Mutated vesicular virus envelope protein and use thereof in lentiviral vector packaging

Abstract

The present invention relates to the field of lentiviral delivery. Specifically provided are a mutated vesicular virus envelope protein and the use thereof in lentiviral vector packaging.

Description

Mutated vesicular stomatitis virus envelope proteins and their use in lentivirus vector packaging

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. 202410702731.4, filed May 31, 2024, the entire contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present application relates to the field of lentivirus delivery, and specifically provides mutated vesicular stomatitis virus envelope proteins and their use in lentivirus vector packaging. BACKGROUND

[0004] In the field of drug delivery, traditional targeted delivery vectors include adenovirus liposome (LNP), Adenovirus (Adv) vector, adeno-associated virus (AAV) vector, lentivirus vector, etc. Among them, the delivery efficiency of lentivirus vector is the highest, and it can integrate the coding sequence of the target gene into the target cell genome and achieve persistent expression in the infected cells and their progeny cells.

[0005] The commonly used lentiviral vector uses vesicular stomatitis virus-derived envelope protein VSV-G as the viral envelope. Vesicular stomatitis virus (VSV) is a non-pathogenic, enveloped, negative-strand RNA virus belonging to the Vesiculovirus genus of the Rhabdoviridae family. It is a mosquito-borne virus that can infect insects, cattle, horses, and pigs, and its glycoprotein G is widely used for viral envelope proteins in gene therapy, including lentiviral vectors. Low-density lipoprotein receptor (LDL-R) is the main entry receptor of VSV. Jovan Nikolic et al. (Nikolic, J. et al. Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein. Nature communications 9, 1029, doi:10.1038 / s41467-018-03432-4 (2018)) reported the crystal structure of VSV G with two different cysteine-rich domains (CR2 and CR3) of LDL-R, showing that their binding sites on G are the same. At the same time, they identified two basic residues lysine K47 and arginine R354 on VSV G, which are essential for the interaction of VSV G with CR2 and CR3 of LDL R, and mutation of these residues will make VSV lose the ability to infect (PCT / EP2018 / 075824). Connor S. Dobson et al. (Dobson, C. S. et al. Antigen identification and high-throughput interaction mapping by reprogramming viral entry. Nature methods 19, 449-460, doi:10.1038 / s41592-022-01436-z (2022)) reported that the co-expression of the above-mentioned mutant VSV G with antibodies, receptors or ligands targeting specific cell surface antigen proteins on the viral membrane surface can achieve cell-specific infection of lentiviral vectors.

[0006] There is still an unmet need in the art for lentiviral vectors with cell-specific infection ability. SUMMARY

[0007] In the present application, the inventors have used structural biology knowledge and through extensive mutation screening, identified novel key amino sites in the VSV G polypeptide sequence that affect lentiviral infection of host cells, and provided lentiviral vectors with cell-specific infection ability. The following aspects are thus provided.

[0008] Mutated vesiculovirus envelope protein

[0009] In one aspect, the present application provides a mutated vesiculovirus envelope protein comprising an amino acid mutation (e.g., a substitution, insertion, or deletion) in at least one (e.g., 1, 2, 3, 4, or 5) of the amino acid positions corresponding to positions 50, 331, 347, 184, 354 with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22.

[0010] In the present context, "an amino acid position corresponding to a given position" means the equivalent position in the sequence under comparison when the sequences are optimally aligned, i.e. when the sequences are aligned to obtain the highest percentage identity. Thus, the corresponding amino acid position can be identified by aligning (e.g. to obtain the highest percentage identity) the target vesiculovirus envelope protein sequence with the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 22. For example, the expression "comprises a substitution in the position corresponding to position 50 with reference to the amino acid residue positions set forth in SEQ ID NO: 3" encompasses an amino acid substitution in the position 50 in the amino acid sequence set forth in SEQ ID NO: 3 or a substitution in the corresponding position in another vesiculovirus envelope protein sequence. For example, the expression "comprises a substitution in the position corresponding to position 50 with reference to the amino acid residue positions set forth in SEQ ID NO: 22" encompasses an amino acid substitution in the position 50 in the amino acid sequence set forth in SEQ ID NO: 22 or a substitution in the corresponding position in another vesiculovirus envelope protein sequence.

[0011] I. Substitution mutations

[0012] In certain embodiments, the amino acid mutation is an amino acid substitution. In certain embodiments, the amino acid mutation is a substitution to an acidic amino acid (e.g., E or D).

[0013] In certain embodiments, the mutated vesiculovirus envelope protein provided by the present application comprises an amino acid substitution in at least one (e.g., 1, 2, 3, or 4) of the amino acid positions corresponding to positions 50, 331, 347, 184 with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22.

[0014] In certain embodiments, the mutated vesicular virus envelope protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from substitution of the amino acid residue at a position corresponding to position 50 with E or D, substitution of the amino acid residue at a position corresponding to position 331 with E or D, substitution of the amino acid residue at a position corresponding to position 347 with E or D, substitution of the amino acid residue at a position corresponding to position 184 with E or D.

[0015] In certain embodiments, the mutated vesicular virus envelope protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from substitution of the amino acid residue at a position corresponding to position 50 with E or D, substitution of the amino acid residue at a position corresponding to position 331 with E or D, substitution of the amino acid residue at a position corresponding to position 347 with E or D, substitution of the amino acid residue at a position corresponding to position 184 with E or D.

[0016] In certain embodiments, the mutated vesicular virus envelope protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from substitution of the amino acid residue at a position corresponding to position 50 with E or D, substitution of the amino acid residue at a position corresponding to position 331 with E or D, substitution of the amino acid residue at a position corresponding to position 347 with E or D, substitution of the amino acid residue at a position corresponding to position 184 with E or D.

[0017] II. Insertion mutations

[0018] In certain embodiments, the amino acid mutation is an amino acid insertion. In this context, the expression "comprises an amino acid insertion at a specified amino acid position" means that an additional amino acid is inserted before that position (i.e., between that position and the preceding position).

[0019] In certain embodiments, the amino acid insertion is an insertion of 1, 2, or 3 contiguous amino acids.

[0020] In certain embodiments, the inserted amino acid is selected from E (Glu), I (Ile), A (Ala), AA (Ala-Ala), GAA (Gly-Ala-Ala), or P (Pro).

[0021] In certain embodiments, the mutated vesicular virus envelope protein of the application comprises an E (Glu) insertion, an I (Ile) insertion, an A (Ala) insertion, an AA (Ala-Ala) insertion, or a GAA (Gly-Ala-Ala) insertion between positions 183 and 184 corresponding to SEQ ID NO: 3 or SEQ ID NO: 22.

[0022] In certain embodiments, the mutated vesicular virus envelope protein of the application comprises an I(Ile) insertion between positions corresponding to positions 183 and 184 of SEQ ID NO: 3 or SEQ ID NO: 22, or an A(Ala) insertion or a P(Pro) insertion between positions corresponding to positions 353 and 354 of SEQ ID NO: 3 or SEQ ID NO: 22.

[0023] In certain embodiments, the mutated vesicular virus envelope protein of the application comprises an I(Ile) insertion between positions corresponding to positions 183 and 184 of SEQ ID NO: 3 or SEQ ID NO: 22, or an A(Ala) insertion or a P(Pro) insertion between positions corresponding to positions 353 and 354 of SEQ ID NO: 3 or SEQ ID NO: 22.

[0024] In certain embodiments, the mutated vesicular virus envelope protein of any of the above embodiments is selected from a vesicular stomatitis virus (VSV) G protein, a Cocal virus G protein, a Maraba virus G protein, a Morreton virus G protein, an Alagoa virus G protein, or a Carajas virus G protein.

[0025] Mutated VSV G protein

[0026] In certain embodiments, the mutated vesicular virus envelope protein of the application is a mutated VSV G protein. In certain embodiments, the wild-type vesicular virus envelope protein from which the mutated vesicular virus envelope protein of the application is derived is a VSV G protein.

[0027] In certain embodiments, the mutated VSV G protein comprises an amino acid substitution at at least one (e.g., 1, 2, 3, or 4) of the amino acid positions corresponding to positions 50, 331, 347, 184, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22, as compared to the corresponding wild-type VSV G protein.

[0028] In certain embodiments, the positions 50, 331, 347, 184 with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22, respectively, correspond to positions 50, 331, 347, 184 of a wild-type VSV Indiana strain G protein (e.g., SEQ ID NO: 34).

[0029] In certain embodiments, the reference is to amino acid position 50, 331, 347, 184 of SEQ ID NO: 3 or SEQ ID NO: 22, which correspond to amino acid position 50, 335, 351, 184, respectively, of the wild-type VSV New Jersey strain G protein (e.g., SEQ ID NO: 38).

[0030] In certain embodiments, the mutated VSV G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from substitution of the amino acid residue at a position corresponding to position 50 with E or D, substitution of the amino acid residue at a position corresponding to position 331 with E or D, substitution of the amino acid residue at a position corresponding to position 347 with E or D, and substitution of the amino acid residue at a position corresponding to position 184 with E or D, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22.

[0031] In certain embodiments, the mutated VSV G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from substitution of the amino acid residue at a position corresponding to position 50 with E or D, substitution of the amino acid residue at a position corresponding to position 331 with E or D, substitution of the amino acid residue at a position corresponding to position 347 with E or D, and substitution of the amino acid residue at a position corresponding to position 184 with E or D, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22.

[0032] In certain embodiments, the mutated VSV G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from substitution of the amino acid residue at a position corresponding to position 50 with E or D, substitution of the amino acid residue at a position corresponding to position 331 with E or D, substitution of the amino acid residue at a position corresponding to position 347 with E or D, and substitution of the amino acid residue at a position corresponding to position 184 with E or D, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22.

[0033] In certain embodiments, the wild-type VSV G protein has the sequence set forth in any one of SEQ ID NOs: 3, 34, 38 or a sequence that is at least 80% (e.g., at least 85%, 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 99%) identical thereto.

[0034] In certain embodiments, the mutated VSV G protein comprises at least one (e.g., 1, 2, 3, or 4) substitution at a position selected from the group consisting of K50, I331, I347, M184, compared to the sequence of the wild-type VSV G protein set forth in SEQ ID NO: 3.

