NK cell costimulatory molecule selected from at least one of the group consisting of: CD160, 4-1BBL, 4-1BB antibody, IL15, IL21, and FLT3L; or

[0317] c) a B cell costimulatory molecule selected from the group consisting of at least one of: CD28, CD40L, CD40 antibody.

[0318] 7. The viral vector according to any one of embodiments 1-6, wherein the immune cell is a T cell.

[0319] 8. The viral vector according to embodiment 7, wherein the immune cell targeting molecule or immune cell activating molecule is a CD3 antibody.

[0320] 9. The viral vector according to embodiment 7 or 8, wherein the specific binding molecule for an immune cell surface adhesion molecule is a LFA-1 specific binding molecule.

[0321] 10. The viral vector according to embodiment 9, wherein the LFA-1 specific binding molecule comprises ICAM-1 or a LFA-1 antibody.

[0322] 11. The viral vector according to any one of embodiments 5-10, wherein the immune cell costimulatory molecule is a T cell costimulatory molecule selected from the group consisting of at least one of: CD80, CD86 and CD28 antibody.

[0323] 12. The viral vector according to any one of embodiments 2-11, wherein the immune cell targeting molecule or immune cell activating molecule is a CD3 antibody; the specific binding molecule for an immune cell surface adhesion molecule is ICAM-1; the immune cell costimulatory molecule is CD80; or

[0324] the immune cell targeting molecule or immune cell activating molecule is a CD3 antibody; the specific binding molecule for an immune cell surface adhesion molecule is a LFA-1 antibody; the immune cell costimulatory molecule is CD80.

[0325] the immune cell targeting molecule or immune cell activating molecule is a CD3 antibody; the specific binding molecule for an immune cell surface adhesion molecule is ICAM-1; the immune cell costimulatory molecule is CD86; or

[0326] the immune cell targeting molecule or immune cell activating molecule is a CD3 antibody; the specific binding molecule for an immune cell surface adhesion molecule is a LFA-1 antibody; the immune cell costimulatory molecule is CD86.

[0327] 13. The viral vector according to any one of embodiments 1-6, wherein the immune cell is an NK cell.

[0328] 14. The viral vector according to embodiment 13, wherein the immune cell targeting molecule or immune cell activating molecule is selected from the group consisting of CD16 antibody, NKp30, NKp44, NKp46, NKG2C, NKG2D.

[0329] 15. The viral vector according to embodiment 13 or 14, wherein the specific binding molecule for an immune cell surface adhesion molecule is a LFA-1 specific binding molecule.

[0330] 16. The viral vector according to embodiment 15, wherein the LFA-1 specific binding molecule comprises ICAM-1 or a LFA-1 antibody.

[0331] 17. The viral vector according to any one of embodiments 13-16, wherein the immune cell costimulatory molecule is an NK cell costimulatory molecule selected from the group consisting of at least one of 4-1BBL, 4-1BB antibody, IL15, IL21 and FLT3L.

[0332] 18. The viral vector according to any one of embodiments 1-17, wherein the viral envelope comprises a membrane-bound cytokine.

[0333] 19. The viral vector according to embodiment 18, wherein the cytokine is selected from the group consisting of at least one of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21.

[0334] 20. The viral vector according to any one of embodiments 1-19, wherein the viral envelope glycoprotein is selected from the group consisting of at least one of vesicular stomatitis virus envelope glycoprotein VSVG and variants thereof, Cocal virus envelope glycoprotein and variants thereof, Maraba virus envelope glycoprotein Maraba and variants thereof, Morreton virus envelope glycoprotein and variants thereof, Alagoa virus envelope glycoprotein and variants thereof, New Jersey virus envelope glycoprotein and variants thereof, Carajas virus envelope glycoprotein and variants thereof, baboon endogenous retrovirus envelope glycoprotein BaEV and variants thereof, feline endogenous retrovirus envelope glycoprotein RD114 and variants thereof, gibbon ape leukemia virus envelope glycoprotein GALV and variants thereof, koala retrovirus envelope glycoprotein KoRV and variants thereof, simian endogenous retrovirus envelope glycoprotein SRV and variants thereof.

[0335] 21. The viral vector of embodiment 20, wherein the viral envelope glycoprotein is selected from the group consisting of: vesicular stomatitis virus glycoprotein (VSVG) and variants thereof, Cocal virus envelope glycoprotein and variants thereof, and Maraba virus envelope glycoprotein Maraba and variants thereof.

[0336] 22. The viral vector of any one of embodiments 1-21, wherein the viral vector is derived from: a retroviral vector, a lentiviral vector, or a virus-like particle (VLP) vector.

[0337] 23. The viral vector of any one of embodiments 1-22, wherein the viral vector further comprises an expression cassette comprising a gene of interest encoding a therapeutic protein or polypeptide of interest.

[0338] 24. The viral vector of embodiment 23, wherein the protein or polypeptide of interest comprises a chimeric antigen receptor (CAR) comprising: an extracellular domain comprising a binding domain that specifically binds to a target molecule; an intracellular signaling domain, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM) and an optional costimulatory signaling domain; and a transmembrane domain linking the extracellular domain and the intracellular signaling domain.

[0339] 25. The viral vector of any one of embodiments 1-24, wherein the viral vector is capable of directly transducing PBMCs or immune cells without prior activation; preferably, the immune cells comprise T cells, NK cells, or B cells.

[0340] 26. The viral vector of any one of embodiments 1-24, wherein the viral vector is capable of transducing immune cells in vivo; preferably, the immune cells comprise T cells, NK cells, or B cells.

[0341] 27. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the viral vector of any one of embodiments 1-26.

[0342] 28. Use of the viral vector of any one of embodiments 1-26 in the manufacture of a medicament for preventing or treating a tumor, a viral infection, aging, or an autoimmune disease.

[0343] 29. A method of transducing immune cells in a subject, comprising administering to the subject the viral vector of any one of embodiments 1-26 or the pharmaceutical composition of embodiment 27.

[0344] 30. A method of making immune cells expressing a gene of interest in a subject, comprising administering to the subject the viral vector of embodiment 23 or 24, or the pharmaceutical composition of embodiment 27.

[0345] 31. A method of making chimeric antigen receptor T cells (CAR-T) in a subject, comprising administering to the subject the viral vector of embodiment 24, or the pharmaceutical composition of embodiment 27.

[0346] 32. A method of treating and / or preventing a disease in a subject, comprising administering to the subject the viral vector of embodiment 23, or the pharmaceutical composition of embodiment 27.

[0347] 33. The method of any one of embodiments 29-32, wherein the viral vector is administered by intraperitoneal, subcutaneous or intranodal injection.

[0348] 34. The method of embodiment 33, wherein the intranodal injection is into a lymph node; preferably, the lymph node is a groin lymph node.

[0349] 35. A kit comprising a container comprising the viral vector of embodiment 23, and / or a pharmaceutically acceptable carrier, and instructions for use in transducing immune cells in vitro or in vivo.

[0350] 36. Use of a Maraba virus envelope in the construction of a pseudotyped viral vector, wherein the pseudotyped viral vector is capable of transducing target cells in a subject.

[0351] 37. The use of embodiment 36, wherein the target cells are immune cells.

[0352] 38. The use of embodiment 36 or 37, wherein the pseudotyped viral envelope comprises at least one heterologous membrane-bound protein on its surface.

[0353] 39. Use of a Maraba virus envelope in the construction of a pseudotyped viral vector, wherein the pseudotyped viral envelope comprises at least one heterologous membrane-bound protein on its surface.

[0354] 40. The use of embodiment 36, wherein the heterologous membrane-bound protein is selected from the group consisting of: an immune cell targeting molecule as defined in any one of embodiments 1-17, a specific binding molecule for an immune cell surface adhesion molecule, an immune cell costimulatory molecule as defined in any one of embodiments 5-17, and a cytokine as defined in embodiment 18.

[0355] 41. The use of either of embodiments 36 or 37, wherein the Maraba virus envelope glycoprotein has mutations at sites corresponding to the following sites of the vesicular stomatitis virus Indiana strain envelope glycoprotein VSVG, compared to the VSVG global: substitution / deletion at H8, substitution / deletion at N9, substitution / deletion at Q10, substitution / deletion at K47, substitution / deletion at K50, substitution / deletion at A51, substitution / deletion at K66, substitution / deletion at S162, substitution / deletion at S183, substitution / deletion at S179, substitution / deletion at N180, substitution / deletion at 1182, substitution / deletion at M184, substitution / deletion at Y209, substitution / deletion at S230, substitution / deletion at 1347, substitution / deletion at T350, substitution / deletion at T352, substitution / deletion at E353, substitution at R354, substitution / deletion at T368, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353, or any combination thereof.

[0356] The present application also relates to the following embodiments:

[0357] 1. A novel viral particle, wherein the viral envelope surface of the viral particle comprises:

[0358] an immune cell targeting molecule; and / or

[0359] an immune cell activating molecule; and

[0360] a specific binding molecule for an immune cell surface adhesion molecule; and

[0361] a heterologous viral envelope glycoprotein that generates a pseudotyped virus.

[0362] 2. The viral particle of embodiment 1, wherein the immune cell targeting molecule comprises a ligand, receptor, or antibody that specifically binds an immune cell marker, wherein the immune cell marker comprises:

[0363] a) a T cell marker selected from the group consisting of: CD3, CD28, 4-1BB, AhR, CD2, CD7, CD4, CD8, CD25, CD44, CD45RA, CD47, CD62L, CD69, CD94, CD95, CD127, CD161, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5), CD193 (CCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, PD-1, TCRa / b, CD5, CD27, CD45RO, CD45RB, CD57, CD103, CD122, P2RX7, LAG-3, TIM-3, IL6ST, gdTCR, Vd1, Vd2, TCR (Va24-Ja18), CD185 (CXCR5), CXCR6, IL-21R, Va7.2, Ja33, CXCR6, IL-18R, and KLRB1 (CD161); or

[0364] b) an NK cell marker selected from the group consisting of: CD56, CD57, NKp30, NKp44, NKp46, CD16, KIR(s), NKG2 proteins, KLRB1 (CD161), KLRD1 (cd94), IL2Rb (CD122), IL-21R, SLAMF6 (CD352), SLAMF7 (CD319), and IL-18R; or

[0365] c) a B cell marker selected from the group consisting of: CD19, CD20, CD21, CD22, CD24, CD38, CD40, CD72, CD32b, CD268, CD269, CD267, CD86, CD80, CD52, CD138, CD27, CD28, CD23, CD84, CD257, CD270, CD37, CD74, and CD269.

[0366] 3. The viral particle according to embodiment 1, characterized in that the immune cell activating molecule comprises:

[0367] a) a T cell activating molecule selected from the group consisting of CD3, CD4, and CD8 antibodies; or

[0368] b) an NK cell activating molecule comprising a ligand or antibody for an NK cell activating receptor selected from the group consisting of: CD16, VLA-4, CD27, CD69, NKG2C, NKG2D, NKG2E, CD223, CD226, CD244, CD319, NKp30, NKp44, NKp46, Ly-49D, Ly-49H, NKR-P1C, NKR-P1A, NKR-P1F, and PILR-beta; or

[0369] c) a B cell activating molecule comprising a ligand or antibody for a B cell activating receptor selected from the group consisting of: CD79, CD19, CD21, CD24, CD81.

[0370] 4. The viral particle of embodiment 1, wherein the specific binding molecule for an immune cell surface adhesion molecule comprises a ligand, receptor or antibody for an immune cell surface adhesion molecule selected from the group consisting of:

[0371] a) a T cell surface adhesion molecule selected from the group consisting of: LFA-1, CD2, VLA-4, CD43, and CD45; or

[0372] b) an NK cell surface adhesion molecule selected from the group consisting of: LFA-1, CD2, pTA1, Mac-1, p150, p95, CD31, CD44, CD162R, and VLA1-6; or

[0373] c) a B cell surface adhesion molecule selected from the group consisting of: ICAM-1 and CD22.

[0374] 5. The viral particle of embodiment 1, wherein the viral envelope further comprises an immune cell costimulatory molecule, which provides a costimulatory signal for immune cell activation.

[0375] 6. The viral particle of embodiment 5, wherein the costimulatory molecule comprises:

[0376] d) a T cell costimulatory molecule selected from the group consisting of at least one of: CD80, CD86, 4-1BBL, B7-H2, CD40, IL2, CD28 antibody, 4-1BB antibody, ICOS antibody, and CD40L antibody; or

[0377] e) an NK cell costimulatory molecule selected from the group consisting of at least one of: CD160, 4-1BBL, 4-1BB antibody, IL15, IL21, and FLT3L; or

[0378] f) a B cell costimulatory molecule selected from the group consisting of at least one of: CD28, CD40L, CD40 antibody.

[0379] 7. The viral particle according to any one of embodiments 1-6, wherein the immune cell is a T cell.

[0380] 8. The viral particle according to embodiment 7, wherein the immune cell targeting molecule or immune cell activating molecule is a CD3 antibody.

[0381] 9. The viral particle according to embodiment 7 or 8, wherein the specific binding molecule for an immune cell surface adhesion molecule is a LFA-1 specific binding molecule.

[0382] 10. The viral particle according to embodiment 9, wherein the LFA-1 specific binding molecule comprises ICAM-1 or a LFA-1 antibody.

[0383] 11. The viral particle according to any one of embodiments 5-10, wherein the immune cell costimulatory molecule is a T cell costimulatory molecule selected from the group consisting of at least one of: CD80, CD86 and CD28 antibody.

[0384] 12. The viral particle according to any one of embodiments 2-11, wherein the immune cell targeting molecule or immune cell activating molecule is a CD3 antibody; the specific binding molecule for an immune cell surface adhesion molecule is ICAM-1; the immune cell costimulatory molecule is CD80; or

[0385] the immune cell targeting molecule or immune cell activating molecule is a CD3 antibody; the specific binding molecule for an immune cell surface adhesion molecule is a LFA-1 antibody; the immune cell costimulatory molecule is CD80.

[0386] the immune cell targeting molecule or immune cell activating molecule is a CD3 antibody; the specific binding molecule for an immune cell surface adhesion molecule is ICAM-1; the immune cell costimulatory molecule is CD86; or

[0387] the immune cell targeting molecule or immune cell activating molecule is a CD3 antibody; the specific binding molecule for an immune cell surface adhesion molecule is a LFA-1 antibody; the immune cell costimulatory molecule is CD86.

[0388] 13. The viral particle according to any one of embodiments 1-6, wherein the immune cell is an NK cell.

[0389] 14. The viral particle according to embodiment 13, wherein the immune cell targeting molecule or immune cell activating molecule is selected from the group consisting of CD16 antibody, NKp30, NKp44, NKp46, NKG2C, NKG2D.

[0390] 15. The viral particle according to embodiment 13 or 14, wherein the specific binding molecule for an immune cell surface adhesion molecule is a LFA-1 specific binding molecule.

[0391] 16. The viral particle according to embodiment 15, wherein the LFA-1 specific binding molecule comprises an ICAM-1 or a LFA-1 antibody.

[0392] 17. The viral particle according to any one of embodiments 13-16, wherein the immune cell costimulatory molecule is an NK cell costimulatory molecule selected from the group consisting of at least one of 4-1BBL, 4-1BB antibody, IL15, IL21 and FLT3L.

[0393] 18. The viral particle according to any one of embodiments 1-17, wherein the viral envelope comprises a membrane-bound cytokine.

[0394] 19. The viral particle according to embodiment 18, wherein the cytokine is selected from the group consisting of at least one of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21.