[0035] In certain embodiments, the mutated VSV G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of K50E, I331E, I347E, M184D, compared to the sequence of the wild-type VSV G protein set forth in SEQ ID NO: 3.

[0036] In certain embodiments, the mutated VSV G protein comprises at least one (e.g., 1, 2, 3, or 4) substitution at a position selected from the group consisting of K50, I331, I347, M184, compared to the sequence of the wild-type VSV G protein set forth in SEQ ID NO: 34.

[0037] In certain embodiments, the mutated VSV G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of K50E, I331E, I347E, M184D, compared to the sequence of the wild-type VSV G protein set forth in SEQ ID NO: 34.

[0038] In certain embodiments, the mutated VSV G protein comprises at least one (e.g., 1, 2, 3, or 4) substitution at a position selected from the group consisting of T50, L335, V351, V184, compared to the sequence of the wild-type VSV G protein set forth in SEQ ID NO: 38.

[0039] In certain embodiments, the mutated VSV G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of T50E, L335E, V351E, V184D, compared to the sequence of the wild-type VSV G protein set forth in SEQ ID NO: 38.

[0040] In certain embodiments, the mutated VSV G protein has a sequence selected from the group consisting of the sequence set forth in any one of SEQ ID NOs: 5-8, a sequence comprising one mutation selected from K50E, I331E, I347E, M184D compared to the sequence set forth in SEQ ID NO: 34, or a sequence comprising one mutation selected from T50E, L335E, V351E, V184D compared to the sequence set forth in SEQ ID NO: 38.

[0041] In certain embodiments, the mutated VSV G protein comprises an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g., an I insertion) between positions 183 and 184, or an A insertion or a P insertion between positions 353 and 354, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22, as compared to the corresponding wild-type VSV G protein.

[0042] In certain embodiments, the positions 183, 184, 353, 354, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22, correspond to positions 183, 184, 353, 354, respectively, of the wild-type VSV Indiana strain G protein (e.g., SEQ ID NO: 34).

[0043] In certain embodiments, the positions 183, 184, 353, 354, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22, correspond to positions 183, 184, 353, 354, respectively, of the wild-type VSV New Jersey strain G protein (e.g., SEQ ID NO: 38).

[0044] In certain embodiments, the mutated VSV G protein has a sequence selected from the group consisting of: an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g., an I insertion) between S183 and M184, or an A insertion or a P insertion between E353 and R354, as compared to the sequence set forth in SEQ ID NO: 3.

[0045] In certain embodiments, the mutated VSV G protein has a sequence selected from the group consisting of: an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g., an I insertion) between S183 and M184, or an A insertion or a P insertion between E353 and R354, as compared to the sequence set forth in SEQ ID NO: 34.

[0046] In certain embodiments, the mutated VSV G protein has a sequence selected from the group consisting of: an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g., an I insertion) between L183 and V184, or an A insertion or a P insertion between V357 and R358, as compared to the sequence set forth in SEQ ID NO: 38.

[0047] Mutated Cocal G protein

[0048] In certain embodiments, the mutated arenavirus envelope protein is a mutated Cocal G protein. In certain embodiments, the wild-type arenavirus envelope protein from which the mutated arenavirus envelope protein is derived is a Cocal G protein.

[0049] In certain embodiments, the mutated Cocal G protein comprises an amino acid substitution at at least one (e.g., 1, 2, 3, or 4) of the amino acid positions corresponding to positions 50, 331, 347, 184 with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22, as compared to the corresponding wild-type Cocal G protein.

[0050] In certain embodiments, positions 50, 331, 347, 184 with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22 correspond to positions 50, 331, 347, 184, respectively, of a wild-type Cocal G protein (e.g., SEQ ID NO: 22).

[0051] In certain embodiments, the mutated Cocal G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from substitution of the amino acid residue at a position corresponding to position 50 with E or D, substitution of the amino acid residue at a position corresponding to position 331 with E or D, substitution of the amino acid residue at a position corresponding to position 347 with E or D, substitution of the amino acid residue at a position corresponding to position 184 with E or D, as compared to the corresponding wild-type Cocal G protein.

[0052] In certain embodiments, the mutated Cocal G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from substitution of the amino acid residue at a position corresponding to position 50 with E or D, substitution of the amino acid residue at a position corresponding to position 331 with E or D, substitution of the amino acid residue at a position corresponding to position 347 with E or D, substitution of the amino acid residue at a position corresponding to position 184 with E or D, as compared to the corresponding wild-type Cocal G protein.

[0053] In certain embodiments, the mutated Cocal G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of: substitution of the amino acid residue at a position corresponding to position 50 with E, substitution of the amino acid residue at a position corresponding to position 331 with E, substitution of the amino acid residue at a position corresponding to position 347 with E, substitution of the amino acid residue at a position corresponding to position 184 with D, substitution of the amino acid residue at a position corresponding to position 353 with A, and substitution of the amino acid residue at a position corresponding to position 354 with P.

[0054] In certain embodiments, the wild-type Cocal G protein has the sequence set forth in SEQ ID NO: 22 or a sequence that has at least 80% (e.g., at least 85%, 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 99%) identity thereto.

[0055] In certain embodiments, the mutated Cocal G protein comprises at least one (e.g., 1, 2, 3, or 4) substitution at a position selected from the group consisting of: K50, I331, I347, T184, T353, and P354, as compared to the wild-type Cocal G protein sequence set forth in SEQ ID NO: 22.

[0056] In certain embodiments, the mutated Cocal G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of: K50E, I331E, I347E, T184D, T353A, and P354P, as compared to the wild-type Cocal G protein sequence set forth in SEQ ID NO: 22.

[0057] In certain embodiments, the mutated Cocal G protein has a sequence selected from the group consisting of: the sequence set forth in any one of SEQ ID NOs: 24-27.

[0058] In certain embodiments, the mutated Cocal G protein comprises an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g., an I insertion) between positions corresponding to positions 183 and 184 of SEQ ID NO: 3 or SEQ ID NO: 22, or an A insertion or a P insertion between positions corresponding to positions 353 and 354 of SEQ ID NO: 3 or SEQ ID NO: 22, as compared to the corresponding wild-type Cocal G protein.

[0059] In certain embodiments, the reference to amino acid position 183, 184, 353, 354 of SEQ ID NO: 3 or SEQ ID NO: 22 corresponds to amino acid position 183, 184, 353, 354, respectively, of wild-type Cocal G protein (e.g., SEQ ID NO: 22).

[0060] In certain embodiments, the mutated Cocal G protein has a sequence set forth in any one of SEQ ID NOs: 41-47. In certain embodiments, the mutated Cocal G protein has a sequence set forth in any one of SEQ ID NOs: 42, 46, 47.

[0061] Mutated Maraba G protein

[0062] In certain embodiments, the mutated water bubble virus envelope protein is a mutated Maraba virus G protein. In certain embodiments, the wild-type water bubble virus envelope protein from which the mutated water bubble virus envelope protein is derived is a Maraba G protein.

[0063] In certain embodiments, the mutated Maraba G protein comprises an amino acid substitution at at least one (e.g., 1, 2, 3, or 4) of the amino acid positions corresponding to positions 50, 331, 347, 184, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22, as compared to the corresponding wild-type Maraba G protein.

[0064] In certain embodiments, the reference to amino acid position 50, 331, 347, 184 of SEQ ID NO: 3 or SEQ ID NO: 22 corresponds to amino acid position 50, 331, 347, 184, respectively, of wild-type Maraba G protein (e.g., SEQ ID NO: 35).

[0065] In certain embodiments, the mutated Maraba G protein comprises an amino acid residue at at least one (e.g., 1, 2, 3, or 4) of the amino acid positions corresponding to positions 50, 331, 347, 184, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22, is substituted to an acidic amino acid (e.g., E or D), as compared to the corresponding wild-type Maraba G protein.

[0066] In certain embodiments, the mutated Maraba G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of: substitution of the amino acid residue at a position corresponding to position 50 with E or D, substitution of the amino acid residue at a position corresponding to position 331 with E or D, substitution of the amino acid residue at a position corresponding to position 347 with E or D, substitution of the amino acid residue at a position corresponding to position 184 with E or D, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22.

[0067] In certain embodiments, the mutated Maraba G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of: substitution of the amino acid residue at a position corresponding to position 50 with E, substitution of the amino acid residue at a position corresponding to position 331 with E, substitution of the amino acid residue at a position corresponding to position 347 with E, substitution of the amino acid residue at a position corresponding to position 184 with D, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22.

[0068] In certain embodiments, the wild-type Maraba G protein has the sequence set forth in SEQ ID NO: 35 or a sequence that is at least 80% (e.g., at least 85%, 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 99%) identical thereto.

[0069] In certain embodiments, the mutated Maraba G protein comprises at least one (e.g., 1, 2, 3, or 4) substitution at a position selected from the group consisting of: K50, I331, M347, V184, as compared to the wild-type Maraba G protein sequence set forth in SEQ ID NO: 35.

[0070] In certain embodiments, the mutated Maraba G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of: K50E, I331E, M347E, V184D, as compared to the wild-type Maraba G protein sequence set forth in SEQ ID NO: 35.

[0071] In certain embodiments, the mutated Maraba G protein has a sequence selected from the group consisting of: a sequence comprising one mutation selected from the group consisting of K50E, I331E, M347E, V184D, as compared to the sequence set forth in SEQ ID NO: 35.

[0072] In certain embodiments, the mutated Maraba G protein comprises an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g., an I insertion) between positions 183 and 184, or an A insertion or a P insertion between positions 353 and 354, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22, as compared to the corresponding wild-type Maraba G protein.

[0073] In certain embodiments, the positions 183, 184, 353, 354, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22, correspond to positions 183, 184, 353, 354, respectively, of a wild-type Maraba G protein (e.g., SEQ ID NO: 35).

[0074] In certain embodiments, the mutated Maraba G protein has a sequence selected from the group consisting of: an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g., an I insertion) between S183 and V184, or an A insertion or a P insertion between E353 and R354, as compared to the sequence set forth in SEQ ID NO: 35.

[0075] Mutated Morreton G protein

[0076] In certain embodiments, the mutated vesicular virus envelope protein is a mutated Morreton virus G protein. In certain embodiments, the wild-type vesicular virus envelope protein from which the mutated vesicular virus envelope protein is derived is a Morreton G protein.

[0077] In certain embodiments, the mutated Morreton G protein comprises an amino acid substitution at at least one (e.g., 1, 2, 3, or 4) of the amino acid positions corresponding to positions 50, 331, 347, 184, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22, as compared to the corresponding wild-type Morreton G protein.

[0078] In certain embodiments, the positions 50, 331, 347, 184, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22, correspond to positions 50, 331, 347, 184, respectively, of a wild-type Morreton G protein (e.g., SEQ ID NO: 36).

[0079] In some embodiments, the mutant Morreton G protein, compared to the corresponding wild-type Morreton G protein, has at least one (e.g., position 1, 2, 3, or 4) amino acid residue replaced with an acidic amino acid (e.g., E or D) at positions corresponding to the amino acid positions shown in SEQ ID NO:3 or SEQ ID NO:22: position 50, position 331, position 347, and position 184.

[0080] In some embodiments, the mutated Morreton G protein, compared to the corresponding wild-type Morreton G protein, contains at least one (e.g., 1, 2, 3, or 4) mutations selected from the following positions, referring to the amino acid positions shown in SEQ ID NO:3 or SEQ ID NO:22: an amino acid residue at position 50 is replaced with E or D, an amino acid residue at position 331 is replaced with E or D, an amino acid residue at position 347 is replaced with E or D, and an amino acid residue at position 184 is replaced with E or D.

[0081] In some embodiments, the mutated Morreton G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the following positions, referring to the amino acid positions shown in SEQ ID NO:3 or SEQ ID NO:22: an amino acid residue at position 50 is replaced with E, an amino acid residue at position 331 is replaced with E, an amino acid residue at position 347 is replaced with E, and an amino acid residue at position 184 is replaced with D.

[0082] In some embodiments, the wild-type Morreton G protein has the sequence shown in SEQ ID NO:36 or a sequence that has at least 80% (e.g., at least 85%, 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 99%) identity with it.

[0083] In some embodiments, the mutated Morreton G protein, compared to the wild-type Morreton G protein sequence shown in SEQ ID NO:36, contains at least one (e.g., 1, 2, 3, or 4) substitutions at positions selected from the following: K50, I331, I347, M184.

[0084] In certain embodiments, the mutated Morreton G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from K50E, I331E, I347E, M184D, compared to the wild-type Morreton G protein sequence set forth in SEQ ID NO: 36.

[0085] In certain embodiments, the mutated Morreton G protein has a sequence selected from a sequence comprising one mutation selected from K50E, I331E, I347E, M184D, compared to the sequence set forth in SEQ ID NO: 36.

[0086] In certain embodiments, the mutated Morreton G protein comprises an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g., an I insertion) between positions 183 and 184, or an A insertion or a P insertion between positions 353 and 354, corresponding to SEQ ID NO: 3 or SEQ ID NO: 22, compared to the corresponding wild-type Morreton G protein.

[0087] In certain embodiments, the positions 183, 184, 353, 354, corresponding to SEQ ID NO: 3 or SEQ ID NO: 22, respectively, correspond to positions 183, 184, 353, 354, respectively, of a wild-type Morreton G protein (e.g., SEQ ID NO: 36).

[0088] In certain embodiments, the mutated Morreton G protein has a sequence selected from a sequence comprising an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g., an I insertion) between S183 and M184, or an A insertion or a P insertion between E353 and R354, compared to the sequence set forth in SEQ ID NO: 36.

[0089] Mutated Alagoa G protein

[0090] In certain embodiments, the mutated vesiculovirus envelope protein is a mutated Alagoa virus G protein. In certain embodiments, the wild-type vesiculovirus envelope protein from which the mutated vesiculovirus envelope protein is derived is an Alagoa G protein.

[0091] In certain embodiments, the mutated Alagoa G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of: substitution of the amino acid residue at the position corresponding to position 50 with E or D, substitution of the amino acid residue at the position corresponding to position 331 with E or D, substitution of the amino acid residue at the position corresponding to position 347 with E or D, and substitution of the amino acid residue at the position corresponding to position 184 with E or D, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22.

[0092] In certain embodiments, the positions 50, 331, 347, and 184 with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22 correspond to positions 50, 331, 347, and 184, respectively, of a wild-type Alagoa G protein (e.g., SEQ ID NO: 37).

[0093] In certain embodiments, the mutated Alagoa G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of: substitution of the amino acid residue at the position corresponding to position 50 with E or D, substitution of the amino acid residue at the position corresponding to position 331 with E or D, substitution of the amino acid residue at the position corresponding to position 347 with E or D, and substitution of the amino acid residue at the position corresponding to position 184 with E or D, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22.

[0094] In certain embodiments, the mutated Alagoa G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of: substitution of the amino acid residue at the position corresponding to position 50 with E or D, substitution of the amino acid residue at the position corresponding to position 331 with E or D, substitution of the amino acid residue at the position corresponding to position 347 with E or D, and substitution of the amino acid residue at the position corresponding to position 184 with E or D, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22.

[0095] In certain embodiments, the mutated Alagoa G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of: substitution of the amino acid residue at the position corresponding to position 50 with E or D, substitution of the amino acid residue at the position corresponding to position 331 with E or D, substitution of the amino acid residue at the position corresponding to position 347 with E or D, and substitution of the amino acid residue at the position corresponding to position 184 with E or D, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22.

[0096] In certain embodiments, the wild-type Alagoa G protein has the sequence set forth in SEQ ID NO: 37 or a sequence at least 80% (e.g., at least 85%, 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 99%) identical thereto.

[0097] In certain embodiments, the mutated Alagoa G protein comprises at least one (e.g., 1, 2, 3, or 4) substitution at a position selected from the group consisting of: K50, L331, V347, T184, as compared to the wild-type Alagoa G protein sequence set forth in SEQ ID NO: 37.

[0098] In certain embodiments, the mutated Alagoa G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of: K50E, L331E, V347E, T184D, as compared to the wild-type Alagoa G protein sequence set forth in SEQ ID NO: 37.

[0099] In certain embodiments, the mutated Alagoa G protein has a sequence selected from the group consisting of: a sequence comprising one mutation selected from the group consisting of K50E, L331E, V347E, T184D, as compared to the sequence set forth in SEQ ID NO: 37.

[0100] In certain embodiments, the mutated Alagoa G protein comprises an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g., an I insertion) between positions corresponding to positions 183 and 184 of SEQ ID NO: 3 or SEQ ID NO: 22, or an A insertion or a P insertion between positions corresponding to positions 353 and 354 of SEQ ID NO: 3, as compared to the corresponding wild-type Alagoa G protein.

[0101] In certain embodiments, the positions 183, 184, 353, 354, respectively, of the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22 correspond to positions 183, 184, 353, 354, respectively, of a wild-type Alagoa G protein (e.g., SEQ ID NO: 37).

[0102] In certain embodiments, the mutated Alagoa G protein has a sequence comprising an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion between S183 and T184 (e.g., an I insertion), or an A insertion or a P insertion between K353 and R354, selected from the group consisting of: SEQ ID NO: 37.

[0103] Mutated Carajas G protein

[0104] In certain embodiments, the mutated water bubble virus envelope protein is a mutated Carajas virus G protein. In certain embodiments, the wild-type water bubble virus envelope protein from which the mutated water bubble virus envelope protein is derived is a Carajas G protein.

[0105] In certain embodiments, the mutated Carajas G protein comprises an amino acid substitution at at least one (e.g., 1, 2, 3, or 4) of the amino acid positions corresponding to positions 50, 331, 347, 184, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22, as compared to the corresponding wild-type Carajas G protein.

[0106] In certain embodiments, positions 50, 331, 347, 184 with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22 correspond to positions 50, 335, 351, 184, respectively, of a wild-type Carajas G protein (e.g., SEQ ID NO: 39).

[0107] In certain embodiments, the mutated Carajas G protein comprises an amino acid residue that is replaced with an acidic amino acid (e.g., E or D) at at least one (e.g., 1, 2, 3, or 4) of the amino acid positions corresponding to positions 50, 331, 347, 184, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22, as compared to the corresponding wild-type Carajas G protein.

[0108] In certain embodiments, the mutated Carajas G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of: substitution of the amino acid residue at a position corresponding to position 50 with E or D, substitution of the amino acid residue at a position corresponding to position 331 with E or D, substitution of the amino acid residue at a position corresponding to position 347 with E or D, substitution of the amino acid residue at a position corresponding to position 184 with E or D, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22.

[0109] In certain embodiments, the mutated Carajas G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of: substitution of the amino acid residue at a position corresponding to position 50 with E, substitution of the amino acid residue at a position corresponding to position 331 with E, substitution of the amino acid residue at a position corresponding to position 347 with E, substitution of the amino acid residue at a position corresponding to position 184 with D, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22.

[0110] In certain embodiments, the wild-type Carajas G protein has the sequence set forth in SEQ ID NO: 39 or a sequence that is at least 80% (e.g., at least 85%, 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 99%) identical thereto.

[0111] In certain embodiments, the mutated Carajas G protein comprises at least one (e.g., 1, 2, 3, or 4) substitution selected from the group consisting of: K50, I335, V351, M184, as compared to the wild-type Carajas G protein sequence set forth in SEQ ID NO: 39.

[0112] In certain embodiments, the mutated Carajas G protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the group consisting of: K50E, I335E, V351E, M184D, as compared to the wild-type Carajas G protein sequence set forth in SEQ ID NO: 39.

[0113] In certain embodiments, the mutated Carajas G protein has a sequence selected from the group consisting of: a sequence comprising one mutation selected from the group consisting of K50E, I335E, V351E, M184D, as compared to the sequence set forth in SEQ ID NO: 39.

[0114] In certain embodiments, the mutated Carajas G protein comprises an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g., an I insertion) between positions 183 and 184 corresponding to SEQ ID NO: 3 or SEQ ID NO: 22, or an A insertion or a P insertion between positions 353 and 354 corresponding to SEQ ID NO: 3 or SEQ ID NO: 22, as compared to the corresponding wild-type Carajas G protein.