[0395] 20. The viral particle according to any one of embodiments 1-19, wherein the viral envelope glycoprotein is selected from the group consisting of at least one of vesicular stomatitis virus envelope glycoprotein VSVG and variants thereof, Cocal virus envelope glycoprotein and variants thereof, Maraba virus envelope glycoprotein Maraba and variants thereof, Morreton virus envelope glycoprotein and variants thereof, Alagoa virus envelope glycoprotein and variants thereof, New Jersey virus envelope glycoprotein and variants thereof, Carajas virus envelope glycoprotein and variants thereof, baboon endogenous retrovirus envelope glycoprotein BaEV and variants thereof, feline endogenous retrovirus envelope glycoprotein RD114 and variants thereof, gibbon ape leukemia virus envelope glycoprotein GALV and variants thereof, koala retrovirus envelope glycoprotein KoRV and variants thereof, simian endogenous retrovirus envelope glycoprotein SRV and variants thereof.

[0396] 21. The viral particle of embodiment 20, wherein the viral envelope glycoprotein is selected from the group consisting of vesicular stomatitis virus envelope glycoprotein VSVG and variants thereof, Cocal virus envelope glycoprotein and variants thereof, and Maraba virus envelope glycoprotein Maraba and variants thereof.

[0397] 22. The viral particle of any one of embodiments 1-21, wherein the viral particle is selected from the group consisting of a retrovirus, a lentivirus, or a virus-like particle (VLP).

[0398] 23. The viral particle of any one of embodiments 1-22, wherein the viral particle further comprises an expression cassette comprising a gene of interest encoding a therapeutic protein or polypeptide of interest.

[0399] 24. The viral particle of embodiment 23, wherein the protein or polypeptide of interest comprises a chimeric antigen receptor (CAR) comprising: an extracellular domain comprising a binding domain that specifically binds to a target molecule; an intracellular signaling domain, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM) and an optional costimulatory signaling domain; and a transmembrane domain linking the extracellular domain and the intracellular signaling domain.

[0400] 25. The viral particle of any one of embodiments 1-24, wherein the viral particle is capable of directly transducing PBMCs or immune cells without the need for prior activation; preferably, the immune cells comprise T cells, NK cells, or B cells.

[0401] 26. The viral particle of any one of embodiments 1-24, wherein the viral particle is capable of transducing immune cells in vivo; preferably, the immune cells comprise T cells, NK cells, or B cells.

[0402] 27. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the viral particle of any one of embodiments 1-26.

[0403] 28. Use of the viral particle of any one of embodiments 1-26 in the manufacture of a medicament for the prevention or treatment of a tumor, a viral infection, aging, or an autoimmune disease.

[0404] 29. A method of transducing immune cells in a subject, comprising administering to the subject the viral particle of any one of embodiments 1-26 or the pharmaceutical composition of embodiment 27.

[0405] 30. A method of making immune cells expressing a gene of interest in a subject, comprising administering to the subject the viral particle of embodiment 23 or 24, or the pharmaceutical composition of embodiment 27.

[0406] 31. A method of making chimeric antigen receptor T cells (CAR-T) in a subject, comprising administering to the subject the viral particle of embodiment 24, or the pharmaceutical composition of embodiment 27.

[0407] 32. A method of treating and / or preventing a disease in a subject, comprising administering to the subject the viral particle of embodiment 23, or the pharmaceutical composition of embodiment 27.

[0408] 33. The method of any one of embodiments 29-32, wherein the viral particle is administered by intraperitoneal, subcutaneous, or intranodal injection.

[0409] 34. The method of embodiment 33, wherein the intranodal injection is into a lymph node; preferably, the lymph node is a groin lymph node.

[0410] 35. A kit comprising a container comprising the viral particle of embodiment 23, and / or a pharmaceutically acceptable carrier, and instructions for transducing immune cells in vitro or in vivo.

[0411] 36. Use of a Maraba virus envelope protein in the construction of a pseudotyped viral vector, wherein the pseudotyped viral vector is capable of transducing a target cell in a subject.

[0412] 37. The use of embodiment 36, wherein the target cell is an immune cell.

[0413] 38. The use of embodiment 36 or 37, wherein the pseudotyped viral envelope further comprises at least one heterologous membrane-bound protein; preferably, the pseudotyped viral envelope further comprises at least two heterologous membrane-bound proteins.

[0414] 39. Use of a Maraba virus envelope protein in the construction of a pseudotyped viral vector, wherein the pseudotyped viral envelope further comprises at least one heterologous membrane-bound protein; preferably, the pseudotyped viral envelope further comprises at least two heterologous membrane-bound proteins.

[0415] 40. The use according to any one of embodiments 36-39, wherein the heterologous membrane-bound protein is selected from the group consisting of an immune cell targeting molecule as defined in any one of embodiments 1-17, an immune cell activating molecule, a specific binding molecule for an immune cell surface adhesion molecule, an immune cell costimulatory molecule as defined in any one of embodiments 5-17, and a cytokine as defined in embodiment 18.

[0416] 41. The use according to any one of embodiments 36-40, wherein the Maraba virus envelope glycoprotein has a mutation at a site corresponding to one or any combination of the following sites of the vesicular stomatitis virus Indiana strain envelope glycoprotein VSVG: substitution / deletion of H8, substitution / deletion of N9, substitution / deletion of Q10, substitution / deletion of K47, substitution / deletion of K50, substitution / deletion of A51, substitution / deletion of K66, substitution / deletion of S162, substitution / deletion of S183, substitution / deletion of S179, substitution / deletion of N180, substitution / deletion of 1182, substitution / deletion of M184, substitution / deletion of Y209, substitution / deletion of S230, substitution / deletion of 1347, substitution / deletion of T350, substitution / deletion of T352, substitution / deletion of E353, substitution of R354, substitution / deletion of T368, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353.

[0417] 42. A viral particle, wherein the envelope of the viral particle comprises a Maraba virus envelope glycoprotein or a mutant thereof.

[0418] 43. The viral particle of embodiment 42, wherein the Maraba virus envelope glycoprotein comprises a mutation at one or any combination of the following positions corresponding to positions H8 substitution / deletion, N9 substitution / deletion, Q10 substitution / deletion, K47 substitution / deletion, K50 substitution / deletion, A51 substitution / deletion, K66 substitution / deletion, S162 substitution / deletion, S183 substitution / deletion, S179 substitution / deletion, N180 substitution / deletion, A182 substitution / deletion, M184 substitution / deletion, Y209 substitution / deletion, S230 substitution / deletion, I347 substitution / deletion, T350 substitution / deletion, T352 substitution / deletion, E353 substitution / deletion, R354 substitution, T368 substitution / deletion, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353, as compared to the vesicular stomatitis virus Indiana strain envelope glycoprotein VSVG universe.

[0419] 44. The viral particle of embodiment 42 or 43, wherein the envelope surface further comprises at least one heterologous membrane-bound protein; preferably, the envelope surface further comprises at least two heterologous membrane-bound proteins.

[0420] 45. The viral particle of any one of embodiments 42-44, further comprising one or more T cell activating ligands on the envelope.

[0421] 46. The viral particle of any one of embodiments 42-45, further comprising one or more adhesion molecules on the envelope.

[0422] 47. The viral particle of any one of embodiments 42-44, further comprising a fusion protein consisting of one or more T cell activating ligands or adhesion molecules on the envelope.

[0423] 48. The viral particle of any one of embodiments 42-47, wherein the viral particle is selected from the group consisting of a retrovirus, a lentivirus, or a virosome.

[0424] 49. The viral particle of any one of embodiments 42-48, wherein the viral particle comprises an expression cassette comprising a gene of interest encoding a therapeutic protein or polypeptide of interest.

[0425] 50. The viral particle of embodiment 49, wherein the protein or polypeptide of interest is selected from a chimeric antigen receptor (CAR) or a T cell receptor (TCR); the chimeric antigen receptor comprises: an extracellular domain comprising a binding domain that specifically binds to a target molecule; an intracellular signaling domain, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM) and an optional costimulatory signaling domain; and a transmembrane domain linking the extracellular domain and the intracellular signaling domain.

[0426] 51. The viral particle of any one of embodiments 42-50, wherein the viral particle is capable of transducing an immune cell in vivo; preferably, the immune cell comprises a T cell.

[0427] 52. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the viral particle of any one of embodiments 42-51.

[0428] 53. Use of the viral particle of any one of embodiments 42-51 in the manufacture of a medicament for preventing and / or treating a tumor, a viral infection, aging, or an autoimmune disease.

[0429] 54. A method of transducing an immune cell in a subject, comprising administering to the subject the viral particle of any one of embodiments 42-51 or the pharmaceutical composition of embodiment 52.

[0430] 55. A method of producing an immune cell expressing a gene of interest in a subject, comprising administering to the subject the viral particle of embodiment 49 or the pharmaceutical composition of embodiment 52.

[0431] 56. A method of producing a chimeric antigen receptor T cell (CAR-T) in a subject, comprising administering to the subject the viral particle of embodiment 50 or the pharmaceutical composition of embodiment 52.

[0432] 57. A method of treating and / or preventing a disease in a subject, comprising administering to the subject the viral particle of embodiment 49 or the pharmaceutical composition of embodiment 52.

[0433] 58. The method of any one of embodiments 54-57, wherein the viral particle is administered by intravenous, intratumoral, intraperitoneal, subcutaneous, or intralymph node injection.

[0434] 59. The method of embodiment 58, wherein the intralymph node is an intralymph node of a groin; preferably, the intralymph node is an inguinal lymph node.

[0435] 60. A kit comprising a container comprising viral particles as defined in any one of embodiments 42-51, and instructions for transducing immune cells in vitro or in vivo.

[0436] 61. A novel Maraba virus envelope glycoprotein comprising at least one mutation in the following positions: substitution / deletion of H8, substitution / deletion of Q10, substitution / deletion of K47, substitution / deletion of A182, substitution / deletion of S183, substitution / deletion of H209, substitution of R354.

[0437] 62. The envelope glycoprotein according to embodiment 61, wherein the substitution / deletion of H8 is selected from: H8A, H8V or H8P.

[0438] 63. The envelope glycoprotein according to embodiment 61, wherein the substitution / deletion of Q10 is Q10A.

[0439] 64. The envelope glycoprotein according to embodiment 61, wherein the substitution / deletion of K47 is selected from: K47A, K47E, K47Q or AK47.

[0440] 65. The envelope glycoprotein according to embodiment 61, wherein the substitution / deletion of A182 is selected from: A182E, A182W, A182F or AA182.

[0441] 66. The envelope glycoprotein according to embodiment 61, wherein the substitution / deletion of S183 is selected from: S183P, S183F or AS183.

[0442] 67. The envelope glycoprotein according to embodiment 61, wherein the substitution / deletion of H209 is selected from: H209A, H209E or AH209.

[0443] 68. The envelope glycoprotein according to embodiment 61, wherein the substitution / deletion of R354 is selected from: R354A, R354E, R354W or AR354.

[0444] 69. The envelope glycoprotein according to embodiment 61, wherein the mutation is selected from at least two combinations of: H8A, Q10A, K47E / K47A / K47Q, A182E, S183P, H209A and R354A.

[0445] 70. The envelope glycoprotein according to embodiment 61, wherein the mutation is selected from at least two combinations of: H8A, K47E / K47A / K47Q, A182E, R354A.

[0446] 71. The envelope glycoprotein of embodiment 61, wherein the mutations comprise H8A, K47E, and A182E.

[0447] 72. The envelope glycoprotein of any one of embodiments 61-71, having at least 95% sequence identity to SEQ ID NO: 33.

[0448] 73. The envelope glycoprotein of any one of embodiments 61-72, wherein the mutation sites correspond to amino acid sites in SEQ ID NO: 33.

[0449] 74. A nucleic acid molecule encoding the envelope glycoprotein of any one of embodiments 61-73.

[0450] 75. A vector comprising the nucleic acid molecule of embodiment 74.

[0451] 76. A viral particle comprising the envelope glycoprotein of any one of embodiments 61-73.

[0452] 77. The viral particle of embodiment 76, further comprising a cell targeting molecule.

[0453] 78. The viral particle of embodiment 76 or 77, wherein the viral particle is a pseudotyped lentivirus.

[0454] 79. The viral particle of any one of embodiments 76-78, wherein the viral particle further comprises a nucleic acid molecule encoding a heterologous molecule of interest.

[0455] 80. The viral particle of embodiment 79, wherein the heterologous molecule of interest is selected from the group consisting of an siRNA, an shRNA, a non-coding RNA (e.g., a guide RNA of a CRISPR system), a polypeptide, a protein, a viral genome, or a combination thereof.

[0456] 81. The viral particle of embodiment 79 or 80, wherein the heterologous molecule of interest is a chimeric antigen receptor.

[0457] 82. The viral particle of any one of embodiments 77-81, wherein the cell to which the cell targeting molecule is capable of binding is selected from the group consisting of: a normal tissue cell, an immune cell, a tumor cell, a stem cell, and a combination thereof.

[0458] 83. The viral particle of embodiment 82, wherein the cell is an immune cell selected from the group consisting of: a T, B, NK, DC, macrophage, NKT cell, PBMC, or a combination thereof.

[0459] 84. The viral particle of embodiment 83, wherein the targeting molecule is a T cell targeting molecule selected from the group consisting of: a ligand, a receptor, or an antibody that binds to a T cell surface specific expression molecule.

[0460] 85. The T cell surface specific expression molecule of embodiment 84 is selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD30, CD39, CD40, CD44, CD45, CD45RA, CD45RO, CD62L, CD70, CD73, OX40, 4-1BB, ICOS, IL2RB, IL7R, IL12R, IL18R, IL21R, LFA-1, VLA-4, PSGL-1, CXCR2, CXCR3, CXCR4, CCR5, CCR7, CCR4, CCR8, or a combination thereof.

[0461] The present application also relates to the following embodiments:

[0462] 1. A novel viral particle, wherein the viral envelope surface of the viral particle comprises:

[0463] an immune cell targeting molecule; and / or an immune cell activating molecule.

[0464] 2. The viral particle of embodiment 1, wherein the immune cell targeting molecule comprises a ligand, a receptor, or an antibody that specifically binds to an immune cell marker comprising:

[0465] a) a T cell marker selected from the group consisting of: CD3, CD28, 4-1BB, AhR, CD2, CD7, CD4, CD8, CD25, CD44, CD45RA, CD47, CD62L, CD69, CD94, CD95, CD127, CD161, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5), CD193 (CCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, PD-1, TCRa / b, CD5, CD27, CD45RO, CD45RB, CD57, CD103, CD122, P2RX7, LAG-3, TIM-3, IL6ST, gdTCR, Vd1, Vd2, TCR (Va24-Ja18), CD185 (CXCR5), CXCR6, IL-21R, Va7.2, Ja33, CXCR6, IL-18R, and KLRB1 (CD161); or

[0466] b) an NK cell marker selected from the group consisting of: CD56, CD57, NKp30, NKp44, NKp46, CD16, KIR(s), NKG2 proteins, KLRB1 (CD161), KLRD1 (cd94), IL2Rb (CD122), IL-21R, SLAMF6 (CD352), SLAMF7 (CD319), and IL-18R; or

[0467] c) a B cell marker selected from the group consisting of: CD19, CD20, CD21, CD22, CD24, CD38, CD40, CD72, CD32b, CD268, CD269, CD267, CD86, CD80, CD52, CD138, CD27, CD28, CD23, CD84, CD257, CD270, CD37, CD74, and CD269.