[0115] In certain embodiments, the reference to positions 183, 184, 353, 354 of the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22 correspond to positions 183, 184, 357, 358, respectively, of a wild-type Carajas G protein (e.g., SEQ ID NO: 39).

[0116] In certain embodiments, the mutated Carajas G protein has a sequence selected from the group consisting of: an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g., an I insertion) between A183 and M184, or an A insertion or a P insertion between S357 and R358, as compared to the sequence set forth in SEQ ID NO: 39.

[0117] In another aspect, the present application provides an isolated nucleic acid molecule encoding a vesiculovirus envelope protein as described herein.

[0118] In another aspect, the present application provides a vector (e.g., an expression vector) comprising an isolated nucleic acid molecule as described above.

[0119] Lentiviral vector

[0120] In another aspect, the present application provides a lentiviral vector comprising an envelope and a viral genome, wherein the envelope comprises a mutated vesiculovirus envelope protein as described herein. In certain embodiments, the lentiviral vector of the present application is pseudotyped by a mutated vesiculovirus envelope protein as described herein.

[0121] Envelope

[0122] The lentiviral vector defined in the present application is a pseudotyped lentiviral vector packaged by a vector particle carrying an envelope protein derived from a virus different from the particular lentivirus providing the lentiviral vector genome, and is also referred to as a lentiviral vector particle. The envelope protein is derived from a virus different from the particular lentivirus providing the lentiviral vector genome.

[0123] In certain embodiments, the lentiviral vector further comprises a non-viral membrane binding protein. In certain embodiments, the envelope of the lentiviral vector further comprises a non-viral membrane binding protein.

[0124] In certain embodiments, the non-viral membrane-bound protein comprises an extracellular targeting domain and a membrane-binding domain.

[0125] In certain embodiments, the extracellular targeting domain comprises a specific binding domain for a target cell. In certain embodiments, the extracellular targeting domain specifically binds to an antigen or receptor on the surface of a target cell. In certain embodiments, the binding interaction between the extracellular targeting domain and an antigen or receptor on the surface of a cell enables entry of the lentivirus into the cell (e.g., an antigen-specific cell, such as a T cell).

[0126] In certain embodiments, the extracellular targeting domain binds to an antigen or receptor present on the surface of a cell of a single T cell or a subset of a population of T cells.

[0127] In certain embodiments, the target cell surface antigen is selected from:

[0128] (1) a T cell marker, such as selected from CD3, CD28, CD80, 4-1BB, AhR, CD3, 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, TIGIT, LAG-3, TIM-3, and IL6ST, or any combination thereof;

[0129] (2) a gd T cell marker, such as selected from gd TCR, Vd1, Vd2, and NKG2D (KLRK1, CD314);

[0130] (3) a NKT cell marker, such as selected from invariant TCR (Va24-Ja18), CD185 (CXCR5), CXCR6, and IL-21R, or any combination thereof;

[0131] (4) a MAIT cell marker, such as selected from Va7.2, Ja33, CXCR6, IL-18R, KLRB1 (CD161), and VLA4 (a4b1 integrin), or any combination thereof;

[0132] (5) a tumor cell or other cell marker, such as selected from the group consisting of TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, DLL3, or combinations thereof.

[0133] In certain embodiments, the extracellular targeting domain is an antibody or a ligand against a target cell surface antigen.

[0134] In certain embodiments, the extracellular targeting domain is an antibody against a target cell surface antigen, e.g., a scFv or a VHH.

[0135] In certain embodiments, the extracellular targeting domain is an anti-CD3 antibody. In certain exemplary embodiments, the anti-CD3 antibody comprises the VH and VL sequences of the scFv set forth in SEQ ID NO: 9, e.g., comprises the scFv set forth in SEQ ID NO: 9. In certain exemplary embodiments, the anti-CD3 antibody comprises the VH and VL sequences of the scFv set forth in SEQ ID NO: 48, e.g., comprises the scFv set forth in SEQ ID NO: 48.

[0136] In certain embodiments, the membrane binding domain is an amino acid sequence that allows the protein or peptide to be fully or partially embedded in the membrane (e.g., envelope) of the lentivirus. In certain embodiments, the membrane binding domain comprises at least a transmembrane domain. In certain embodiments, the membrane binding domain comprises an intracellular domain and a transmembrane domain.

[0137] In certain embodiments, the membrane binding domain comprises a transmembrane region of a transmembrane protein, e.g., a CD8 transmembrane region, a CD4 transmembrane region, a CD28 transmembrane region, or a NKG2D transmembrane region, etc. In certain embodiments, the membrane binding domain comprises a human CD8 transmembrane region. In certain embodiments, the membrane binding domain comprises the sequence set forth in SEQ ID NO: 12.

[0138] In certain embodiments, the non-viral membrane binding protein further comprises a hinge region between the extracellular targeting domain and the membrane binding domain. In certain embodiments, the hinge region can be a hinge region of a naturally occurring protein or a portion thereof (e.g., a fragment of at least 15, 20, 25, 30, 35, or 40 contiguous amino acids). In certain embodiments, the hinge region is a hinge region of CD8 or CD28 or a portion thereof. In certain embodiments, the hinge region comprises the sequence set forth in SEQ ID NO: 11.

[0139] In certain embodiments, the non-viral membrane binding protein further comprises a signal peptide at its N-terminus. In certain embodiments, the function of the signal peptide is to translocate the non-viral membrane binding protein to the membrane (or envelope) of the lentivirus. In certain embodiments, the signal peptide is a CD8 signal peptide, a GM-CSF receptor signal peptide, an IgG signal peptide, etc. In certain embodiments, the signal peptide is a human CD8 signal peptide. In certain embodiments, the signal peptide comprises the sequence set forth in SEQ ID NO: 10.

[0140] In certain exemplary embodiments, the non-viral membrane binding protein has the following structure: [signal peptide]-[extracellular targeting domain]-[hinge region]-[membrane binding domain].

[0141] In certain exemplary embodiments, the non-viral membrane binding protein comprises an amino acid sequence set forth in SEQ ID NO: 40. In certain exemplary embodiments, the non-viral membrane binding protein comprises an amino acid sequence set forth in SEQ ID NO: 49.

[0142] In certain exemplary embodiments, the C-terminus of the non-viral membrane binding protein can further comprise an additional peptide sequence (e.g., a peptide linker), such as a peptide linker comprising one or more G and / or S, such as GSG.

[0143] In certain exemplary embodiments, the envelope of the lentiviral vector is encoded by a nucleic acid molecule having the following structure: [non-viral membrane binding protein coding sequence]-[linker]-[mutated vesicular stomatitis virus envelope protein coding sequence], the linker being selected from a cleavable linker, such as a 2A peptide (e.g., T2A, P2A, E2A, or F2A) coding sequence or an IRES sequence. In certain exemplary embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding an additional peptide sequence (e.g., a peptide linker) between the [non-viral membrane binding protein coding sequence] and the [linker], such as a peptide linker comprising one or more G and / or S, such as GSG. In certain exemplary embodiments, the nucleic acid molecule comprises a nucleotide sequence selected from a nucleotide sequence encoding an amino acid sequence set forth in any one of SEQ ID NOs: 17-20, 30-33, or a degenerate sequence thereof. In certain exemplary embodiments, the nucleic acid molecule comprises a nucleotide sequence selected from a nucleotide sequence encoding an amino acid sequence set forth in any one of SEQ ID NOs: 52-58 (preferably, SEQ ID NOs: 53, 57, 58), or a degenerate sequence thereof.

[0144] Vector genome

[0145] The lentiviral vector comprises a vector genome which is essentially devoid of the original lentiviral sequences encoding lentiviral proteins, or when present, are modified and in particular prevent expression of the biologically active pol antigens as well as optionally further lentiviral structural and / or accessory and / or regulatory proteins. The biologically active pol antigens comprise the viral enzymatic proteins protease (RT), reverse transcriptase (RT and RNase H), and integrase (IN) produced by cleavage of the gag-pol polyprotein. The pol antigens are not biologically active when the biological activity of at least one of these enzymes is not activated. The vector genome further comprises a polynucleotide of interest or transgene (i.e. a nucleic acid of interest).

[0146] In certain embodiments, the polynucleotide or transgene in the vector genome lacks a functional pol gene, in particular does not contain an intact pol gene.

[0147] The vector genome as defined herein contains, in addition to the heterologous polynucleotide of interest placed under the control of appropriate regulatory sequences, lentiviral genomic sequences which are non-coding regions of the genome and which are essential to provide recognition signals for DNA or RNA synthesis and processing. These sequences are cis-acting sequences. The structure and composition of the vector genome used to prepare the lentiviral vector of the application are based on the principles in the prior art.

[0148] In certain embodiments, the vector genome comprises a polynucleotide of interest located between two long terminal repeats (LTRs). In certain embodiments, the vector genome can be a replacement vector wherein the viral protein coding sequences between the two long terminal repeats (LTRs) are replaced by the polynucleotide of interest.

[0149] In certain embodiments, the lentiviral vector genome comprises a nucleic acid of interest and flanking LTRs (5' LTR and 3' LTR).

[0150] In certain embodiments, the lentiviral vector is a replication incompetent lentiviral vector, which is a result of the fact that the gag and pol functional genes are provided absolutely in trans and thus are not present in the vector genome. In this case, when the lentiviral vector is administered to a host, it is unable to replicate in the host cell. Thus, it provides the polynucleotide of interest into the host cell for expression but does not form further lentiviral vector particles. This replication incompetent state of the lentiviral vector is achieved particularly when the lentiviral gag, pol, env genes are not provided in the vector genome or are not provided as functional genes. By "functional" it is meant that the genes are properly transcribed, and / or properly expressed. Thus, the lentiviral vector genome of the application in this embodiment contains at least one of the gag, pol and env genes that is not transcribed or is incompletely transcribed; by "incompletely transcribed" it is meant that the transcript gag, gag-pro or gag-pro-pol is altered, one or several of these are not transcribed. Other sequences involved in the replication of the lentivirus can also be mutated in the vector genome to achieve this state.

[0151] In certain embodiments, in the vector genome, the 3' LTR sequence of the lentiviral vector genome lacks at least the promoter of the activator (enhancer) and possibly the U3 region. In certain embodiments, the 3' LTR region lacks the U3 region (delta U3).

[0152] In certain embodiments, the lentiviral vector of the application is derived from HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedi virus (VMV) virus; caprine arthritis- encephalitis virus (CAEV); equine infectious anaemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); simian immunodeficiency virus (SIV) and the like.

[0153] In certain embodiments, an HIV-based vector backbone (i.e. HIV cis-acting sequence elements) is used.

[0154] In certain embodiments, the lentiviral vector particle is a human immunodeficiency virus-1 (HIV-1).

[0155] Lentiviral packaging envelope plasmid

[0156] In another aspect, the application provides one or more vectors comprising a first nucleotide sequence encoding a mutated vesiculovirus envelope protein as described herein and a second nucleotide sequence encoding a non-viral membrane-bound protein; wherein the non-viral membrane-bound protein is as defined in the preceding aspect.

[0157] In certain embodiments, the first and second nucleotide sequences are located on the same vector.

[0158] In certain embodiments, the first and second nucleotide sequences are linked by a 2A peptide (e.g., T2A, P2A, E2A, or F2A) encoding sequence or an IRES sequence. When a single promoter is used to drive the expression of multiple ORFs, each ORF separated by an internal ribosome entry site (IRES) or a 2A peptide in a single mRNA transcript will result in multiple proteins. Furthermore, one of skill in the art understands that the C-terminus of the upstream protein separated by a 2A peptide can have some additional 2A peptide residues added, and the N-terminus of the downstream protein can also have additional residues added, but this does not affect the function of the upstream and downstream proteins, respectively.

[0159] In certain embodiments, the first and second nucleotide sequences are linked by a 2A peptide (e.g., T2A, P2A, E2A, or F2A) encoding sequence. In certain exemplary embodiments, the first and second nucleotide sequences are linked by a T2A peptide (e.g., SEQ ID NO: 14) encoding sequence.

[0160] In certain exemplary embodiments, when the first and second nucleotide sequences are on the same vector, they have the following structure: [second nucleotide sequence]-[linker]-[first nucleotide sequence], the linker is selected from a cleavable linker, such as a 2A peptide (e.g., T2A, P2A, E2A, or F2A) encoding sequence or an IRES sequence. In certain exemplary embodiments, the vector comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 17-20, 30-33, or a degenerate sequence thereof. In certain exemplary embodiments, the vector comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 52-58 (preferably, SEQ ID NOs: 53, 57, 58), or a degenerate sequence thereof. In certain exemplary embodiments, the [second nucleotide sequence] and [linker] further comprise a nucleotide sequence encoding an additional peptide sequence (e.g., a peptide linker), such as a peptide linker comprising one or more G and / or S, such as GSG.

[0161] In certain embodiments, the first and second nucleotide sequences are on different vectors.

[0162] In certain embodiments, the vector is an expression vector.

[0163] In certain embodiments, the vector is used for an envelope plasmid in lentivirus vector packaging.

[0164] Packaging of lentivirus

[0165] Methods of lentivirus packaging are known in the art. Typically, the lentiviral vectors of the present application are replication-incompetent lentiviral vectors, and thus, in order to obtain a lentiviral vector of the present application, the vector genome has to be packaged into a particle or pseudoparticle. Thus, lentiviral proteins, except for the envelope protein, have to be provided in trans to the vector genome in the production system, in particular in the producer cell.

[0166] In another aspect, the present application provides a lentiviral vector packaging system comprising:

[0167] a first nucleic acid molecule encoding a mutated vesicular stomatitis virus envelope protein as described herein;

[0168] a second nucleic acid molecule encoding a non-viral membrane binding protein as described herein;

[0169] a third nucleic acid molecule encoding gag and pol;

[0170] a fourth nucleic acid molecule encoding rev; and

[0171] a fifth nucleic acid molecule, i.e. a lentiviral vector genome, comprising a nucleic acid of interest.

[0172] In certain embodiments, the first, second, third, fourth, fifth nucleic acid molecules are present on one or more expression vectors. In certain embodiments, the first, second nucleic acid molecules are present on one expression vector. In certain embodiments, the third, fourth nucleic acid molecules are present on one expression vector, or on two expression vectors, respectively. In certain embodiments, the fifth nucleic acid molecule is present on one expression vector.

[0173] In certain embodiments, the lentiviral vector packaging system comprises an envelope plasmid, a packaging plasmid, and a packaging plasmid, wherein:

[0174] the envelope plasmid comprises the first nucleic acid molecule and the second nucleic acid molecule;

[0175] the packaging plasmid comprises the third nucleic acid molecule and the fourth nucleic acid molecule; and

[0176] the transfer plasmid comprises the fifth nucleic acid molecule.

[0177] In certain embodiments, the envelope plasmid encodes only viral envelope proteins.

[0178] In certain embodiments, the packaging plasmid encodes only the lentiviral proteins necessary for viral particle synthesis. Accessory genes present in the plasmid that can lead to safety concerns are thus removed. The viral proteins brought in trans, exemplified for HIV-1, are: 1. Gag proteins for the construction of the matrix (MA, with an apparent molecular weight of pi 7), the capsid (CA, p24), and the nucleocapsid (NC, p6); 2. Pol-encoded enzymes: integrase, protease, and reverse transcriptase; 3. Tat and Rev-encoding regulatory proteins, Tat being required for initiation of LTR-mediated transcription; it can be omitted if the U3 region of the 5' LTR is replaced by a promoter that drives tat-independent transcription.

[0179] In certain embodiments, the transfer plasmid comprises a vector genome, wherein the vector genome comprises a nucleic acid of interest but lacks viral coding sequences and / or cis-acting genetic elements required for particle formation.

[0180] In certain embodiments, the vector genome comprises a 5' LTR and a 3' LTR, which is optionally deleted in the U3 region, without interfering with functions required for gene transfer.

[0181] In certain embodiments, the vector genome comprises one or more (e.g., all) of: a 5' promoter (e.g., for controlling expression of the entire packaged RNA), a 5' LTR (e.g., which includes R (polyadenylation tail signal) and / or U5 including a primer activation signal), a primer binding site, a psi packaging signal, an RRE element for nuclear export, a promoter directly upstream of the nucleic acid of interest to control expression of the nucleic acid of interest, the nucleic acid of interest, a polypurine tract, and a 3' LTR (e.g., which includes a mutated U3, R, and U5). In certain embodiments, the vector genome further comprises one or more of a cPPT, a WPRE, and / or an insulator element.

[0182] In certain embodiments, the lentiviral vectors of the application are derived from HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedi virus (VMV) virus; caprine arthritis- encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); simian immunodeficiency virus (SIV), and the like.

[0183] In certain embodiments, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is used.

[0184] In certain embodiments, the lentiviral vector is a human immunodeficiency virus-1 (HIV-1).

[0185] In another aspect, the present application provides a method of producing a lentiviral vector, comprising expressing a lentiviral vector packaging system described herein in a host cell.

[0186] In certain embodiments, the method comprises introducing into the host cell an envelope plasmid, a packaging plasmid, and a transfer plasmid comprised by the lentiviral vector packaging system. Methods of introducing vectors into host cells are well known to those skilled in the art, for example transfection, such as chemical transfection (calcium phosphate, liposomes or cationic polymers), electroporation, photoporation, and the like. The transfection can be transient or stable.

[0187] In certain embodiments, the host cell can be any prokaryotic (bacterial) or eukaryotic (yeast, insect, or animal including mammal, especially human) cell. In certain embodiments, the host cell is a mammalian cell, for example a human cell, for example an isolated human cell, "isolated" meaning outside its natural environment. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC 5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH 3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In certain embodiments, the host cell is a 293 cell, 293T cell, or A549 cell.

[0188] In certain embodiments, the method further comprises recovering viral particles from the culture of the cells.

[0189] Delivery of nucleic acids

[0190] In another aspect, the present application provides a method of delivering a nucleic acid of interest to a target cell, comprising:

[0191] (i) providing a lentiviral vector described herein;

[0192] (ii) contacting the lentiviral vector with the target cell, thereby delivering the nucleic acid of interest to the target cell.

[0193] In certain embodiments, the nucleic acid is delivered to the cell when the lentivirus enters or infects the cell during step (ii). In certain embodiments, the method does not require a transfection agent (e.g., a lipophilic transfection agent, such as Lipofectin).

[0194] In certain embodiments, the method is performed in vitro.

[0195] In certain embodiments, the method is performed in vivo, the method comprising administering to a subject a lentiviral vector described herein, thereby delivering a nucleic acid of interest to a target cell in vivo. In certain embodiments, the subject is a mammal, e.g., a human.

[0196] In certain embodiments, the method is used to transgenically modify the target cell.

[0197] In certain embodiments, the method is used to genetically edit the target cell.

[0198] In certain embodiments, the nucleic acid of interest comprises a foreign gene. The foreign gene encodes, e.g., a therapeutic protein (e.g., a protein that compensates for a disease condition of the subject) or an antigen (such as a pathogen antigen), a gene editing tool (e.g., a Cas protein and / or gRNA of a CRISPR / Cas system), or a gene silencing tool (e.g., an shRNA).

[0199] In certain embodiments, the nucleic acid of interest encodes an mRNA molecule, optionally wherein the mRNA is a foreign gene as described above.

[0200] In certain embodiments, the nucleic acid of interest encodes a double-stranded RNA, an antisense RNA, a microRNA, or any other RNA molecule.

[0201] In certain embodiments, the target cell surface comprises an antigen or receptor that can be targeted by a non-viral membrane-bound protein comprised by a lentiviral vector described herein.