[0468] 3. The viral particle according to embodiment 1, wherein the immune cell activating molecule comprises:

[0469] a) a T cell activating molecule selected from the group consisting of CD3, CD4, and CD8 antibodies; or

[0470] b) an NK cell activating molecule comprising a ligand or antibody for an NK cell activating receptor selected from the group consisting of: CD16, VLA-4, CD27, CD69, NKG2C, NKG2D, NKG2E, CD223, CD226, CD244, CD319, NKp30, NKp44, NKp46, Ly-49D, Ly-49H, NKR-P1C, NKR-P1A, NKR-P1F, and PILR-beta; or

[0471] c) a B cell activating molecule comprising a ligand or antibody for a B cell activating receptor selected from the group consisting of: CD79, CD19, CD21, CD24, CD81.

[0472] 4. The viral particle according to any one of embodiments 1-3, wherein the viral envelope surface further comprises a specific binding molecule for an immune cell surface adhesion molecule.

[0473] 5. The viral particle according to embodiment 4, wherein the specific binding molecule for an immune cell surface adhesion molecule comprises a ligand, receptor, or antibody for an immune cell surface adhesion molecule; preferably, the immune cell surface adhesion molecule comprises:

[0474] a) a T cell surface adhesion molecule selected from the group consisting of: LFA-1, CD2, VLA-4, CD43, and CD45; or

[0475] b) an NK cell surface adhesion molecule selected from the group consisting of: LFA-1, CD2, pTA1, Mac-1, p150, p95, CD31, CD44, CD162R, and VLA1-6; or

[0476] c) a B cell surface adhesion molecule selected from the group consisting of: ICAM-1 and CD22.

[0477] 6. The viral particle according to any one of embodiments 1-5, wherein the viral envelope surface further comprises an immune cell costimulatory molecule, the costimulatory molecule providing a costimulatory signal for immune cell activation.

[0478] 7. The viral particle according to embodiment 6, wherein the costimulatory molecule comprises:

[0479] a) a T cell costimulatory molecule selected from the group consisting of: at least one of CD80, CD86, 4-1BBL, B7-H2, CD40, IL2, CD28 antibody, 4-1BB antibody, ICOS antibody, and CD40L antibody; or

[0480] b) an NK cell costimulatory molecule selected from the group consisting of: at least one of CD160, 4-1BBL, 4-1BB antibody,

[0481] IL15, IL21, and FLT3L; or

[0482] c) a B cell costimulatory molecule selected from the group consisting of: at least one of CD28, CD40L, CD40 antibody.

[0483] 8. The viral particle according to any one of embodiments 1-7, wherein the immune cell is a T cell.

[0484] 9. The viral particle according to embodiment 8, wherein the immune cell marker is a T cell marker selected from the group consisting of: at least one of TCRa / b, TCRy / d, CD3, and CD7.

[0485] 10. The viral particle according to embodiment 9, wherein the viral envelope surface comprises an antibody to TCRa / b, TCRy / d, or CD3; and a CD7 antibody.

[0486] 11. The viral particle according to embodiment 9 or 10, wherein the viral envelope surface comprises a TCRa / b antibody, a TCRy / d antibody or a CD3 antibody; and a CD7 antibody; and a specific binding molecule for a T cell surface adhesion molecule selected from the group consisting of at least one of LFA-1, CD2, VLA-4, CD43 and CD45.

[0487] 12. The viral particle according to embodiment 11, wherein the specific binding molecule for a T cell surface adhesion molecule is selected from the group consisting of at least one of ICAM-1, LFA-1 antibody, CD58, CD2 antibody, VCAM-1, Fibronectin, VLA-4 antibody, E-selectin, SIGLEC1, ERM protein, CD43 antibody, Galectin-3, PP14 / PAEP, CD45 antibody.

[0488] 13. The viral particle according to embodiment 12, wherein the viral envelope surface comprises a TCRa / b antibody, a CD7 antibody and ICAM-1; or a TCRa / b antibody, a CD7 antibody and a LFA-1 antibody.

[0489] 14. The viral particle according to embodiment 12, wherein the viral envelope surface comprises a CD3 antibody, a CD7 antibody and ICAM-1; or a CD3 antibody, a CD7 antibody and a LFA-1 antibody.

[0490] 15. The viral particle according to embodiment 12, wherein the viral envelope surface comprises a TCRa / b antibody, a CD7 antibody and CD58; or a TCRa / b antibody, a CD7 antibody and a CD2 antibody.

[0491] 16. The viral particle according to embodiment 12, wherein the viral envelope surface comprises a CD3 antibody, a CD7 antibody and CD58; or a CD3 antibody, a CD7 antibody and a CD2 antibody.

[0492] 17. The viral particle according to embodiment 8, wherein the immune cell is a T cell and the T cell targeting or activating molecule is a CD3 antibody.

[0493] 18. The viral particle according to embodiment 17, wherein the specific binding molecule for a T cell surface adhesion molecule is a LFA-1 specific binding molecule.

[0494] 19. The viral particle according to embodiment 18, wherein the LFA-1 specific binding molecule comprises ICAM-1 or a LFA-1 antibody.

[0495] 20. The viral particle of any one of embodiments 6-19, wherein the immune cell costimulatory molecule is a T cell costimulatory molecule selected from at least one of: CD80, CD86, and CD28 antibodies.

[0496] 21. The viral particle of any one of embodiments 6-20, wherein the viral envelope surface comprises: CD3 antibodies, ICAM-1, and CD80; or CD3 antibodies, LFA-1 antibodies, and CD80; or CD3 antibodies, ICAM-1, and CD86; or CD3 antibodies, LFA-1 antibodies, and CD86; or TCRa / b antibodies, CD7 antibodies, ICAM-1, and CD80; or TCRa / b antibodies, CD7 antibodies, LFA-1 antibodies, and CD80; or CD3 antibodies, CD7 antibodies, ICAM-1, and CD80; or CD3 antibodies, CD7 antibodies, LFA-1 antibodies, and CD80; or TCRa / b antibodies, CD7 antibodies, CD58, and CD80; or TCRa / b antibodies, CD7 antibodies, CD2 antibodies, and CD80; or CD3 antibodies, CD7 antibodies, CD58, and CD80; or CD3 antibodies, CD7 antibodies, CD2 antibodies, and CD80; or a CD80 alteration in the above combinations to CD86 or CD28 antibodies.

[0497] 22. The viral particle of any one of embodiments 1-7, wherein the immune cell is an NK cell.

[0498] 23. The viral particle of embodiment 22, wherein the immune cell targeting molecule or immune cell activating molecule is selected from CD16 antibodies, NKp30, NKp44, NKp46, NKG2C, NKG2D.

[0499] 24. The viral particle of embodiments 22 or 23, wherein the specific binding molecule for an immune cell surface adhesion molecule is a LFA-1 specific binding molecule.

[0500] 25. The viral particle of embodiment 24, wherein the LFA-1 specific binding molecule comprises ICAM-1 or LFA-1 antibodies.

[0501] 26. The viral particle of any one of embodiments 22-25, wherein the immune cell costimulatory molecule is an NK cell costimulatory molecule selected from at least one of: 4-1BBL, 4-1BB antibodies, IL15, IL21, and FLT3L.

[0502] 27. The viral particle according to any one of embodiments 1 to 26, characterized in that the viral envelope comprises a membrane-bound cytokine.

[0503] 28. The viral particle according to embodiment 27, characterized in that the cytokine is selected from at least one of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21.

[0504] 29. The viral particle according to any one of embodiments 1 to 28, characterized in that the viral envelope glycoprotein is selected from at least one of the vesicular stomatitis virus envelope glycoprotein VSVG and variants thereof, Cocal virus envelope glycoprotein and variants thereof, Maraba virus envelope glycoprotein Maraba and variants thereof, Morreton virus envelope glycoprotein and variants thereof, Alagoa virus envelope glycoprotein and variants thereof, New Jersey virus envelope glycoprotein and variants thereof, Carajas virus envelope glycoprotein and variants thereof, baboon endogenous retrovirus envelope glycoprotein BaEV and variants thereof, feline endogenous retrovirus envelope glycoprotein RD114 and variants thereof, gibbon ape leukemia virus envelope glycoprotein GALV and variants thereof, koala retrovirus envelope glycoprotein KoRV and variants thereof, simian endogenous retrovirus envelope glycoprotein SRV and variants thereof.

[0505] 30. The viral particle according to embodiment 29, characterized in that the viral envelope glycoprotein is selected from the vesicular stomatitis virus envelope glycoprotein VSVG and variants thereof, Cocal virus envelope glycoprotein and variants thereof, and Maraba virus envelope glycoprotein Maraba and variants thereof; preferably, the Maraba virus envelope glycoprotein comprises at least one mutation of the following sites: substitution / deletion of H8, substitution / deletion of Q10, substitution / deletion of K47, substitution / deletion of A182, substitution / deletion of S183, substitution / deletion of H209, substitution / deletion of R354.

[0506] 31. The viral particle according to any one of embodiments 1 to 30, characterized in that the viral particle is selected from a retrovirus, a lentivirus, or a virosome (VLP).

[0507] 32. The viral particle according to any one of embodiments 1 to 31, characterized in that the viral particle further comprises an expression cassette comprising a gene of interest encoding a therapeutic protein or polypeptide of interest.

[0508] 33. The viral particle of embodiment 32, wherein the protein or polypeptide of interest comprises a chimeric antigen receptor (CAR) comprising: an extracellular domain comprising a binding domain that specifically binds to a target molecule; an intracellular signaling domain, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM) and an optional costimulatory signaling domain; and a transmembrane domain linking the extracellular domain and the intracellular signaling domain.

[0509] 34. The viral particle of any one of embodiments 1-33, wherein the viral particle is capable of directly transducing PBMCs or immune cells without prior activation; preferably, the immune cells comprise T cells, NK cells, or B cells.

[0510] 35. The viral particle of any one of embodiments 1-33, wherein the viral particle is capable of transducing immune cells in vivo; preferably, the immune cells comprise T cells, NK cells, or B cells.

[0511] 36. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the viral particle of any one of embodiments 1-35.

[0512] 37. Use of the viral particle of any one of embodiments 1-35 in the manufacture of a medicament for preventing or treating a tumor, a viral infection, aging, or an autoimmune disease.

[0513] 38. A method of transducing immune cells in a subject, comprising administering to the subject the viral particle of any one of embodiments 1-35 or the pharmaceutical composition of embodiment 36.

[0514] 39. A method of producing immune cells expressing a gene of interest in a subject, comprising administering to the subject the viral particle of embodiment 32 or 33 or the pharmaceutical composition of embodiment 36.

[0515] 40. A method of producing chimeric antigen receptor T cells (CAR-T) in a subject, comprising administering to the subject the viral particle of embodiment 33 or the pharmaceutical composition of embodiment 36.

[0516] 41. A method of treating and / or preventing a disease in a subject, comprising administering to the subject the viral particle of embodiment 32 or the pharmaceutical composition of embodiment 36.

[0517] 42. The method of any one of embodiments 38-41, wherein the viral particle is administered by intraperitoneal, subcutaneous, or intranodal injection.

[0518] 43. The method according to embodiment 42, wherein the node is a lymph node; preferably, the lymph node is a inguinal lymph node.

[0519] 44. A kit comprising a container comprising the viral particle according to embodiment 32, and / or a pharmaceutically acceptable carrier, and instructions for transducing immune cells in vitro or in vivo.

[0520] 45. Use of a Maraba virus envelope protein for constructing a pseudotyped viral vector, wherein the pseudotyped viral vector is capable of transducing target cells in a subject.

[0521] 46. The use according to embodiment 45, wherein the target cells are immune cells.

[0522] 47. The use according to embodiment 45 or 46, wherein the pseudotyped viral envelope further comprises at least one heterologous membrane-bound protein; preferably, the pseudotyped viral envelope further comprises at least two heterologous membrane-bound proteins.

[0523] 48. Use of a Maraba virus envelope protein for constructing a pseudotyped viral vector, wherein the pseudotyped viral envelope further comprises at least one heterologous membrane-bound protein; preferably, the pseudotyped viral envelope further comprises at least two heterologous membrane-bound proteins.

[0524] 49. The use according to any one of embodiments 45 to 48, wherein the heterologous membrane-bound protein is selected from the group consisting of an immune cell targeting molecule as defined in any one of embodiments 1 to 17, an immune cell activating molecule, a specific binding molecule for an immune cell surface adhesion molecule, an immune cell costimulatory molecule as defined in any one of embodiments 5 to 17, and a cytokine as defined in embodiment 18.

[0525] 50. The use according to any one of embodiments 45 to 49, wherein the Maraba virus envelope glycoprotein comprises a mutation in at least one of the following positions: substitution / deletion of H8, substitution / deletion of Q10, substitution / deletion of K47, substitution / deletion of A182, substitution / deletion of S183, substitution / deletion of H209, substitution / deletion of R354.

[0526] 51. A novel pseudotyped viral particle, wherein the envelope of the viral particle comprises a Maraba virus envelope glycoprotein or a mutant thereof.

[0527] 52. The novel pseudotyped viral particle according to embodiment 51, comprising at least one mutation in the following positions: substitution / deletion of H8, substitution / deletion of Q10, substitution / deletion of K47, substitution / deletion of A182, substitution / deletion of S183, substitution / deletion of H209, substitution / deletion of R354.

[0528] 53. The novel pseudotyped viral particle according to embodiment 51 or 52, wherein the envelope surface further comprises at least one heterologous membrane-bound protein; preferably, the envelope surface further comprises at least two heterologous membrane-bound proteins.

[0529] 54. The novel pseudotyped viral particle according to any one of embodiments 51-53, further comprising one or more immune cell activating molecules on the envelope.

[0530] 55. The novel pseudotyped viral particle according to any one of embodiments 51-54, further comprising one or more specific binding molecules for immune cell surface adhesion molecules on the envelope.

[0531] 56. The novel pseudotyped viral particle according to embodiment 54 or 55, further comprising a fusion protein consisting of the one or more immune cell activating molecules and the one or more specific binding molecules for immune cell surface adhesion molecules on the envelope.

[0532] 57. A novel Marabavirus envelope glycoprotein comprising at least one mutation in the following positions: substitution / deletion of H8, substitution / deletion of Q10, substitution / deletion of K47, substitution / deletion of A182, substitution / deletion of S183, substitution / deletion of H209, substitution / deletion of R354.

[0533] 58. The envelope glycoprotein according to embodiment 57, wherein the substitution / deletion of H8 is selected from: H8A, H8V or H8P.

[0534] 59. The envelope glycoprotein according to embodiment 57, wherein the substitution / deletion of Q10 is Q10A.

[0535] 60. The envelope glycoprotein according to embodiment 57, wherein the substitution / deletion of K47 is selected from: K47A, K47E, K47Q or AK47.

[0536] 61. The envelope glycoprotein according to embodiment 57, wherein the substitution / deletion of A182 is selected from: A182E, A182W, A182F or AA182.

[0537] 62. The envelope glycoprotein of embodiment 57, wherein the substitution / deletion of S183 is selected from: S183P, S183F, or AS183.

[0538] 63. The envelope glycoprotein of embodiment 57, wherein the substitution / deletion of H209 is selected from: H209A, H209E, or AH209.

[0539] 64. The envelope glycoprotein of embodiment 57, wherein the substitution / deletion of R354 is selected from: R354A, R354E, R354W, or AR354.

[0540] 65. The envelope glycoprotein of embodiment 57, wherein the mutations are selected from at least two combinations of: H8A, Q10A, K47E / K47A / K47Q, A182E, S183P, H209A, and R354A.

[0541] 66. The envelope glycoprotein of embodiment 57, wherein the mutations are selected from at least two combinations of: H8A, K47E / K47A / K47Q, A182E, R354A.

[0542] 67. The envelope glycoprotein of embodiment 57, wherein the mutations comprise H8A, K47E, and A182E.

[0543] 68. The envelope glycoprotein of any one of embodiments 57-67, having at least 95% sequence identity to SEQ ID NO: 33.

[0544] 69. The envelope glycoprotein of any one of embodiments 57-68, wherein the mutation sites correspond to amino acid sites in SEQ ID NO: 33.

[0545] 70. A nucleic acid molecule encoding the envelope glycoprotein of any one of embodiments 57-69.

[0546] 71. A vector comprising the nucleic acid molecule of embodiment 70.

[0547] 72. A viral particle comprising the envelope glycoprotein of any one of embodiments 57-69.

[0548] 73. The viral particle of embodiment 72, further comprising a cell targeting molecule.

[0549] 74. The viral particle of embodiment 72 or 73, wherein the viral particle is a pseudotyped lentivirus.

[0550] 75. The viral particle of any one of embodiments 72-74, wherein the viral particle further comprises a nucleic acid molecule encoding a heterologous molecule of interest.

[0551] 76. The viral particle of embodiment 75, wherein the heterologous molecule of interest is selected from the group consisting of siRNA, shRNA, non-coding RNA (e.g., guide RNA of CRISPR system), polypeptide, protein, viral genome, or a combination thereof.

[0552] 77. The viral particle of embodiment 75 or 76, wherein the heterologous molecule of interest is a chimeric antigen receptor.

[0553] 78. The viral particle of any one of embodiments 73-77, wherein the cell targeted by the cell targeting molecule is selected from the group consisting of: normal tissue cell, immune cell, tumor cell, stem cell, and a combination thereof.

[0554] 79. The viral particle of embodiment 78, wherein the cell is an immune cell selected from the group consisting of: T, B, NK, DC, macrophage, NKT cell, PBMC, or a combination thereof.

[0555] 80. The viral particle of embodiment 79, wherein the targeting molecule is a T cell targeting molecule selected from the group consisting of: a ligand, a receptor, or an antibody that binds to a T cell surface specifically expressed molecule.

[0556] 81. The T cell surface specifically expressed molecule of embodiment 80 is selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD30, CD39, CD40, CD44, CD45, CD45RA, CD45RO, CD62L, CD70, CD73, OX40, 4-1BB, ICOS, IL2RB, IL7R, IL12R, IL18R, IL21R, LFA-1, VLA-4, PSGL-1, CXCR2, CXCR3, CXCR4, CCR5, CCR7, CCR4, CCR8, or a combination thereof.

[0557] The embodiments of the present application will be described in detail in connection with the following examples. It should be understood that these examples are for illustrative purposes only and are not meant to limit the scope of the present application. The experimental methods in the following examples, where no specific conditions are mentioned, are preferably performed according to the instructions given in the present application, but can also be performed according to the protocols or general conditions known in the art, or according to the conditions suggested by the manufacturer of the reagents used, if any.

[0558] In the following detailed examples, the amount of the raw material components are described in terms of measurement parameters, which can vary within a range of measurement accuracy unless otherwise specified. In terms of temperature and time parameters, acceptable variations are permitted due to instrument testing or operator error. Examples

[0559] Example 1. Construction of Target Lentiviral Envelope Plasmid

[0560] To directly activate T cells in transduced PBMC cells, membrane-bound OKT3 (mb-OKT3, which is a CD3 antibody, herein denoted as aCD3, and unless otherwise specified, aCD3 used in the examples is membrane-bound OKT3 with hinge and transmembrane region derived from PDGFR, i.e. mb-OKT3 structure OKT3 scFv-PDGFR stalk-PDGFR TM), CD80 and other ligands are expressed on the surface of the viral envelope. Thus, first, each amino acid sequence is obtained from Uniport, the gene fragment is obtained by gene synthesis, after PCR amplification, it is inserted into the backbone vector pMD2.G (Addgene, #12259) which is single-enzyme cut by EcoRI through Gibson recombination, and the ligation product after homologous recombination is transformed into E. coli competent cells by heat shock method, and the next day, single colonies are picked for Sanger sequencing. The correct sequencing is the successful construction of the envelope plasmid, which is used for subsequent viral packaging. Figure 1 is a schematic diagram of the viral envelope structure, and the relevant protein sequences shown therein are as follows: unless otherwise specified, the CDR of the antibody in the present application is defined using the Kabat numbering scheme and definition scheme.

[0561] Full-length amino acid sequence of VSVG wild-type envelope protein (VSVG Env) SEQ ID NO: 1

[0562] VSVG mature sequence SEQ ID NO: 119 (without signal peptide)

[0563] Protein sequence of aCD3 (OKT3 scFv-PDGFR stalk-PDGFR TM) SEQ ID NO: 2

[0564] Membrane-bound protein signal peptide SEQ ID NO: 3

[0565] OKT3 scFv SEQ ID NO: 4

[0566] OKT3 HCDR1 SEQ ID NO: 5

[0567] OKT3 HCDR2 SEQ ID NO:6

[0568] OKT3 HCDR3 SEQ ID NO:7

[0569] OKT3 LCDR1 SEQ ID NO:8

[0570] OKT3 LCDR2 SEQ ID NO:9

[0571] OKT3 LCDR3 SEQ ID NO:10

[0572] OKT3 VH SEQ ID NO:11

[0573] OKT3 VL SEQ ID NO:12

[0574] PDGFR stalk-PDGFR TM SEQ ID NO:13

[0575] CD80 protein sequence (1-34 is signal peptide sequence) SEQ ID NO: 14

[0576] 41BBL protein sequence SEQ ID NO: 15

[0577] CD58 protein sequence (1-28 is signal peptide sequence) SEQ ID NO: 16

[0578] mb-Natalizumab protein sequence SEQ ID NO: 17

[0579] ICAM-1 protein sequence (1-27 is signal peptide sequence) SEQ ID NO: 18

[0580] VCAM-1 protein sequence (1-24 is signal peptide sequence) SEQ ID NO: 19

[0581] P2A protein sequence SEQ ID NO:20

[0582] Example 2. Lentivirus packaging

[0583] The constructed envelope plasmid was co-transfected with helper packaging plasmids pRSV-Rev (Addgene, #12253), pMDLg-pRRE (Addgene, #12251) and a shuttle plasmid with CAR2 (CLDN18.2-28z) (sequence synthesized by Genscript) into HEK293FT suspension cells via pEI. After 48 hours, the culture supernatant was collected, and lentivirus was obtained by ultracentrifugation (10000g, 2h, 4°C). The obtained lentivirus was resuspended in PBS for titer determination and stored at -80°C for standby. The full-length CAR plasmid was synthesized by Genscript, and the extracellular domain of CAR2 structure was VHH targeting CLDN18.2, the hinge region and transmembrane region were CD28 hinge and CD28 TM, and the intracellular costimulatory region was CD28 costimulatory domain and CD3 zeta, i.e. CLDN18.2VHH-CD28 hinge (hinge region)-CD28 TM (transmembrane region)-CD28 ICD (costimulatory signal region)-CD3 zeta. CAR2 amino acid sequence SEQ ID NO:21

[0584] Example 3. PBMC cells infected with different envelope lentiviruses

[0585] After the PBMC cells were resuscitated, they were plated in a 24-well plate at 1E6 cells / well. The harvested virus was directly added to the PBMC cells for infection at a MOI of 15. After 2 days of incubation, the residual virus was washed away with PBS and fresh culture medium was added for continued culture. The proportion of CD3 cells and the proportion of CAR2 (CLDN18.2-28z) positive cells in each group were detected on Day 3 and Day 7, respectively.

[0586] As shown in FIGS. 2 and 3, the flow detection results showed that the lentivirus packaged with the P24018 (ICAM-1 group) envelope structure was superior to other virus envelopes in terms of transduction efficiency, and the CAR2 positive cells of P24018 (ICAM-1 group) proliferated the fastest. This result showed that the ICAM-1 protein was superior to other adhesion proteins, such as CD58, in promoting the adhesion of the virus envelope to T cells. Meanwhile, the proliferation multiple of T cells after P24018 infection was also superior to other virus envelope structures (FIG. 4).

[0587] Example 4. Construction of a target lentivirus envelope plasmid with a membrane-bound mb-Efalizumab

[0588] The above results show that ICAM-1 adhesion protein is superior to other adhesion proteins in the transduction efficiency and cell expansion capacity of T cells in unactivated PBMC, ICAM-1 adheres to LFA-1 on the surface of T cells, and if an antibody fragment such as membrane-bound Efalizumab (mb-Efalizumab, structure: Efalizumab scFv-PDGFR stalk-PDGFR TM) that also binds LFA-1 is displayed on the surface of the virus envelope, whether it has the same effect on transduction efficiency. To this end, the Efalizumab amino acid sequence was first obtained through NCBI, the gene fragment was obtained by gene synthesis, and after PCR amplification, it was also inserted into the EcoRI single enzyme-digested backbone vector pMD2.G (Addgene, #12259) through Gibson recombination, and the ligation product after homologous recombination was transformed into E. coli competent cells by heat shock method. The next day, single colonies were picked for Sanger sequencing. The correct sequencing is the successful construction of the envelope plasmid, which is used for subsequent virus packaging. Figure 5 is a schematic diagram of the virus envelope structure for this comparison.

[0589] Efalizumab scFv-PDGFR stalk-PDGFR TM protein sequence SEQ ID NO:22

[0590] Membrane-bound protein signal peptide SEQ ID NO:23

[0591] Efalizumab scFv SEQ ID NO:24

[0592] Efalizumab HCDR1 SEQ ID NO:25

[0593] Efalizumab HCDR2 SEQ ID NO:26

[0594] Efalizumab HCDR3 SEQ ID NO:27

[0595] Efalizumab LCDR1 SEQ ID NO:28

[0596] Efalizumab LCDR2 SEQ ID NO:29

[0597] Efalizumab LCDR3 SEQ ID NO:30

[0598] Efalizumab VH SEQ ID NO:31

[0599] Efalizumab VL SEQ ID NO:32

[0600] Example 5. Lentivirus packaging with mb-Efalizumab envelope

[0601] The constructed envelope plasmid was co-transfected with helper packaging plasmids pRSV-Rev (Addgene, #12253), pMDLg-pRRE (Addgene, #12251) and a shuttle plasmid with CAR2 (CLDN18.2-28z) into HEK293FT suspension cells by pEI, and the culture supernatant was collected 48 hours later. The lentivirus was obtained by ultracentrifugation (10000g, 2h, 4°C), and the obtained lentivirus was resuspended in PBS for titer determination and stored at -80°C for standby. The full-length CAR plasmid was synthesized by Genscript, and the extracellular domain of the CAR structure was VHH targeting CLDN18.2, the hinge region and transmembrane region were CD28 hinge and CD28 TM, and the intracellular costimulatory region was CD28 costimulatory domain and CD3 zeta, i.e. CLDN18.2VHH-CD28hinge (hinge region)-CD28TM (transmembrane region)-CD28ICD (costimulatory signal region)-CD3 zeta.

[0602] Example 6. Infection of PBMC cells by different envelope lentiviruses

[0603] After the PBMC cells were resuscitated, they were plated in a 24-well plate at 1E6 cells / well. The harvested virus was directly added to the PBMC cells for infection at a MOI of 15, and after 2 days of incubation, the residual virus was washed away with PBS and fresh culture medium was added for continued culture. The proportion of CD3 cells and the positive proportion of CAR2 (CLDN18.2-28z) in each group were detected on Day 3 and Day 7, respectively.

[0604] As shown in FIGS. 6 and 7, the results of flow cytometry detection showed that P24005 (with mb-Efalizumab) envelope structure packaged lentivirus was superior to other viral envelopes in terms of transduction efficiency, including CD58; at the same time, the CAR positive cell expansion rate of P24005 (with mb-Efalizumab) was the fastest (FIG. 8). This result showed that mb-Efalizumab protein was superior to other adhesion proteins, such as CD58, in promoting the adhesion of viral envelope to T cells; at the same time, the expansion fold of CAR2 (CLDN18.2-28z) positive cells after P24005 infection was also superior to other viral envelope structures (FIG. 8).

[0605] Example 7. Construction of Maraba-G envelope plasmid

[0606] VSVG lentivirus envelope is the most commonly used lentivirus envelope, but the viral titer is reduced after adding a targeting ligand, so it is explored whether other viral envelopes, such as Maraba virus (Maraba virus) envelope, are superior to VSVG viral envelope in terms of virus titer and transduction efficiency.

[0607] First, the amino acid sequence of the envelope protein of Maraba virus (Maraba-G) was obtained from Uniport (SEQ ID NO: 34), the target fragment was obtained by gene synthesis, and after PCR amplification, it was inserted into the EcoRI single enzyme-digested backbone vector pMD2.G (Addgene, #12259) by Gibson recombination, and the ligation product after homologous recombination was transformed into E. coli competent cells by heat shock method, and single colonies were picked up for Sanger sequencing the next day. The correct sequencing is the successfully constructed envelope plasmid, which is used for subsequent virus packaging. FIG. 9 is a schematic diagram of the viral envelope structure compared this time.

[0608] Wild type Maraba-G envelope glycoprotein mature sequence SEQ ID NO: 33 (without signal peptide)

[0609] Wild type Maraba-G envelope glycoprotein full-length sequence SEQ ID NO: 34

[0610] Wild type Maraba-G envelope glycoprotein signal peptide sequence SEQ ID NO: 35

[0611] Example 8. Lentivirus packaging of different envelopes

[0612] The constructed envelope plasmid and helper packaging plasmids pRSV-Rev (Addgene, #12253), pMDLg-pRRE (Addgene, #12251) and the shuttle plasmid with GFP or CAR were co-transfected into HEK293FT suspension cells by pEI. After 48 hours, the culture supernatant was collected, and the lentivirus was obtained by ultracentrifugation (10000g, 2h, 4°C). The obtained lentivirus was resuspended in PBS and stored at -80°C for later use. The structure of the shuttle plasmid with CAR is shown in Figure 10. CAR1 (P21002) is EpCAM scFv-28z CAR, i.e., the extracellular region of the CAR structure is an scFv targeting EpCAM, followed by the hinge region and transmembrane region of CD28, and the costimulatory region is the costimulatory region of CD28 and CD3 zeta, i.e., EpCAM scFv-CD28 hinge (hinge region)-CD28TM (transmembrane region)-CD28ICD (costimulatory signal region)-CD3 zeta; CAR2 (P21047) is CLDN18.2 VHH-28z CAR, the extracellular region of the CAR structure is a VHH targeting CLDN18.2, the hinge region and transmembrane region are CD28 hinge and CD28TM, and the intracellular costimulatory region is the CD28 costimulatory domain and CD3 zeta, i.e., CLDN18.2VHH-CD28hinge (hinge region)-CD28TM (transmembrane region)-CD28ICD (costimulatory signal region)-CD3 zeta; The GFP plasmid is used as a control, and the GFP replaces the CAR to obtain the control plasmid used.