[0202] In certain embodiments, the target cell can be any prokaryotic (bacterial) or eukaryotic (yeast, insect, or animal including mammal, especially human) cell. In certain embodiments, the target cell is a human, mouse, rat, or non-human primate cell. In certain embodiments, the target cell is a somatic or germ cell. In certain embodiments, the target cell is an epithelial cell, a neural cell, a hormone secreting cell, an immune cell, a secretory cell, a blood cell, an interstitial cell, or a germ cell. In certain embodiments, the target cell is an antigen-specific cell (e.g., a cell that binds a particular antigen). In certain embodiments, the antigen-specific cell is an immune cell. In certain embodiments, the antigen-specific cell is a B cell or a T cell.

[0203] In certain embodiments, the target cell is a cell used for cell therapy.

[0204] Therapeutic applications

[0205] In another aspect, the present application provides a pharmaceutical composition comprising a lentiviral vector described herein, or a target cell obtained from a method described herein for delivering a nucleic acid of interest to a target cell, and a pharmaceutically acceptable carrier and / or excipient.

[0206] In certain embodiments, the pharmaceutical composition comprises an effective amount (e.g., a therapeutically or prophylactically effective amount) of the lentiviral vector or target cell.

[0207] In certain embodiments, the nucleic acid of interest comprises a foreign gene.

[0208] In certain embodiments, the foreign gene encodes a therapeutic protein (e.g., a protein that compensates for a disease condition of the subject) or an antigen (e.g., a pathogen antigen), for example.

[0209] In certain embodiments, the foreign gene encodes a gene editing tool (e.g., a Cas protein and / or a gRNA of a CRISPR / Cas system) or a gene silencing tool (e.g., an shRNA).

[0210] In certain embodiments, the pharmaceutical composition is a nucleic acid vaccine (e.g., an mRNA vaccine), the lentiviral vector carries a nucleic acid of interest that is an antigen, e.g., a pathogen antigen.

[0211] In another aspect, the present application provides a method for gene editing or gene therapy, comprising administering to a subject in need thereof an effective amount of a lentiviral vector described herein or a target cell obtained from a method described herein for delivering a nucleic acid of interest.

[0212] In certain embodiments, the nucleic acid of interest comprises a foreign gene.

[0213] In certain embodiments, the foreign gene encodes a therapeutic protein (e.g., a protein that compensates for a disease condition of the subject) or an antigen (e.g., a pathogen antigen), for example.

[0214] In certain embodiments, the foreign gene encodes a gene editing tool (e.g., a Cas protein and / or a gRNA of a CRISPR / Cas system) or a gene silencing tool (e.g., an shRNA).

[0215] In certain embodiments, the subject is a mammal, e.g., a human.

[0216] In another aspect, the present application provides use of a lentiviral vector described herein, a lentiviral vector packaging system, or a target cell obtained from a method described herein for delivering a nucleic acid of interest, in the manufacture of a medicament for gene editing or gene therapy, or for gene editing or gene therapy.

[0217] In another aspect, the present application provides a method for inducing an immune response in a subject (e.g., a human), comprising administering to a subject in need thereof an effective amount of a lentiviral vector or a pharmaceutical composition described herein. In certain embodiments, the target nucleic acid carried by the lentiviral vector is an antigen, e.g., a pathogen antigen. In certain embodiments, the subject is administered a pharmaceutical composition described herein, wherein the pharmaceutical composition is a nucleic acid vaccine (e.g., an mRNA vaccine). In certain embodiments, the subject is a mammal, e.g., a human.

[0218] In another aspect, the present application provides use of a lentiviral vector or a lentiviral vector packaging system described herein in the manufacture of, or as a nucleic acid vaccine (e.g., an mRNA vaccine). In certain embodiments, the target nucleic acid carried by the lentiviral vector is an antigen, e.g., a pathogen antigen.

[0219] Definitions of terms

[0220] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. For better understanding of the present application, the definitions and explanations of relevant terms are provided below.

[0221] When the terms “for example,” “e.g.,” “such as,” “including,” “containing,” “for instance,” or “including” are used in this disclosure, they are to be interpreted as “by way of illustration,” only and not as an exhaustive list of the items being recited.

[0222] The terms “a” and “an” and “the” and similar referents in the context of describing the application (especially in the context of the following claims) are to be interpreted in an inclusive rather than an exclusive manner, unless otherwise indicated or clearly contradicted by context.

[0223] The term “corresponding to” with respect to the position of a protein / polypeptide, such as recitation of a nucleotide or amino acid position “corresponding to” a nucleotide or amino acid position in a disclosed sequence (such as shown in the sequence listing), refers to the nucleotide or amino acid position identified upon alignment with the disclosed sequence based on a structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm. For example, the corresponding residues of a similar sequence (e.g., a fragment or a species variant) can be determined by aligning the reference sequence using a structural alignment method. By aligning the sequences, one of skill in the art can identify the corresponding residues, e.g., using conserved and identical amino acid residues as guides.

[0224] As used herein, the term "identity" is used in reference to the match between sequences of two polypeptides or two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = # of identical positions / total # of positions x 100%). In certain embodiments, the two sequences are the same length.

[0225] Determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated in the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0226] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, and directs the expression of elements of genetic material it carries in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can contain a replication origin.

[0227] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0228] As used herein, the term "vector genome" refers to a nucleic acid, i.e., a nucleic acid of lentiviral origin, which constitutes the genome of a lentiviral vector particle. Thus, the term relates to any suitable nucleic acid, i.e., DNA or RNA, double-stranded or single-stranded, including forms containing DNA flap, as a triplex sequence. The nature of the nucleic acid (DNA, RNA) and its organization depend on the stage of the particle cycle, including the transfer plasmid (for co-transfecting cells with packaging and envelope plasmids to express viral particles), or the RNA genome of the viral particle, or various forms of this nucleic acid in the transduced cells of the host to which the viral particle is administered (including genomic mRNA transcripts, linear unintegrated DNA reverse transcripts, or unintegrated one or two LTR DNA circular forms or integrated provirus), including the vector integration pre-complex.

[0229] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are compatible, in pharmacology and / or physiology, with the subject and the active ingredient, which are well known in the art, and include but are not limited to: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents include but are not limited to phosphate buffer. Surfactants include but are not limited to cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include but are not limited to sodium chloride. Preservatives include but are not limited to various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Agents to maintain osmotic pressure include but are not limited to sugars, NaCl and the like. Agents to delay absorption include but are not limited to monostearate and gelatin. Diluents include but are not limited to water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc.

[0230] As used herein, the term "prevention" refers to a method undertaken in order to stop or delay the occurrence of a disease or disorder or symptoms thereof in a subject. As used herein, the term "treatment" refers to a method undertaken in order to obtain a beneficial or desired clinical result. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0231] As used herein, the term "effective amount" means an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease means an amount that is sufficient to prevent, stop, or delay the occurrence of the disease; an effective amount for treating a disease means an amount that is sufficient to cure or at least partially arrest the disease and its complications in a patient already having the disease. Determining such effective amounts is well within the capability of those skilled in the art. For example, an amount effective for therapeutic purposes will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight, and gender, the mode of administration of the drug, and other therapies that the patient may be undergoing, etc.

[0232] Advantages of the invention

[0233] The present invention identifies key amino acid sites that affect lentiviral targeted infection, mutation of which can significantly inhibit lentiviral infection of target cells, while at the same time utilizing a redirected targeting molecule (e.g. antibody) to achieve specific cell targeted infection of lentivirus with mutant membrane protein. Thus, the present invention provides lentiviral vectors with cell-specific infection ability, which has broad application prospects and important clinical significance. BRIEF DESCRIPTION OF DRAWINGS

[0234] Figure 1: Three lentiviral structures used for infectivity analysis.

[0235] Figure 2: Test of the infectivity of lentivirus with different VSV G envelope protein mutations on CD3 + Jurkat cells.

[0236] Figure 3: Test of the infectivity of lentivirus with different VSV G envelope protein mutations on CD3 - Raji cells.

[0237] Figures 4A-4C: Test of infectivity of lentivirus with different VSV G envelope protein mutations on T cells, NK cells and B cells in PBMC. A: anti-CD3 redirected lentivirus; B: lentivirus without anti-CD3 redirection; C: statistical results of infectivity test.

[0238] Figure 5: Test of infectivity of lentivirus with different Cocal G envelope protein mutations on CD3 + Jurkat cells.

[0239] Figure 6: Test of infectivity of lentivirus with different Cocal G envelope protein mutations on CD3 - Raji cells.

[0240] Figure 7: Test of infectivity of lentivirus with different Cocal G envelope protein insertion mutations on CD3 + human primary cells, CD3 + Jurkat cells, and CD3 - Raji cells. A: Test of infectivity of lentivirus with Cocal G T184 site pre-insertion mutation envelope protein on the above-mentioned cells; B: Test of infectivity of lentivirus with Cocal G R354 site pre-insertion mutation envelope protein on the above-mentioned cells.

[0241] Explanation of sequence information

[0242] The information of the sequences involved in the present application is provided in Table 1.

[0243] Table 1: Sequence information Examples

[0244] The present application will now be described with reference to the following examples, which are intended to illustrate the present application (but not to limit the present application).

[0245] It is known to those skilled in the art that the examples describe the present application by way of illustration, and are not intended to limit the scope of the present application claimed in the present application. The experimental methods in the examples are conventional methods, unless otherwise specified. When no specific conditions are indicated in the examples, conventional conditions or conditions recommended by the manufacturer are used. When no manufacturer is indicated for the reagents or instruments used, they are conventional products that can be obtained commercially.

[0246] Example 1: Packaging of lentiviral vectors

[0247] 1. G protein mutants

[0248] Using structural biology knowledge and through extensive mutation screening, four new key amino sites in the VSV G polypeptide sequence were identified that affect lentiviral infection of host cells, which are lysine at position 50 (K50), isoleucine at position 331 (I331), isoleucine at position 347 (I347), and methionine at position 184 (M184) for the wild-type VSV G sequence shown in SEQ ID NO: 3. The specific mutations are lysine at position 50 mutated to glutamic acid (K50E), isoleucine at position 331 mutated to glutamic acid (I331E), isoleucine at position 347 mutated to glutamic acid (I347E), and methionine at position 184 mutated to aspartic acid (M184D). The amino acid sequences of the four mutant VSV Gs, VSV G-K50E, VSV G-I331E, VSV G-I347E, and VSV G-M184D, are shown in SEQ ID NOs: 5-8, respectively.