[0613] CAR1 amino acid sequence SEQ ID NO: 36

[0614] Example 9. Lentivirus titer detection

[0615] First, the virus concentrate was diluted 100 times with PBS for standby use. Jurkat cells were plated in a 24-well plate at 2E5 / well, and the helper infection reagent polybrean was added at a ratio of 1 / 2000. Then, the virus diluent was added to the corresponding wells in a gradient of 2μl, 10μl, and incubated in a 37°C incubator for 48h.

[0616] After 48h, the 24-well plate was removed, and FACS was used to detect gene expression. The virus titer was calculated according to the positive proportion. The calculation formula is: cell number*positive proportion (0-20%) / virus volume (ml)*dilution multiple. As shown in Figure 11, the Maraba-G lentivirus titer is significantly higher than that of the VSVG envelope packaged lentivirus. In addition, for packaging non-CAR structures, such as GFP, the Maraba-G lentivirus titer is still significantly higher than that of the VSVG envelope packaged lentivirus.

[0617] Example 10. Infection of T cells with different envelope lentiviruses

[0618] The day before viral infection, 6-well plates were prepared by adding 2 ml PBS to each well, and 3 ul of CD3 (1 ug / ul), 3 ul of CD28 (1 ug / ul), and 48 ul of RetroNectin (1 ug / ul) were dissolved in one well and stored in a 4°C refrigerator.

[0619] The next day, after the PBMC cells were resuscitated, the T cells were sorted by magnetic beads, and 3E6 cells / well were added to the RetroNectin-coated 6-well plates. After 24 hours of activation, the packaged viruses were added to the corresponding wells at MOI = 1 and MOI = 3, and after 2 days of incubation, the residual viruses were washed away with PBS and fresh culture medium was added for continued culture. The CAR-positive proportion of each group was detected on Day 3 and Day 7, respectively.

[0620] As shown in FIGS. 12 to 15, the flow detection results and data statistics show that the Maraba-G lentivirus (lentivirus packaged with P24069 envelope plasmid) is superior to the VSVG envelope packaged lentivirus in terms of transduction efficiency. In addition, for CARs of different structures and lengths, the Maraba-G lentivirus is significantly superior to the traditional VSVG envelope packaged lentivirus in terms of titer and infection efficiency.

[0621] Example 11. Killing detection of CAR-T cells prepared with different envelopes on NUGC4-luc

[0622] CAR-T cells were taken to detect the CAR+ positive rate (detected one day in advance or on the same day). Well-grown target cells (NUGC4-luc) were taken, digested, and terminated, and the cells were collected. 1.2-2 times the amount of target cells required for the experiment were taken, centrifuged at 1500 rpm / min for 5 min, resuspended in physiological saline, washed once, resuspended in killing medium (X VIVO-15 + 5% FBS), counted, and the cell number was adjusted to 5x10E5 / mL, 100ul / well, i.e. 5x10E4 / well, as needed, and plated into a U-shaped plate (3 wells of target cells were also plated for spontaneous release); CAR-T cells were taken, counted, and about 2 times the amount of effector cells required for the experiment were taken, centrifuged at 1500 rpm / min for 5 min, washed twice with physiological saline, resuspended in physiological saline containing 2 uM CFSE, with a density of 1E7-1E8 / ml, and incubated at 37°C for 20 min; 10 times the volume of killing medium was added, washed twice, resuspended in killing medium, and the cell density was adjusted to 2.25x10E6 / mL.

[0623] The treated effector cells and target cells were added to the corresponding experimental groups at 3:1 and 1:1, 100ul / well, and the target cells in the spontaneous release group were supplemented with 100ul / well to 200ul / well, gently mixed, centrifuged at 200g for 2min, and placed in the incubator for 4h before removal.

[0624] After the culture plate was removed, the cells were dispersed by blowing. Centrifugation at 500g for 5min, remove supernatant, add 200ul physiological saline to each well, wash twice. 100ul binding buffer+5ul Annexin V and 5ul PI per well; gently mix, incubate at room temperature for 15min, then resuspend in 200ul / well binding buffer before loading; calculate the percentage of Annexin V and PI double negative cell population of target cell population in experimental wells and corresponding target cell spontaneous release wells, and calculate the killing efficiency:

[0625] As shown in Figure 16, the Maraba-G and VSVG envelope packaged lentivirus of two structural CARs, respectively, and the CAR-T cells prepared by the four lentiviruses killed NUGC4-luc cells, and the CAR-T cells prepared by Maraba-G lentivirus had stronger killing ability of target cells than the CAR-T cells prepared by VSVG lentivirus. The above results show that Maraba-G is superior to VSVG envelope in preparing lentivirus titer, improving the positive rate of CAR-T cells and killing effect, etc.

[0626] Example 12. Different envelope lentivirus packaging carrying activation molecules and adhesion molecules

[0627] Different envelope plasmids carrying activating molecules and adhesion molecules were co-transfected with helper packaging plasmids pRSV-Rev (Addgene, #12253), pMDLg-pRRE (Addgene, #12251) and a shuttle plasmid with CAR2 (P21047) into HEK293FT suspension cells by pEI. After 48 hours, the culture supernatant was collected, and the lentivirus was obtained by ultracentrifugation (10000g, 2h, 4°C). The obtained lentivirus was resuspended in PBS and stored at -80°C for later use. The extracellular domain of CAR2 structure is VHH targeting CLDN18.2, the hinge region and transmembrane region are CD28 hinge and CD28 TM, and the intracellular costimulatory region is CD28 costimulatory domain and CD3 zeta, i.e. CLDN18.2VHH-CD28 hinge (hinge region)-CD28 TM (transmembrane region)-CD28 ICD (costimulatory signal region)-CD3 zeta. The structure of different envelope plasmids carrying activating molecules and adhesion molecules is shown in Figure 17. In addition, as shown in Figure 18, Maraba-G envelope carrying activating molecules and adhesion molecules can still package lentivirus with very high titer, which is significantly higher than VSVG.

[0628] Example 13. Preparation of CAR-T cells by different envelope lentivirus carrying activating molecules and adhesion molecules in vitro

[0629] The day before virus infection, 6-well plates were prepared, 2ml PBS was added to each well, 3ul of CD3 (1ug / ul), 3ul of CD28 (1ug / ul) and 48ul of RetroNectin (1ug / ul) were dissolved in 1 well and placed in a 4°C refrigerator for standby. The next day, after the PBMC cells were resuscitated, the T cells were sorted by magnetic beads, and 3E6 cells / well were added to the RetroNectin coated 6-well plates. After 24 hours of activation, the packaged virus was added to the corresponding well plates at MOI=1 and MOI=3, and after 2 days of incubation, the residual virus was washed away with PBS and fresh culture medium was added for continuous culture. The CAR positive proportion of each group was detected on Day 3 and Day 7, respectively.

[0630] As shown in Figure 19, the flow detection results and data statistics show that the transduction efficiency of P24077 (Maraba-G) lentivirus is better than that of VSVG envelope packaged lentivirus. The Maraba-G lentivirus is significantly better than the traditional VSVG envelope packaged lentivirus in terms of infection efficiency.

[0631] Example 14. Packaging of different envelope lentivirus carrying activating molecules and adhesion molecules

[0632] The constructed envelope plasmids carrying different forms of ligands were co-transfected with helper packaging plasmids pRSV-Rev (Addgene, #12253), pMDLg-pRRE (Addgene, #12251) and a shuttle plasmid with CAR2 (CLND8.2-28z) via pEI into HEK293FT suspension cells. Among them, P23383 virus is co-expressed with VSVG envelope protein and ligand through P2A connection; P24077 is co-expressed with Maraba-G envelope protein and ligand through P2A connection; PZ202+P24117 is VSVG envelope and ligand on two different plasmids, and the ligand (activating molecule and adhesion molecule) is expressed in the form of a fusion protein, and the plasmid structure is shown in Figure 20; P24069+P24117 is Maraba-G envelope protein and ligand on two different plasmids, and the ligand (activating molecule and adhesion molecule) is expressed in the form of a fusion protein, and the plasmid structure is shown in Figure 20. The culture medium supernatant was collected after 48 hours, and the lentivirus was obtained by ultracentrifugation (10000g, 2h, 4h). The obtained lentivirus was resuspended in PBS for titer determination and stored at -80°C for standby.

[0633] Example 15. Preparation of CAR-T cells carrying fusion activating molecules and adhesion molecules in different envelope lentivirus in vitro

[0634] The day before virus infection, a 6-well plate was taken, 2ml PBS was added to each well, 3ul of CD3 (1ug / ul), 3ul of CD28 (1ug / ul) and 48ul of RetroNectin (1ug / ul) were dissolved in 1 well, and placed in a 4°C refrigerator for standby. The next day, after the PBMC cells were resuscitated, the T cells were sorted by magnetic beads, and were added to the RetroNectin coated 6-well plate at 3E6 cells / well. After 24 hours of activation, the packaged virus was added to the corresponding well plate at MOI=1 and MOI=3, and incubated for 2d. Then the residual virus was washed away with PBS and fresh culture medium was added for continuous culture. The CAR positive proportion of each group was detected on Day 3 and Day 7, respectively.

[0635] As shown in Figures 21 and 22, the flow detection results and data statistics show that the lentivirus packaged by the virus envelope structure carrying the fusion activating molecule and adhesion molecule is superior to the lentivirus packaged by the virus envelope structure carrying the ordinary activating molecule and adhesion molecule in terms of transduction efficiency. In addition, the Maraba-G carrying the fusion activating molecule and adhesion molecule is superior to the VSVG envelope in terms of transduction efficiency on unactivated PBMC.

[0636] Example 16. Generation of CAR-T for treating B cell tumors in vivo by Maraba-G virus

[0637] NCG-MHC-dKO mice (Nanjing Jiqie Yaoke Biotechnology Co., Ltd.) were inoculated with Nalm6-luc cells (Nanjing Jiqie Yaoke Biotechnology Co., Ltd.) via the tail vein, and the inoculation day was recorded as D-4; 2x10 7 cells / 200 μL / each PBMC (Miaoshun Shanghai Biotechnology Co., Ltd.); On the first day after PBMC inoculation, when the average number of tumor photons reached about 5E3-1E4 (p / sec / cm2 / sr), the mice were grouped according to the number of tumor photons and the level of reconstruction, and were divided into a solvent control group and a Maraba-G (P24069+P24117) group; the grouping day was defined as D0; on D0, the drugs were injected intraperitoneally, and 200 uL of solvent and Maraba-G (P24069+P24117) carrying T cell targeting modules (TCM) were injected, respectively. The CAR targeted by the shuttle plasmid carried by Maraba-G (P24069+P24117) targets CD19, the extracellular region is FMC63, the hinge region and transmembrane region are CD28 hinge and CD28 TM, and the intracellular costimulatory region is CD28 ICD and CD3 zeta, i.e., FMC63 scFv-CD28 hinge region-CD28 transmembrane region-CD28 costimulatory signal region-CD3 zeta, and the specific sequence is as follows (SEQ ID NO: 37). The total amount of virus was 1x10 9 TU. After administration, the body weight was measured regularly and the live imaging was performed. As shown in Figures 23 and 24, compared with the solvent control group, the tumor growth of the virus injection group was significantly inhibited, indicating that Maraba-G envelope virus can effectively generate CAR-T in vivo and produce an anti-tumor effect. During the experiment, the body weight of the mice in the Maraba-G virus group was stable, and the mice were well tolerated, while the body weight of the mice in the lysis control group decreased in the later stage of the experiment, and the results are shown in Figure 25.

[0638] On day 25 after administration (D25), the content of CAR-T in the peripheral blood was detected using a flow cytometry method, and the results showed that CAR-T can be successfully generated in vivo after Maraba-G virus administration and can persist for a long time, as shown in Figures 26 and 27.

[0639] CD19 CAR amino acid sequence SEQ ID NO: 37

[0640] Example 17. Construction of Maraba-G single mutant

[0641] To improve the targeting specificity of Maraba-G envelope, i.e. only rely on TCM to target and infect T cells, point mutations were made to some amino acids of the envelope to eliminate the binding of Maraba-G and the corresponding receptors. The mutation site information is shown in Figure 28, a total of 25 amino acid sites, including H8, H9, Q10, K47, S48, H49, K50, A51, H60, H132, A182, S183, I331, V184, D185, H209, M347, R329, T350, T351, T352, E353, R354, H407, H409 (the sites shown are relative to SEQ ID NO: 33). The asterisk in Figure 28 indicates mutations that can reduce or eliminate the binding of Maraba-G and the receptor.

[0642] The mutant envelope plasmid was constructed by Genewiz using P24069 (Maraba-G) as a template, and then the mutant envelope plasmid and P24069 (Maraba-G) were used to package lentivirus, respectively, and the GOI was GFP.

[0643] Example 18. In vitro specificity comparison of Maraba-G single mutants

[0644] Each Maraba-G envelope mutant plasmid, helper packaging plasmid pRSV-Rev (Addgene, #12253), pMDLg-pRRE (Addgene, #12251), GFP plasmid, and TCM plasmid (a total of 5 plasmids) or without TCM plasmid (a total of 4 plasmids) were co-transfected into HEK293FT suspension cells with pEI, and after 24 hours, the feed was added, and after 48 hours, the culture supernatant was collected, filtered through a 0.45 μm filter membrane, and the P24 titer was detected.

[0645] Each mutant virus was used to infect Jurkat cells at 0.05 μg of lentivirus particles, and after 4 days, FACS was used to detect the GFP positive rate. The results are shown in Figure 29, and the K47G, K47T, K47W, K47P, K47E, K47A, K47Q, A182E, I331E, H209E, M347E, T352E, R354A, R354G, R354S, R354E, R354W mutation sites can significantly reduce the infection ability of Maraba-G while not affecting its ability to infect Jurkat cells through TCM. At the same time, among all the sites, K47E, K47A, K47Q have the most obvious elimination effect, and the lentivirus without ligand almost cannot infect Jurkat. The TCM used in the single mutant test is also called MDF sequence, and the structure is shown in Figure 20.

[0646] To confirm the mutation of amino acid at K47 from the above results, K47E, K47A, K47Q and AK47 (K47 deletion) were selected as mutant envelope to package lentivirus, and the GOI was GFP reporter gene. Each mutant virus was used to infect Jurkat cells at 0.05 pg lentivirus particles, and the GFP positive rate was detected by FACS after 4 days. The results are shown in Figure 30. K47E mutation showed excellent performance in eliminating Maraba-G and receptor binding, and in infecting Jurkat cells through TCM. K47E was used as the main mutation site for subsequent testing.

[0647] The amino acid sequences of some Maraba-G envelope single mutant are as follows.

[0648] Maraba-G-K47E SEQ ID NO: 38

[0649] Maraba-G-K47A SEQ ID NO: 39

[0650] Maraba-G-K47Q SEQ ID NO: 40

[0651] Maraba-G-A182E SEQ ID NO: 41

[0652] Maraba-G-M347E SEQ ID NO: 42

[0653] Maraba-G-T352E SEQ ID NO: 43

[0654] Maraba-G-K47G SEQ ID NO: 44

[0655] Maraba-G-K47T SEQ ID NO: 45

[0656] Maraba-G-K47W SEQ ID NO: 46

[0657] Maraba-G-K47P SEQ ID NO: 47

[0658] Maraba-G- AK47 SEQ ID NO: 48

[0659] Maraba-G- I331E SEQ ID NO: 49

[0660] Maraba-G- H209E SEQ ID NO: 50

[0661] Maraba-G- R354A SEQ ID NO: 51

[0662] Maraba-G- R354G SEQ ID NO: 52

[0663] Maraba-G- R354S SEQ ID NO: 53

[0664] Maraba-G- R354E SEQ ID NO: 54

[0665] Maraba-G- R354W SEQ ID NO: 55

[0666] P24117-MDF sequence SEQ ID NO: 56

[0667] Example 19. Maraba-G combination mutants and their in vitro specificity test

[0668] Based on K47E mutant, further introduced A182E, R354A, H8A mutations, respectively with single, double and triple mutant virus envelope, with and without TCM and TCM ligand (P24117) to package GFP lentivirus, to infect Jurkat cells with 0.05 μg P24 virus particles, and detect GFP expression after 4 days. The results are shown in Figure 31A, the single, double and triple mutants tested can effectively eliminate the binding of Maraba-G and receptor, and can effectively depend on TCM to infect Jurkat cells. Among them, the triple mutant H8A / K47E / A182E is more obvious than K47E mutant in eliminating the binding of Maraba-G and receptor, and the ability to depend on TCM to infect Jurkat cells is not inferior to wild type Maraba-G.

[0669] The specificity of the combined mutant envelope was verified repeatedly, as shown in Figure 31B, different concentrations of P24 viral particles infected Jurkat cells, and the same Maraba-G (H8A / K47E / A182E) infected Jurkat cells with the lowest efficiency without TCM ligand, and could effectively infect Jurkat cells under TCM ligand conditions. Therefore, Maraba-G (H8A / K47E / A182E) was selected as a representative to carry out subsequent research.

[0670] The sequences of Maraba-G double mutant and triple mutant are as follows.

[0671] Maraba-G (K47E / A182E) SEQ ID NO: 57

[0672] Maraba-G (K47E / R354A) SEQ ID NO: 58

[0673] Maraba-G (H8A / K47E / A182E) SEQ ID NO: 59

[0674] Maraba-G (K47E / A182E / R354A) SEQ ID NO: 60

[0675] Maraba-G (K47E / S183P) SEQ ID NO: 61

[0676] Example 20. Maraba-G (H8A / K47E / A182E) can reduce the sensitivity to serum

[0677] The lentiviral envelope can be recognized by neutralizing antibodies in the blood, which can reduce its inventory in the human body and cause the drug efficacy to be reduced, and Maraba-G (H8A / K47E / A182E) can reduce the sensitivity to neutralizing antibodies. Freshly collected fresh plasma was inactivated at 56°C for 30 min, placed on ice for 20 min, and then centrifuged at 1000g for 10 min, and the supernatant was taken; X VIVO-15 resuspended Jurkat was added to a 24-well plate, 1E5 / well; the virus was directly incubated with 20 times the volume of plasma for 1 h, and then a certain amount was added to the corresponding well plate at MOI = 5 and 50, and mixed; 48-72 h later, flow cytometry was used to detect the expression of GFP in each well. The results are shown in Figure 32, and the Maraba-G triple mutant is less sensitive to serum than VSVG, and can still effectively infect target cells in serum.

[0678] Example 21. Maraba-G(H8A / K47E / A182E) mutation significantly improves lentivirus targeting specificity

[0679] Maraba-G(H8A / K47E / A182E) or wild type Maraba-G envelope and T cell targeted TCM packaged lentivirus were used to infect different cell lines at different MOI. As shown in Figure 33, Maraba-G(H8A / K47E / A182E) + TCM only effectively infected Jurkat cells, but not other cell lines, while Maraba-G infection was not selective and could infect almost all cell lines shown in the figure, indicating that Maraba-G(H8A / K47E / A182E) mutant in combination with T cell targeted TCM can significantly improve the targeting specificity of lentivirus, and can be used as an effective delivery vector for in-vivo CAR-T. The TCM used here is aCD7(23279-G4S-23286)-aCD3-ICAM1, aCD7 is a membrane-bound CD7 antibody, containing two VHH (antibody numbers are 23279 and 23286) connected by G4S (GGGGS, SEQ ID NO: 62), anchored to the surface of the virus envelope by CD8 hinge and transmembrane region. From this example, in the subsequent results, aCD7 refers to aCD7(23279-G4S-23286)-CD8hinge-CD8TM unless otherwise specified.

[0680] Example 22. Comparison of Maraba-G(H8A / K47E / A182E) targeting specificity

[0681] In addition, Maraba-G(H8A / K47E / A182E) envelope and VSVG(I182E / T214N / T352A) and VSVG(K47Q / R354A) targeting specificity were also compared. The three mutant envelopes were used to package lentivirus without ligand, with CD7 ligand aCD7-aCD3-ICAM1 and with MDF ligand (as shown in Figure 20) respectively, and after centrifugal concentration, Jurkat and Raji cells were infected with 500 ng / ml P24 respectively, and 48 h later, the GFP expression of each cell was detected by flow cytometry. The results are shown in Figure 34. The three mutant envelopes did not infect Raji cells; while on Jurkat cells, Maraba-G(H8A / K47E / A182E) envelope and VSVG(I182E / T214N / T352A) were superior to VSVG(K47Q / R354A) in specificity without ligand, and Maraba-G(H8A / K47E / A182E) and VSVG(I182E / T214N / T352A) were comparable; all three could effectively infect Jurkat cells after adding different ligands targeting Jurkat cells, and the infection efficiency reached more than 90%.

[0682] The above results show that Maraba-G(H8A / K47E / A182E) has both targeting specificity and infection effectiveness, and can be used as an ideal delivery vector for in-vivo CAR-T.

[0683] Example 23. Screening of Targeting Molecule Combinations

[0684] To improve the specific activation and infection ability of Maraba-G envelope on T cells, different targeting molecule combinations were added to the mutant Maraba-G. The molecule combinations consisted of one or more of T cell activation molecules, adhesion molecules and specific protein antibodies such as aCD3, CD80, CD58, aCD7 and ICAM1.

[0685] In all examples, the targeting combination molecules can be constructed on the same plasmid with the virus envelope protein through 2A cleavable connection or G4S non-cleavable connection, or on two or more plasmids, and two or more plasmids are added simultaneously during virus packaging, or expressed on the packaging cells. First, the virus containing CD19 CAR (CD19 CAR is the same as in Example 16) was packaged with the virus envelope combination as shown in Figure 35, and the infection titer was detected. As shown in Figure 35, the targeting molecule combinations of aCD7-aCD3, aCD7-aCD3-CD80, aCD7-aCD3-G4S-CD80, aCD7-CD58, aCD7-aCD3-CD58 and aCD7-MDF could all effectively infect Jurkat cells.

[0686] In this and subsequent examples, two structural elements are connected by P2A instead of G4S, for example: aCD7-aCD3 is aCD7-P2A-aCD3, aCD7-aCD3-CD80 is aCD7-P2A-aCD3-P2A-CD80, aCD7-aCD3-G4S-CD80 is aCD7-P2A-aCD3-G4S-CD80. Maraba-G* means 6 mutants, including H8A, Q10A, K47Q, A182E, H209A and R354A mutations compared to wild type Maraba-G (sequence shown in SEQ ID NO: 33), and the following Maraba-G* refers to 6 mutants.

[0687] Maraba-G* amino acid sequence SEQ ID NO: 63

[0688] Example 24. Specific detection of enveloped combination infection

[0689] Different combinations of targeting molecules were added on the basis of the triple mutant Maraba-G (H8A / K47E / A182E), including aCD3-G4S-CD80-ICAM1, aCD3-G4S-CD80-CD58, aCD7-CD80-ICAM1, aCD7-aCD3-ICAM1, MDF, and Maraba-G*-aCD3-CD80-ICAM1 and Maraba-G-WT-MDF as controls. These different enveloped combinations were packaged into lentivirus in 293FT cells with GFP as GOI. P24 titers of different viruses were detected using P24 detection kit, and infection titers were also detected at the same time.

[0690] First, P24 titer 1 ug / mL, 0.1 ug / mL, 0.01 ug / mL, 0.001 ug / mL infected Jurkat cells and Raji cells, 2 days after detection of the positive rate of GFP. Results as shown in Figure 36, Jurkat cells, all viruses can infect Jurkat cells, in 1 ug / mL, 0.1 ug / mL concentration, most of the virus infection efficiency reached saturation, in 0.01 ug / mL and 0.001 ug / mL concentration, Maraba-G-WT-MDF, Maraba-G(H8A / K47E / A182E)-MDF, Maraba-G(H8A / K47E / A182E)-αCD7-CD80-ICAM1, Maraba-G(H8A / K47E / A182E)-αCD7-αCD3-ICAM1 infection efficiency is higher; Raji cells, only Maraba-G-WT-MDF virus containing wild type Maraba-G envelope can infect at all concentrations and the positive rate is very high, other viruses containing mutant Maraba envelope have almost no infection at 0.1 ug / mL, 0.01 ug / mL, 0.001 ug / mL concentration, at 1 ug / mL concentration, only Maraba-G(H8A / K47E / A182E)-αCD7-CD80-ICAM1 virus has very low infection efficiency. The results show that based on Maraba-G* and Maraba-G(H8A / K47E / A182E), increasing αCD3-G4S-CD80-ICAM1, αCD3-G4S-CD80-CD58, αCD7-CD80-ICAM1, αCD7-αCD3-ICAM1 has good T cell infection efficiency, and the specificity is better, almost no infection of Raji cells.

[0691] Example 25. Comparison of the activation and infection ability of T cells by different envelope combinations (I)

[0692] Figure 37 shows the activation and infection ability of T cells with different combinations of targeting molecules and envelope proteins. Most of the targeting molecules were packaged based on Maraba-G (H8A / K47E / A182E), and a few were packaged based on Maraba-G* or wild-type Maraba-G (Maraba-WT). Different envelope plasmids, master plasmids containing CD19 CAR, and helper plasmids were added to 293FT cells for virus packaging. The harvested virus was incubated with PBMCs at an MOI of 5, and T cell activation (CD25+) and transfection efficiency were detected at day 2 and day 6, respectively. As shown in Figure 37, the activation efficiency of aCD3, CD80, CD86, aCD7-CD80-ICAM1 was low, and other molecular combinations could effectively activate T cells. Among the mutant envelopes of Maraba, aCD7-aCD3-ICAM1, MDF, aCD3-G4S-CD80-CD58, aCD3-G4S-CD80-ICAM1, aCD3-CD80-ICAM1 had higher activation efficiency.

[0693] Figure 38 shows the positive rate of CD19 CAR in T cells. The positive rates of aCD7-aCD3-ICAM1, MDF, aCD3-G4S-CD80-CD58, aCD3-G4S-CD80-ICAM1, aCD3-CD80-ICAM1, aCD7-CD80-ICAM1 were higher at day 2. At day 6, the positive rates of most virus infections decreased compared to day 2, and the virus containing aCD7-aCD3-ICAM1 in the mutant envelope still had a high positive rate at day 6.

[0694] CAR-T cells generated by Maraba-G (H8A / K47E / A182E) + aCD7-aCD3-ICAM1 and Cocal + MDF were taken at day 7, and each group of cells was incubated with target cells Raji at different effector-target ratios. The apoptosis rate of Raji cells was detected 4 hours later to represent the killing ability of different groups of CAR-T cells. The results are shown in Figure 39A. Each group can effectively kill target cells, and Maraba-G (H8A / K47E / A182E) + aCD7-aCD3-ICAM1 and Cocal + MDF have higher killing efficiency. Raji was used to activate different groups of cells, and the exhaustion phenotype of CAR-T cells was detected before and after activation. The results are shown in Figure 39B. The proportion of exhaustion markers in the Maraba-G (H8A / K47E / A182E) + aCD7-aCD3-ICAM1 group after target cell activation was lower than that in the Cocal + MDF group.

[0695] In the Raji killing experiment, the supernatant was collected and the cytokine secretion was detected, and the results are shown in Figure 40. Each group can effectively secrete cytokines.

[0696] CD86 amino acid sequence (1-23 is the signal peptide sequence) SEQ ID NO: 64

[0697] Cocal virus envelope glycoprotein full-length amino acid sequence SEQ ID NO: 120

[0698] Cocal virus envelope glycoprotein mature sequence SEQ ID NO: 121 (without signal peptide)

[0699] Example 26. Comparison of the activation and infection ability of T cells by different envelope combinations (two)

[0700] In order to further compare different envelope combinations, the plasmids containing the targeting molecules as shown in Figure 41, Maraba-G (H8A / K47E / A182E) envelope plasmid, CD19 CAR plasmid and helper plasmid were added to 293FT cells to package viruses containing different envelope combinations. The titer was detected and incubated with PBMC at MOI = 5, and two groups of conventional CD3 and CD28 antibody activated PBMC were set as controls, namely the unt (pre-activated PBMC) group and the aCD7-aCD3-ICAM1 (pre-activated PBMC) group. The activation of T cells and the positive rate of CD19 CAR were detected on the 2nd day and the 6th day, respectively. The results are shown in Figure 41. The conventional antibody coated group can effectively activate T cells. Among the groups only added with viruses, the viruses containing different envelope combinations can effectively activate T cells, and the activation efficiency of the MDF, aCD7-aCD3-ICAM1, aCD7-aCD3-CD58, aCD3-CD58 groups is the highest.

[0701] As shown in Figure 42, the conventional antibody activated group (containing aCD7-aCD3-ICAM1) has a higher CAR positive rate. Among the groups only added with viruses, aCD7-aCD3-ICAM1, aCD7-aCD3-CD58, aCD7-aCD3-CD80, aCD7-aCD3 groups can effectively infect T cells.

[0702] Example 27. Specificity detection of viruses with different envelopes on primary cells and tumor cells

[0703] To test the infection of virus on normal tissue primary cells and tumor cells, Maraba-G(H8A / K47E / A182E)+aCD7-aCD3-ICAM1 envelope, Cocal+MDF envelope and VSVG(K47Q / R354A)+aCD3-CD80 envelope plasmids were combined with CD19 CAR master plasmid and helper plasmid into 293FT cells for virus packaging. The packaged virus was added to primary cells and tumor cells as shown in FIG. 43 at MOI=2 and MOI=0.5, and the positive rate of CD19 CAR was detected after 2 days. The results showed that at MOI=2 and 0.5, the three viruses could effectively infect Jurkat cells, and the infection efficiency of Maraba-G(H8A / K47E / A182E)+aCD7-aCD3-ICAM1 was higher than that of Cocal+MDF and VSVG(K47Q / R354A)+aCD3-CD80. Maraba-G(H8A / K47E / A182E)+aCD7-aCD3-ICAM1 hardly infected primary cells, while Cocal+MDF and VSVG(K47Q / R354A)+aCD3-CD8 detected a certain degree of infection in intrahepatic bile duct epithelial cells and retinal pigment epithelial cell line-19. These results showed that Maraba-G(H8A / K47E / A182E)+aCD7-aCD3-ICAM1 had better tumor cell infection specificity.

[0704] Example 28. Transduction efficiency verification of aCD7-aCD3-ICAM1 targeting module in different envelopes

[0705] To test the infection efficiency of lentivirus prepared by aCD7-aCD3-ICAM1 targeting module and different virus envelopes on PBMC cells, aCD7-aCD3-ICAM1 ligand plasmid was combined with Maraba-G(H8A / K47E / A182E), Cocal-K47E and VSVG(K47Q / R354A) envelope plasmids, and CD19 CAR master plasmid and helper plasmid were added to 293FT cells for virus packaging. At the same time, aCD7-aCD3-ICAM1 ligand plasmid was replaced by MDF or aCD3-CD80 ligand plasmid for synchronous testing. The packaged virus was added to PBMC cells at MOI=1, 5, 25, and the positive rate of CD19 CAR was detected after 3 days. The results showed that as shown in FIG. 44, the infection efficiency of lentivirus prepared by all mutant envelopes and aCD7-aCD3-ICAM1 ligand on PBMC was higher.