[0249] The Cocal virus G protein, which is structurally similar to VSV G with a sequence homology of 72.93%, only has some differences in the flexible region of the membrane proximal domain (MP), and can effectively resist neutralization reactions in serum. Therefore, the above mutation sites were also introduced into the wild-type Cocal G protein (SEQ ID NO: 22), and the specific mutations in Cocal G are lysine at position 50 mutated to glutamic acid (K50E), isoleucine at position 331 mutated to glutamic acid (I331E), isoleucine at position 347 mutated to glutamic acid (I347E), and threonine at position 184 mutated to aspartic acid (T184D). The amino acid sequences of the four mutant Cocal Gs, Cocal G-K50E, Cocal G-I331E, Cocal G-I347E, and Cocal G-T184D, are shown in SEQ ID NOs: 24-27, respectively.

[0250] In addition, the above mutation sites are introduced into other homologous viral envelope proteins to obtain corresponding mutant proteins, including: VSV Indiana strain G protein (SEQ ID NO: 34), VSV New Jersey strain G protein (SEQ ID NO: 38), Maraba virus G protein (SEQ ID NO: 35), Morreton virus G protein (SEQ ID NO: 36), Alagoa virus G protein (SEQ ID NO: 37), Carajas virus G protein (SEQ ID NO: 39), and the specific mutant forms are as follows:

[0251] The specific mutant forms in Indiana G are respectively lysine at position 50 mutated to glutamic acid (K50E), isoleucine at position 331 mutated to glutamic acid (I331E), isoleucine at position 347 mutated to glutamic acid (I347E), and methionine at position 184 mutated to aspartic acid (M184D).

[0252] The specific mutant forms in New Jersey G are respectively threonine at position 50 mutated to glutamic acid (T50E), leucine at position 335 mutated to glutamic acid (L335E), valine at position 351 mutated to glutamic acid (V351E), and valine at position 184 mutated to aspartic acid (V184D).

[0253] The specific mutant forms in Maraba G are respectively lysine at position 50 mutated to glutamic acid (K50E), isoleucine at position 331 mutated to glutamic acid (I331E), methionine at position 347 mutated to glutamic acid (M347E), and valine at position 184 mutated to aspartic acid (V184D).

[0254] The specific mutant forms in Morreton G are respectively lysine at position 50 mutated to glutamic acid (K50E), isoleucine at position 331 mutated to glutamic acid (I331E), isoleucine at position 347 mutated to glutamic acid (I347E), and methionine at position 184 mutated to aspartic acid (M184D).

[0255] The specific mutant forms in Alagoa G are respectively lysine at position 50 mutated to glutamic acid (K50E), leucine at position 331 mutated to glutamic acid (L331E), valine at position 347 mutated to glutamic acid (V347E), and threonine at position 184 mutated to aspartic acid (T184D).

[0256] The specific mutant forms in Carajas G are lysine at position 50 mutated to glutamic acid (K50E), isoleucine at position 335 mutated to glutamic acid (I335E), valine at position 351 mutated to glutamic acid (V351E), and methionine at position 184 mutated to aspartic acid (M184D), respectively.

[0257] 2. Plasmid / Sequence

[0258] The lentivirus packaging system includes an envelope plasmid, a packaging plasmid, and a transfer plasmid.

[0259] The envelope plasmid carries (i) a G protein coding sequence or (ii) a G protein and a targeting molecule construct, wherein: the G protein and the targeting molecule construct has the structure as shown below: [signal peptide]-[CD3 scFv (aCD3)]-[hinge region]-[transmembrane region]-[linker]-[T2A]-[G protein], the full-length amino acid sequence corresponding to the nucleic acid construct is shown in SEQ ID NOs: 15-20, 28-33. Those skilled in the art understand that a self-cleaving peptide (such as T2A) is used to divide multiple proteins in a single transcript, and the two proteins connected by the self-cleaving peptide (such as T2A) upstream and downstream will be cleaved to form two independent proteins during translation, thereby generating an independent G protein and a targeting molecule (aCD3).

[0260] The above-mentioned envelope plasmid construction is all entrusted to GenScript Biotech Corporation to complete. The lentivirus transfer plasmid Len-E carrying EGFP and the lentivirus packaging helper plasmid pSPAX2 encoding gag-pol-rev are both prepared by GenScript Biotech Corporation.

[0261] 3. Cells

[0262] HEK 293T17 (Nanjing Kebai) cells were maintained in DMEM containing 10% FBS, 1% Pen / Strep, 1% L-glutamine.

[0263] 4. Packaging and concentration of lentivirus

[0264] HEK 293T17 cells were transfected with packaging plasmids using PEI (Polysciences) to produce recombinant lentiviral particles of the three structures shown in Figure 1. Three plasmids were co-transfected: (1) a plasmid expressing VSV-G glycoprotein and / or targeting molecule (aCD3), (2) a plasmid encoding gag-pol-rev (pSPAX2), (3) a lentiviral transfer plasmid carrying EGFP. After 30-48 h of transfection, the culture supernatant was collected, and fresh DMEM medium pre-warmed with 5% FBS was added. The supernatant was stored at 4°C. After 72 h of transfection, the culture supernatant was collected and mixed with the supernatant collected at 30-48 h. The virus in the cell supernatant was concentrated by PEG8000 and resuspended in serum-free RPMI 1640. The lentiviral particle titer was determined using the Lenti-X p24 rapid titer kit (Takara Bio). The specific test steps are as follows:

[0265] 1) 1.7 x 10 7 HEK 293T cells were cultured in 20 mL of DMEM (Hyclone, SH30243.01) containing 10% FBS (Gibco, 10099-141C) medium, mixed well, and cultured at 37°C overnight.

[0266] 2) On the second day, when the HEK 293T (ATCC, CRL-3216) cells reached about 90% confluence, the medium was replaced with 5% FBS DMEM medium.

[0267] 3) Prepare a plasmid complex, the amount of each plasmid is: 21 μg plasmid DNA, 14 μg psPAX2 and 7 μg pMD2g, dissolved in 1 mL of opti-MEM (Gibco, 31985-070) and add 126 μL of PEI (Polysciences, 24765-2), vortex for 20 s, stand at room temperature for 15 min, then gently add the mixture to the HEK293T culture medium along the edge, continue to culture at 37°C.

[0268] 4) After 30-48 h of transfection, the culture supernatant was collected, and fresh DMEM medium pre-warmed with 5% FBS was added. The supernatant was stored at 4°C.

[0269] 5) After 72 h of transfection, the culture supernatant was collected and mixed with the supernatant collected at 30-48 h. Centrifuge at 1000 g for 10 min, filter the supernatant with a 0.45 μm filter, then mix well with a final concentration of 5.3% PEG8000 (Sigma, 89510-1KG-F) and a final concentration of 0.27 M NaCl (Sigma, S5150-1L) at 4°C overnight;

[0270] 6) After centrifugation of virus supernatant at 4°C, 4000g for 40 minutes, the supernatant was discarded and the precipitate was dissolved with 400 μL of serum-free DMEM and stored in -80°C refrigerator.

[0271] 7) Lentivirus particle titer was determined using Lenti-X p24 rapid titer kit (Takara Bio).

[0272] Example 2: Lentivirus infection of Jurkat and Raji cells based on VSV G envelope protein mutation

[0273] 1. Cells

[0274] Jurkat cells were maintained in PRMI 1640 medium containing 10% FBS, 1% Pen / Strep, 1% L-glutamine. Raji cells were maintained in PRMI 1640 medium containing 10% FBS, 1% Pen / Strep, 1% L-glutamine.

[0275] 2. Test procedure for lentivirus infection of Jurkat cells and Raji cells as follows:

[0276] 1) Jurkat tumor cell line of T cell origin or Raji cell line of B cell origin was taken in 96-well U-bottom plate, respectively, with a cell number of 3 x 10 4 cells / well, resuspended in PRMI 1640 medium containing 10% FBS, 1% Pen / Strep, 1% L-glutamine, with a volume of 50 μL / well;

[0277] 2) According to the p24 quantitative results of concentrated lentivirus titer, 2-fold gradient dilution of lentivirus (50 μL / well) and 12 μg / mL protamine (Sigma, P4005) were added to infect the target cells in the 96-well U-bottom plate containing Jurkat or Raji cells, respectively, and 100 μL / well of PRMI 1640 medium containing 10% FBS, 1% Pen / Strep, 1% L-glutamine was added after 8 h;

[0278] 3) After 48 h of lentivirus infection, the supernatant of the infected target cells was discarded after centrifugation at 300g for 5 minutes, and the proportion of EGFP positive cells was analyzed on CytoFLEX flow cytometer (Beckman) after resuspension with PBS solution.

[0279] The experimental results are shown in Figures 2 and 3. Compared with the lentivirus packaged by wild-type VSV G envelope protein, the lentivirus packaged by VSV G envelope protein with K50E, I331E, I347E or M184D mutation all showed significantly reduced infectivity to target cells. However, when the infectivity of lentivirus packaged by various mutant envelope proteins was redirected using anti-CD3 antibody (introducing G protein and targeting molecule construct), all redirected lentivirus recovered the infectivity to CD3 antigen positive Jurkat cells, while the infectivity to CD3 antigen negative Raji cells was weak. In summary, the four sites of K50, I331, I347 and M184 of VSV G all play a similar key role in the targeted infection of VSV G.