[0706] In summary, the combination of aCD7-aCD3-ICAM1 ligand and Maraba-G(H8A / K47E / A182E) performed well in targeting specificity and infection efficiency, and is a better delivery vector for In-Vivo CAR-T therapy.

[0707] Example 29. aCD7-aCD3-ICAM1 and Maraba-G(H8A / K47E / A182E) envelope preparation of CAR-T targeting different antigens

[0708] To verify that the lentivirus of aCD7-aCD3-ICAM1 and Maraba-G(H8A / K47E / A182E) envelope can prepare CAR-T targeting different target points, the aCD7-aCD3-ICAM1 plasmid, Maraba-G(H8A / K47E / A182E) envelope plasmid, helper plasmid, and BCMACAR (CAR structure extracellular region is the scFv sequence of B38M, hinge region is CD8 hinge region, transmembrane region is CD8 transmembrane region, costimulatory region is 41BB and CD3 zeta) or CLDN18.2 CAR (CAR2) plasmid were co-transfected into virus packaging cells 293FT, and GFP group (GFP plasmid instead of CAR plasmid) was used as a control. The prepared lentivirus was used to infect PBMC cells (MOI=5), and was named BCMACAR, CLDN18.2 CAR or GFP, respectively. After 3 days, the positive rate of BCMACAR, CLDN18.2 or GFP in each group of T cells was detected, and the results are shown in Figures 45A and 45C. Each group can effectively infect T cells to generate CAR-positive or GFP-positive cells. To detect the killing of different groups of CAR-T cells to target cells, BCMACAR-T cells were co-incubated with target cells H929 at an effector-to-target ratio of 3:1, 1:1 and 0.3:1 in a 96-well plate, and the GFP group was used as a negative control; at the same time, CLDN18.2 CAR-T cells were co-incubated with target cells NUGC4 at an effector-to-target ratio of 3:1, 1:1 and 0.3:1, and the GFP group was used as a negative control. After 4h, the killing efficiency of each group of CAR-T to target cells was calculated by the proportion of target cell apoptotic cells, as shown in Figures 45B and 45D. BCMACAR-T can effectively kill H929 tumor cells, and CLDN18.2 CAR-T can effectively kill NUGC4 tumor cells, and both have dose-dependent, indicating that aCD7-aCD3-ICAM1 and Maraba-G(H8A / K47E / A182E) envelope can be effectively used to prepare CAR-T cells targeting different target points or antigens.

[0709] BCMACAR amino acid sequence SEQ ID NO:65

[0710] Example 30. Viruses produced by aCD7-aCD3-ICAMl and Maraba-G(H8A / K47E / A182E) envelope generate CD19 CAR-T treatment of B cell tumors directly in vivo

[0711] To verify the efficacy of aCD7-aCD3-ICAMl and Maraba-G(H8A / K47E / A182E) envelope produced viruses in mice, aCD7-aCD3-ICAMl plasmid, Maraba-G(H8A / K47E / A182E) envelope plasmid, helper plasmid, CD19 CAR plasmid were transfected into 293FT to package aCD7-aCD3-ICAMl virus. At the same time, in order to compare with other ligands, MDF(24117) plasmid, Maraba-G(H8A / K47E / A182E) envelope plasmid, helper plasmid, CD19 CAR plasmid were transfected into 293FT to package MDF virus. The two viruses were used for in vivo efficacy test.

[0712] M-NSG (derived from Shanghai Southern Model Organism Technology Co., Ltd.) mice were inoculated with Nalm6-luc (constructed by Shanghai Southern Model Organism Technology Co., Ltd. from Nalm6 cells from ATCC) cells through the tail vein, and the inoculation day was recorded as D-7; on D-1, 1x10 7cells / only PBMC; 1 day after PBMC inoculation, the mice were grouped according to the number of tumor photons. The group injected with PBS only was the vehicle control group (Vehicle control); the group injected with PBMC only on D-1 was the PBMC control group (PBMC control). The experimental groups were injected with PBMC and one kind of virus, specifically, MDF low dose (1E6), MDF medium dose (5E6), MDF high dose (25E6), aCD7-aCD3-ICAM1 low dose (1E6), aCD7-aCD3-ICAM1 medium dose (5E6), aCD7-aCD3-ICAM1 high dose (25E6). The day of grouping was defined as DO, and the drugs were injected intravenously on DO, 200 uL of lentivirus was injected according to the above-mentioned doses. Body weight and live imaging were performed regularly after administration. As shown in FIG. 46A, compared with the control groups, the three dose groups of aCD7-aCD3-ICAM1 virus injection group all had obvious tumor inhibition effect, and the tumor inhibition effect was enhanced with the increase of the dose; while the high dose group of MDF injection showed a certain tumor inhibition effect, but the inhibition effect was much lower than that of the aCD7-aCD3-ICAM1 group. The mice in the two control groups, MDF low dose group and MDF high dose group started to have obvious weight loss on the 13th day after administration due to large tumor load, and the mice in the aCD7-aCD3-ICAM1 group had relatively better tolerance. After administration, the mouse blood was collected every week for CAR-T cell number detection, as shown in FIG. 46B, both viruses can generate CAR-T in vivo.

[0713] In summary of the above results, the aCD7-aCD3-ICAM1 and Maraba-G(H8A / K47E / A182E) packaged CD19 CAR virus can effectively generate CAR-T in vivo, and has a relatively strong tumor inhibition effect.

[0714] Example 31. Comparison of in vivo efficacy of viruses prepared by aCD7-aCD3-ICAM1 and Maraba-G(H8A / K47E / A182E) envelope and other combination viruses

[0715] The aCD7-aCD3-ICAM1 plasmid, Maraba-G(H8A / K47E / A182E) envelope plasmid, helper plasmid, and CD19 CAR plasmid were transfected into 293FT to package Maraba-G(H8A / K47E / A182E)+aCD7-aCD3-ICAM1 virus. The aCD3-CD80 plasmid, VSV-G(K47Q / R354A) envelope plasmid, helper plasmid, and CD19 CAR plasmid were transfected into 293FT to package VSV-G(K47Q / R354A)+aCD3-CD80 virus. The aCD7 plasmid, VSV-G(I182E / T214N / T352A) envelope plasmid, helper plasmid, and CD19 CAR plasmid were transfected into 293FT to package VSV-G(I182E / T214N / T352A)+aCD7 virus. The MDF plasmid, Cocal-G envelope plasmid, helper plasmid, and CD19 CAR plasmid were transfected into 293FT to package Cocal-G+MDF virus. These viruses all contain CD19 CAR and can target CD19-positive tumor cells.

[0716] M-NSG (Shanghai Southern Model Organism Technology Co., Ltd.) mice were inoculated with Nalm6-luc cells (constructed by Shanghai Southern Model Organism Technology Co., Ltd. from Nalm6 cells from ATCC) via the tail vein, and the day of inoculation was recorded as D-7; on D-1, 1×10 7 cells / only PBMC were injected intravenously, and the mice were grouped according to the number of tumor photons 1 day after PBMC inoculation. The PBS injection group was the vehicle control group (Vehicle control); the group injected only with PBMC was the PBMC control group (PBMC control). The experimental groups were injected with PBMC and one of the viruses, and the dose of each virus was 2×10 7 TU. The day of grouping was defined as D0, and the drugs were injected intravenously on D0, and 200 uL of lentivirus was injected at the above-mentioned dose. Body weight was measured and live imaging was performed regularly after administration.

[0717] As shown in FIG. 47A, the efficacy of the Maraba-G(H8A / K47E / A182E)+aCD7-aCD3-ICAM1 virus group was the most obvious, and it could significantly inhibit tumor growth. The VSV-G(I182E / T214N / T352A)+aCD7 virus had a certain tumor inhibition effect, and the other groups had no obvious tumor inhibition effect. During the experiment, the body weight of the vehicle control group of mice decreased, and some mice died. The mice in the Cocal-G+MDF group had a slight decrease in body weight about 20 days after administration, and the other groups had no significant change. The results are shown in FIG. 47B.

[0718] Example 32. Viruses packaged by aCD7-aCD3-ICAM1 and Maraba-G(H8A / K47E / A182E) envelope generate Bcma CAR-T therapy for multiple myeloma in vivo

[0719] To verify the function of aCD7-aCD3-ICAM1 and Maraba-G(H8A / K47E / A182E) envelope system on Bcma CAR, aCD7-aCD3-ICAM1 plasmid, Maraba-G(H8A / K47E / A182E) envelope plasmid, helper plasmid, Bcma CAR plasmid were transfected into 293FT to package Maraba-G(H8A / K47E / A182E)+aCD7-aCD3-ICAM1+Bcma CAR virus, referred to as Invivo Bcma CAR.

[0720] M-NSG (from Shanghai South Model Organism Technology Co., Ltd.) mice were inoculated with 5x10 6 H929-luc (constructed by Shanghai South Model Organism Technology Co., Ltd. from H929 cells from ATCC) cells, when the tumor fluorescence increased to 10 6 around, recorded as D-1; on D-1, except for the solvent control group, the other groups were intravenously inoculated with 1x10 7 cells / mouse PBMC, and D0 was dosed. Only injected with PBS was the solvent control group (Vehicle control); only injected with PBMC was the PBMC control group (PBMC control). The experimental groups were all injected with PBMC on D-1, and 2x10 6 TU or 1x10 7 TU Invivo Bcma CAR lentivirus on D0. Regular body weight and live imaging were performed after dosing.

[0721] As shown in Figure 48, relative to the control group, the two groups injected with virus can effectively inhibit H929-luc tumor cells. On the 7th and 14th day after dosing, the proportion of CAR-T generated in the peripheral blood was detected, and the results are shown in Figure 49. CAR-positive T cells were detected in the two groups injected with Invivo Bcma CAR virus on D14, indicating that CAR-T was successfully generated in vivo. In summary, aCD7-aCD3-ICAM1 and Maraba-G(H8A / K47E / A182E) packaged Bcma CAR virus can effectively generate CAR-T in vivo, and has a relatively strong tumor inhibition effect.

[0722] Example 33. In vivo CLDN18.2 CAR virus directly generates CLDN18.2 CAR-T in vivo to treat gastric cancer tumor

[0723] To verify the function of the Invivo system on CLDN18.2 CAR, the alphaCD7-alphaCD3-ICAM1 plasmid, Maraba-G(H8A / K47E / A182E) envelope plasmid, helper plasmid, CLDN18.2 CAR plasmid were transfected into 293FT to package Maraba-G(H8A / K47E / A182E)+alphaCD7-alphaCD3-ICAM1+CLDN18.2 CAR virus, abbreviated as Invivo CLDN18.2 CAR-1. At the same time, the alphaCD7-alphaCD3 plasmid, Maraba-G(H8A / K47E / A182E) envelope plasmid, helper plasmid, CLDN18.2 CAR plasmid were transfected into 293FT to package Maraba-G(H8A / K47E / A182E)+alphaCD7-alphaCD3+CLDN18.2 CAR virus, abbreviated as Invivo CLDN18.2 CAR-2.

[0724] M-NSG-MHCII / B2M dKO (from Shanghai South Model Organisms Technology Co., Ltd.) mice were inoculated with 1x10 7 NUGC4-luc (Shanghai Penglisi Biomedical Research Co., Ltd.) cells were grouped on the 13th day after tumor inoculation, recorded as D-1; 1x10 7 cells / one PBMC, D0 for administration. Only PBMC injection on D-1 is the PBMC control group (PBMC control). The experimental groups were injected with PBMC on D-1, and 5x10 6 TU Invivo CLDN18.2 CAR-1, 2x10 7 TU Invivo CLDN18.2 CAR-1, 5x10 6 TU Invivo CLDN18.2 CAR-2, 2x10 7 TU Invivo CLDN18.2 CAR-2. Body weight and live imaging were performed regularly after administration.

[0725] As shown in Figure 50, relative to the PBMC control group, the group injected with the Invivo virus had a certain inhibitory effect on tumor cells. Peripheral blood was collected every week after administration, and the proportion and number of CAR-T generated in the peripheral blood were detected. The results are shown in Figure 51. The group injected with the Invivo virus can effectively generate CAR-T. In combination with the above results, the aCD7-aCD3-ICAM1 or aCD7-aCD3 and Maraba-G(H8A / K47E / A182E) packaged CLDN18.2 CAR virus can effectively generate CAR-T in vivo, and has a relatively strong inhibitory effect on tumors.

[0726] Example 34. Transduction efficiency and in vitro functional verification of different T cell activation antibody sequences used in aCD7-aCD3-ICAM1 structure

[0727] In previous studies, mb-OKT3 was used as the targeting antibody (aCD3) of CD3. In this experiment, aCD3 in aCD7-aCD3-ICAM1 was replaced with different T cell activation antibodies (targeting CD3 or TCR) to construct. Specifically, CD3 antibody OKT3 (as described above, membrane-bound type), CD3 antibody UCHT1 (UCHT1 was constructed as a membrane-bound antibody, and the structure is UCHT1 scFv-PDGFR stalk-PDGFR TM), and a TCR activation antibody (VHH membrane-bound antibody, TCR VHH-IgG4 scaffold, targeting TCR a / b) were used. Different ligands were combined, Maraba-G(H8A / K47E / A182E) envelope, helper plasmid and CD19 CAR plasmid were co-transfected into virus packaging cells 293FT, and the prepared lentivirus was used to infect PBMC cells, MOI=5. Four days later, the T cell activation efficiency (CD25 proportion) and the positive rate of CD19 CAR in T cells were detected. The results are shown in Figures 52A and 52B. Each group can effectively activate infected T cells and generate CAR-positive cells.

[0728] In order to detect the killing of different groups of CAR-T cells to target cells Raji, each group of CAR-T cells was incubated with Raji at an effector-to-target ratio of 3:1, 1:1 and 0.3:1 in a 96-well plate. Four hours later, the killing efficiency of each group of CAR-T to Raji was calculated by the proportion of target cell apoptotic cells. As shown in Figure 53, each group can effectively kill Raji tumor cells.

[0729] The above results show that aCD3 in the aCD7-aCD3-ICAM1 targeting molecule combination can be replaced with other molecules targeting TCR complexes, such as antibodies targeting TCR, and different targeting molecules (such as different antibody sequences) can achieve similar effects for the same target.

[0730] UCHT1 scFv-PDGFR stalk-PDGFR TM amino acid sequence SEQ ID NO: 66

[0731] UCHT1 scFv amino acid sequence SEQ ID NO: 67

[0732] UCHT1 HCDR1 SEQ ID NO: 68

[0733] UCHT1 HCDR2 SEQ ID NO: 69

[0734] UCHT1 HCDR3 SEQ ID NO: 70

[0735] UCHT1 LCDR1 SEQ ID NO: 71

[0736] UCHT1 LCDR2 SEQ ID NO: 72

[0737] UCHT1 LCDR3 SEQ ID NO: 73

[0738] UCHT1 VH SEQ ID NO: 74

[0739] UCHT1 VL SEQ ID NO: 75

[0740] TCR VHH-IgG4 scaffold SEQ ID NO: 76

[0741] Membrane binding protein signal peptide SEQ ID NO: 77

[0742] TCR VHH HCDR1 SEQ ID NO: 78

[0743] TCR VHH HCDR2 SEQ ID NO: 79

[0744] TCR VHH HCDR3 SEQ ID NO: 80

[0745] TCR VHH SEQ ID NO:81

[0746] IgG4 scaffold SEQ ID NO:82

[0747] Example 35. Transduction efficiency and in vitro functional validation of different aCD7 antibody sequences in aCD7-aCD3-ICAM1 construct

[0748] In previous examples, aCD7 in the form of 23279-G4S-23286 was used. In this experiment, different aCD7 antibodies were used to construct aCD7-aCD3-ICAM1. The different aCD7 (all membrane-bound) are numbered as 23277, 23279, 23281, 23279-G4S-23286 (23279 and 23286 are connected by G4S linker), 25373, 25374, 25365, 25376. Different aCD7 antibody-derived aCD7-aCD3-ICAM1, Maraba-G (H8A / K47E / A182E) envelope, helper plasmid and CD19 CAR plasmid were co-transfected into virus packaging cells 293FT to prepare lentivirus, which was used to infect PBMC cells at MOI = 5. After 3 days, the positive rate of CD19 CAR in T cells was detected, and the results are shown in Figures 54A and 55A. Each group can effectively infect T cells to generate CAR-positive cells.

[0749] To detect the killing of different groups of CAR-T cells to target cells Raji, each group of CAR-T cells was co-incubated with Raji at effector to target ratios of 3:1, 1:1 and 0.3:1 in a 96-well plate. After 4 hours, the killing efficiency of each group of CAR-T to Raji was calculated by the proportion of target cell apoptotic cells, as shown in Figures 54B and 55B. Each group can effectively kill Raji tumor cells.

[0750] The above results show that for aCD7-aCD3-ICAM1 targeting molecule combination, different targeting molecules (such as different antibody sequences) can achieve similar effects for the same target.

[0751] aCD7 (25373) VHH-CD8hinge-CD8 TM SEQ ID NO:83

[0752] aCD7 (25373) VHH SEQ ID NO:84

[0753] aCD7 (25373) HCDR1 SEQ ID NO: 85

[0754] aCD7 (25373) HCDR2 SEQ ID NO: 86

[0755] aCD7 (25373) HCDR3 SEQ ID NO: 87

[0756] aCD7 (25374) VHH-CD8 hinge-CD8 TM SEQ ID NO: 88

[0757] aCD7 (25374) VHH SEQ ID NO: 89

[0758] aCD7 (25374) HCDR1 SEQ ID NO: 90

[0759] aCD7 (25374) HCDR2 SEQ ID NO: 91

[0760] aCD7 (25374) HCDR3 SEQ ID NO: 92

[0761] aCD7 (25375) scFv-CD8 hinge-CD8 TM SEQ ID NO: 93

[0762] aCD7 (25375) scFv SEQ ID NO: 94

[0763] aCD7 (25375) HCDR2 SEQ ID NO: 96

[0764] aCD7 (25375) HCDR3 SEQ ID NO: 97

[0765] aCD7 (25375) LCDR1 SEQ ID NO: 98

[0766] aCD7 (25375) LCDR2 SEQ ID NO: 99

[0767] aCD7 (25375) HCDR3 SEQ ID NO: 100

[0768] aCD7 (25375) VHH-CD8hinge-CD8 TM SEQ ID NO: 101

[0769] aCD7 (25375) VL SEQ ID NO: 102

[0770] aCD7 (25376) scFv-CD8hinge-CD8 TM SEQ ID NO: 103

[0771] aCD7 (25376) scFv SEQ ID NO: 104

[0772] aCD7 (25376) HCDR1 SEQ ID NO: 105

[0773] aCD7 (25376) HCDR2 SEQ ID NO: 106

[0774] aCD7 (25376) HCDR3 SEQ ID NO: 107

[0775] aCD7 (25376) LCDR1 SEQ ID NO: 108

[0776] aCD7 (25376) LCDR2 SEQ ID NO: 109

[0777] aCD7 (25376) LCDR3 SEQ ID NO: 110

[0778] aCD7 (25376) VHH-CD8hinge-CD8 TM SEQ ID NO: 111

[0779] aCD7 (25376) VL SEQ ID NO: 112

[0780] aCD7 (23279) VHH-CD8hinge-CD8 TM SEQ ID NO: 113

[0781] aCD7 (23279) VHH SEQ ID NO: 114

[0782] aCD7 (23279) HCDR1 SEQ ID NO: 115

[0783] aCD7 (23279) HCDR2 SEQ ID NO: 116

[0784] aCD7 (23279) HCDR3 SEQ ID NO: 117

[0785] CD8 hinge-CD8TM SEQ ID NO: 118

Claims

1. A variant of the Maraba virus envelope glycoprotein (Maraba-G) characterized in that, The Maraba-G variant has reduced or eliminated binding ability to low density lipoprotein receptor (LDLR) on the surface of T cells compared to wild-type Maraba-G comprising the amino acid sequence of SEQ ID NO:33, and the Maraba-G variant comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.3%, or at least 99.5% sequence identity to SEQ ID NO:

33.

2. The Maraba-G variant of claim 1, wherein the variant comprises an amino acid substitution or deletion at one or more positions selected from H8, Q10, K47, A182, S183, H209, R354, I331, M347, and T352 relative to wild-type Maraba-G comprising the amino acid sequence of SEQ ID NO:33; preferably, the variant comprises one or more of the following amino acid substitutions or deletions relative to wild-type Maraba-G: H8A, Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, S183P, H209A, H209E, R354A, R354G, R354S, R354E, R354W, I331E, M347E, and T352E.

3. The Maraba-G variant of claim 2, wherein the variant comprises two or more of the following amino acid substitutions or deletions relative to wild-type Maraba-G: H8A, Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, S183P, H209A, H209E, R354A, R354G, R354S, R354E, R354W, I331E, M347E, and T352E.

4. The Maraba-G variant of claim 2, wherein the variant comprises three or more of the following amino acid substitutions or deletions relative to wild-type Maraba-G: H8A, Q10A, K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, S183P, H209A, H209E, R354A, R354G, R354S, R354E, R354W, I331E, M347E, and T352E.

5. A Maraba virus envelope glycoprotein (Maraba-G) variant comprising an amino acid substitution or deletion at one or more positions selected from H8, Q10, K47, A182, S183, H209, and R354 relative to wild-type Maraba-G comprising the amino acid sequence of SEQ ID NO:

33.

6. The Maraba-G variant of claim 5, wherein the variant comprises one or more amino acid substitutions or deletions relative to wild-type Maraba-G selected from the group consisting of: H8A, H8V, H8P, Q10A, K47Q, K47E, K47A, AK47, A182E, A182W, A182F, AA182, S183P, S183F, AS183, H209A, H209E, AH209, R354A, R354E, R354W, and AR354.

7. The Maraba-G variant of claim 5 or 6, wherein the variant comprises at least two mutations relative to wild-type Maraba-G selected from the group consisting of: H8A, Q10A, K47E / K47A / K47Q, A182E, S183P, H209A, and R354A.

8. The Maraba-G variant of claim 5 or 6, wherein the variant comprises at least two mutations relative to wild-type Maraba-G selected from the group consisting of: H8A, K47E / K47A / K47Q, A182E, and R354A.

9. The Maraba-G variant of any one of claims 1-5, wherein the variant comprises an amino acid substitution or deletion at the K47 position; preferably, the variant comprises an amino acid substitution or deletion at the K47 position selected from the group consisting of: K47G, K47T, K47W, K47P, K47Q, K47E, K47A, and AK47; more preferably, the variant comprises a K47E or K47Q amino acid substitution at the K47 position.

10. The Maraba-G variant of claim 9, wherein the variant further comprises an amino acid substitution or deletion at one or more positions selected from the group consisting of: H8, Q10, A182, S183, H209, and R354; preferably, the variant further comprises one or more amino acid substitutions selected from the group consisting of: H8A, Q10A, A182E, S183P, H209A, and R354A.

11. The Maraba-G variant of any one of claims 1-10, wherein the variant comprises an amino acid substitution or deletion or substitution combination selected from the group consisting of: K47G, K47T, K47W, K47P, K47Q, K47E, K47A, AK47, A182E, I331E, H209E, M347E, T352E, R354A, R354G, R354S, R354E, R354W, K47E / R354A, K47E / A182E, K47E / S183P, H8A / K47E / A182E, K47E / A182E / R354A, and H8A / Q10A / K47Q / A182E / H209A / R354A; preferably, the variant comprises the H8A / K47E / A182E amino acid substitution combination.

12. The Maraba-G variant of any one of claims 1-11, wherein the variant comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.3%, at least 99.5%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 38-55, 57-61, and 63.

13. The Maraba-G variant of any one of claims 1-12, wherein the N-terminus of the variant further comprises a signal peptide; preferably, the signal peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% sequence identity to SEQ ID NO: 35; more preferably, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO:

35.

14. A nucleic acid molecule comprising a nucleotide sequence encoding the Maraba-G variant of any one of claims 1-13.

15. A vector comprising the nucleic acid molecule of claim 14.

16. A vector combination comprising the vector of claim 15, and one or more helper packaging plasmids for assisting lentiviral packaging; optionally, the vector combination further comprises a vector carrying a gene of interest.

17. A host cell comprising the nucleic acid molecule of claim 14, the vector of claim 15, or the vector combination of claim 16.

18. A viral particle comprising a wild-type Maraba-G envelope glycoprotein or the Maraba-G variant of any one of claims 1-13.

19. The viral particle of claim 18, wherein the viral particle is selected from a retroviral particle, a lentiviral particle, or a virus-like particle (VLP); preferably, the viral particle is a lentiviral particle.

20. The viral particle of claim 18 or 19, wherein the viral particle further comprises a nucleic acid molecule carrying a gene of interest.

21. The viral particle of claim 20, wherein the gene of interest encodes a molecule selected from any one or a combination of: an siRNA, an shRNA, a non-coding RNA (e.g., a guide RNA of a CRISPR system), a polypeptide, and a protein.

22. The viral particle of claim 21, wherein the gene of interest encodes a chimeric antigen receptor (CAR).

23. The viral particle of any one of claims 18-22, further comprising one or more cell targeting molecules.

24. The viral particle of claim 23, wherein the cell targeting molecule is capable of binding to one or more cells selected from: a normal tissue cell, an immune cell, a tumor cell, and a stem cell.

25. The viral particle of claim 24, wherein the immune cell is selected from one or more of the following: a T cell, a B cell, an NK cell, a dendritic cell, a macrophage, an NKT cell, and a peripheral blood mononuclear cell (PBMC); preferably, the immune cell is a T cell.

26. The viral particle of claim 25, wherein the cell-targeting molecule is a T cell- targeting molecule selected from a ligand, a receptor, or an antibody that binds to a molecule specifically expressed on the surface of a T cell.

27. The viral particle of claim 26, wherein the molecule specifically expressed on the surface of a T cell is selected from one or more of the following: CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD30, CD39, CD40, CD44, CD45, CD45RA, CD45RO, CD62L, CD70, CD73, OX40, 4-1BB, ICOS, IL2RB, IL7R, IL12R, IL18R, IL21R, LFA-1, VLA-4, PSGL-1, CXCR2, CXCR3, CXCR4, CCR5, CCR7, CCR4, CCR8, and TCR; preferably, the molecule specifically expressed on the surface of a T cell is selected from one or more of the following: CD3, TCR, CD7, LFA-1, and CD28.

28. The viral particle of claim 27, wherein the T cell-targeting molecule is selected from one or more of the following: a CD3 antibody, a TCRa / β antibody, a TCRy / d antibody, a CD7 antibody, a LFA-1 antibody, a LFA-1 ligand (e.g., ICAM1), and a CD28 ligand (e.g., CD80 and / or CD86).

29. The viral particle of claim 28, wherein the T cell-targeting molecule comprises a combination selected from the following: (1) a CD3 antibody + a CD7 antibody; (2) a CD3 antibody + a CD7 antibody + ICAM1; (3) a CD3 antibody + a CD7 antibody + a LFA-1 antibody; and (4) a combination in which the CD3 antibody in the foregoing combination is replaced with a TCRa / β antibody or a TCRy / d antibody.

30. The viral particle of claim 28, wherein the T cell-targeting molecule comprises a combination selected from the following: (1) a CD3 antibody + CD80 + ICAM-1; (2) a CD3 antibody + CD80 + a LFA-1 antibody; (3) a CD3 antibody + CD86 + ICAM-1; and (4) a CD3 antibody + CD86 + a LFA-1 antibody.

31. A composition comprising the viral particle of any one of claims 18-30; optionally, the composition further comprises a pharmaceutically acceptable carrier.

32. A kit comprising the viral particle of any one of claims 18-30 and / or a pharmaceutically acceptable carrier, and instructions for use in transducing target cells in vitro or in vivo.

33. Use of a wild-type Maraba-G envelope glycoprotein or a Maraba-G variant of any one of claims 1-13 in the manufacture of a pseudotyped virus.

34. The use of claim 33, wherein the pseudotyped virus is a pseudotyped lentivirus.

35. The use of claim 33 or 34, wherein the pseudotyped virus is capable of specifically transducing a target cell in a subject; preferably, the target cell is selected from one or more of the following: a T cell, a B cell, a NK cell, a dendritic cell, a macrophage, a NKT cell, and a peripheral blood mononuclear cell (PBMC); preferably, the target cell is a T cell.

36. The use of any one of claims 33-35, wherein the pseudotyped virus further comprises one or more cell targeting molecules; preferably, the cell targeting molecule comprises a T cell targeting molecule as defined in any one of claims 26-30.

37. A method of making a target cell expressing a gene of interest in a subject, the method comprising administering to the subject a viral particle of any one of claims 18-30 or a composition of claim 31.

38. The method of claim 37, wherein the gene of interest encodes a therapeutic nucleic acid, a therapeutic polypeptide, or a therapeutic protein; preferably, the gene of interest encodes a CAR.

39. The method of claim 37 or 38, wherein the target cell is selected from one or more of the following: a T cell, a B cell, a NK cell, a dendritic cell, a macrophage, a NKT cell, and a peripheral blood mononuclear cell (PBMC); preferably, the target cell is a T cell.

40. A method of treating and / or preventing a disease in a subject, the method comprising administering to the subject a viral particle of any one of claims 18-30 or a composition of claim 31.

41. The method of claim 40, wherein the disease is a cancer.

42. The method of claim 41, wherein the cancer is selected from the group consisting of an immune system cancer, a breast cancer, a lung cancer, a melanoma, a bladder cancer, a gastric cancer, an esophageal cancer, a colorectal cancer, a cervical cancer, an ovarian cancer, an endometrial cancer, a kidney cancer, a brain cancer, a small intestine cancer, a liver cancer, a pancreatic cancer, a hepatobiliary tumor, a germ cell cancer, a prostate cancer, a Merkel cell carcinoma, a head and neck cancer, a thyroid cancer, and a sarcoma.

43. The method of claim 42, wherein the immune system cancer is selected from the group consisting of a lymphoma, a leukemia, and a myeloma, such as multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma, primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, B-lymphoblastic leukemia (B-ALL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies.

44. The method of any one of claims 37-43, wherein the viral particle is administered by intraperitoneal injection, subcutaneous injection, intravenous injection, or intranodal injection.

45. The method of claim 44, wherein the intranodal is intralymph node; preferably, the lymph node is a inguinal lymph node.

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