[0280] Example 3: Lentivirus infection of PBMC cells based on VSV G envelope protein mutation

[0281] 1) Commercial PBMC (Shanghai Heyousheng Biological) cells were cultured in X-Vivo 15 (Lonza) medium containing 1% human AB serum, 1% HSA, 1% Pen / Strep, 1% L-glutamine, with an initial cell density of 1 x 10 6 / mL;

[0282] 2) T cell Transact (Miltenyi biotec, 130-111-160) was added at a ratio of 10 ul / ml, and 1000 IU / mL of IL-2 (Sijiqing Biological, GMP) was added to activate T cell expansion;

[0283] 3) After 48 hours of cell activation, the PMBC was transferred to a 96-well U-bottom plate, with a cell number of 3 x 10 4 / mL, and a volume of 50 ul / well; 500 ng of concentrated lentivirus with a P24 concentration and 12 ug / mL protamine (Sigma, P4005) were added to each well for infection. After 12 hours of infection, 100 ul / well of fresh culture medium was added,

[0284] 4) On the 4th day of lentivirus infection, the transduced cells were analyzed by flow cytometry to determine the proportion of GFP positive cells. Specifically, the cells were stained with anti-CD3 antibody to detect CD3 positive cells (BV421 mouse anti-human CD3, clone UCHT1, BD) and GFP expression; the cells were stained with anti-CD19 antibody to detect CD19 positive cells (PE mouse anti-human CD19, clone 4G7, Abeam) and GFP expression; the cells were stained with anti-CD56 antibody to detect CD56 positive cells (APC mouse anti-human CD56, clone TULY56, eBioscience) and GFP expression.

[0285] The results of the experiment are shown in Figure 4, which shows that the VSV-G pseudotyped lentivirus carrying the targeting molecule CD3 can transduce the CD3-positive T cell population, hardly transduce the CD19-positive B cell population, and a small amount of the CD56-positive NK cell population, which is likely due to the expression of CD3e on the surface of NK cells. Lentivirus without the anti-CD3 redirecting molecule showed no transduction ability in the three cell populations.

[0286] Example 4: Lentivirus infection of Jurkat and Raji cells based on mutations in the Cocal G envelope protein

[0287] The VSV G used in this study is highly homologous to the G protein of the Indiana VSV strain, with only 2 amino acid differences. In addition, the Cocal virus G protein is also highly similar in structure to the VSV G used in this study, with a sequence homology of 72.93%, with only some differences in the flexible region of the membrane proximal domain (MP), and is effective in resisting neutralization reactions in serum. Therefore, we also tested the effect of the above-mentioned mutation sites in the Cocal G protein on the specificity of lentivirus infection. The results of the experiment are shown in Figures 5 and 6, which show that the corresponding Cocal G mutant proteins can also block the target cell infection ability of lentivirus.

[0288] The above examples have verified that the four sites K50, I331, I347, and M184 on VSV G and their corresponding sites on Cocal G protein can block the target cell infection ability of lentivirus, and thus those skilled in the art understand that the above-mentioned mutation sites are also applicable to the G proteins of other VSV strains (such as the Indiana strain and the New Jersey strain), and are also applicable to the G proteins of other vesiculoviruses such as Maraba, Morreton, Alagoa, and Carajas, which are highly homologous to the VSV G protein in this study. The corresponding mutation sites or their combined mutation forms of the G proteins of these VSV strains should also be within the scope of protection of the present application.

[0289] Example 5: Lentivirus infection of Jurkat and Raji cells based on insertion mutations in the Cocal G envelope protein

[0290] In addition to the four new key amino acid sites and the corresponding substitution mutation forms identified in the above examples, it was also found that insertion mutations at specific amino acid sites can also weaken or eliminate the target cell infection ability of lentivirus.

[0291] In this study, E(Glu), I(Ile), A(Ala), AA(Ala-Ala) or GAA(Gly-Ala-Ala) were inserted before T184 site (i.e. between D183 and T184) of Cocal virus G protein, or P(Pro) or A(Ala) were inserted before R354 site (i.e. between E353 and R354) of Cocal virus G protein, to form the corresponding insertion mutant proteins of Cocal virus G protein, the sequences of which are shown in SEQ ID NOs:41-47. The constructs of G protein and targeting molecules were constructed and lentivirus was packaged according to the method described in Example 1, the corresponding full-length amino acid sequences of the nucleic acid constructs are shown in SEQ ID NOs:52-58, and then the lentivirus infection ability was detected according to the method described in Example 2.

[0292] The results are shown in Figure 7, and it can be seen that insertion of Ile before T184 site or insertion of Pro or Ala before R354 site of Cocal G protein can weaken or eliminate the infectivity of the corresponding mutant proteins packaged lentivirus.

[0293] It is understood by those skilled in the art that the above-mentioned insertion mutations are also applicable to the G proteins of VSV strain, VSV Indiana strain, VSV New Jersey strain, and other vesiculoviruses such as Maraba, Morreton, Alagoa, and Carajas, which have high homology with the Cocal G protein in this study.

[0294] The T184 site of Cocal G protein corresponds to M184 of VSV G protein (SEQ ID NO:3), M184 of VSV Indiana strain G protein (SEQ ID NO:34), V184 of VSV New Jersey strain G protein (SEQ ID NO:38), V184 of Maraba virus G protein (SEQ ID NO:35), M184 of Morreton virus G protein (SEQ ID NO:36), T184 of Alagoa virus G protein (SEQ ID NO:37), and M184 of Carajas virus G protein (SEQ ID NO:39).

[0295] The R354 position of the Cocal G protein corresponds to R354 of the VSV G protein (SEQ ID NO: 3), R354 of the VSV Indiana strain G protein (SEQ ID NO: 34), R358 of the VSV New Jersey strain G protein (SEQ ID NO: 38), R354 of the Maraba virus G protein (SEQ ID NO: 35), R354 of the Morreton virus G protein (SEQ ID NO: 36), R354 of the Alagoa virus G protein (SEQ ID NO: 37), R358 of the Carajas virus G protein (SEQ ID NO: 39).

[0296] The corresponding insertion mutations of the G proteins of these VSV strains should also be within the scope of protection of the present application.

Claims

1. A mutated Vesiculovirus envelope protein comprising an amino acid mutation (e.g. substitution, insertion or deletion) in at least one (e.g. 1, 2, 3, 4 or 5) of the amino acid positions corresponding to: position 50, position 331, position 347, position 184, position 354, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO:

22.

2. The mutant vesiculovirus envelope protein of claim 1, wherein, the amino acid mutation is an amino acid substitution; preferably the amino acid mutation is a substitution to an acidic amino acid (e.g. E or D).

3. The mutated Vesiculovirus envelope protein of claim 2, wherein the amino acid residue in at least one (e.g. 1, 2, 3 or 4) of the amino acid positions corresponding to: position 50, position 331, position 347, position 184, with reference to the amino acid positions set forth in SEQ ID NO: 3 or SEQ ID NO: 22, is substituted to an acidic amino acid (e.g. E or D).

4. The mutant vesiculovirus envelope protein of claim 1, wherein, the amino acid mutation is an amino acid insertion; preferably the amino acid insertion is an insertion of 1, 2 or 3 contiguous amino acids; preferably the inserted amino acid is selected from E (Glu), I (lie), A (Ala), AA (Ala-Ala), GAA (Gly-Ala-Ala) or P (Pro).

5. The mutated Vesiculovirus envelope protein of claim 4, comprising an insertion of E (Glu), I (lie), A (Ala), AA (Ala-Ala) or GAA (Gly-Ala-Ala) between positions 183 and 184 of SEQ ID NO: 3 or SEQ ID NO: 22, or an insertion of A (Ala) or P (Pro) between positions 353 and 354 of SEQ ID NO: 3 or SEQ ID NO:

22. preferably the mutated Vesiculovirus envelope protein comprises an insertion of I (lie) between positions 183 and 184 of SEQ ID NO: 3 or SEQ ID NO: 22, or an insertion of A (Ala) or P (Pro) between positions 353 and 354 of SEQ ID NO: 3 or SEQ ID NO:

22.

6. The mutated Vesiculovirus envelope protein of any one of claims 1-5, selected from the group consisting of Vesicular Stomatitis Virus (VSV) G protein, Cocal virus G protein, Maraba virus G protein, Morreton virus G protein, Alagoa virus G protein or Carajas virus G protein; preferably the mutated Vesiculovirus envelope protein comprises the mutation as defined in any one of claims 1-5 compared to the wild-type protein from which it is derived; preferably wherein: a wild-type VSV G protein has the sequence set forth in any one of SEQ ID NOs: 3, 34, 38 or a sequence at least 80% identical thereto; a wild-type Cocal G protein has the sequence set forth in SEQ ID NO: 22 or a sequence at least 80% identical thereto; a wild-type Maraba G protein has the sequence set forth in SEQ ID NO: 35 or a sequence at least 80% identical thereto; a wild-type Morreton G protein has the sequence set forth in SEQ ID NO: 36 or a sequence at least 80% identical thereto; a wild-type Alagoa G protein has the sequence set forth in SEQ ID NO: 37 or a sequence at least 80% identical thereto; and / or, a wild-type Carajas G protein has the sequence set forth in SEQ ID NO: 39 or a sequence at least 80% identical thereto.

7. The vesiculovirus envelope protein of any one of claims 1 to 6 having a sequence selected from the group consisting of: (i) the sequence set forth in any one of SEQ ID NOs: 5-8, or a sequence comprising one mutation selected from K50E, I331E, I347E and M184D compared to the sequence set forth in SEQ ID NO: 34, or a sequence comprising one mutation selected from T50E, L335E, V351E and V184D compared to the sequence set forth in SEQ ID NO: 38; (ii) the sequence set forth in any one of SEQ ID NOs: 24-27; (iii) a sequence comprising one mutation selected from K50E, I331E, M347E and V184D compared to the sequence set forth in SEQ ID NO: 35; (iv) a sequence comprising one mutation selected from K50E, I331E, I347E and M184D compared to the sequence set forth in SEQ ID NO: 36; (v) a sequence comprising one mutation selected from K50E, L331E, V347E and T184D compared to the sequence set forth in SEQ ID NO: 37; (vi) a sequence comprising one mutation selected from K50E, I335E, V351E and M184D compared to the sequence set forth in SEQ ID NO:

39.

8. The vesiculovirus envelope protein of any one of claims 1 to 6 having a sequence selected from the group consisting of: (i) the sequence set forth in any one of SEQ ID NOs: 41-47; for example the sequence set forth in any one of SEQ ID NOs: 42, 46, 47; (ii) comprises an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g. an I insertion) between S183 and M184, or an A insertion or a P insertion between E353 and R354, compared to the sequence set forth in SEQ ID NO: 3; or, comprises an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g. an I insertion) between S183 and M184, or an A insertion or a P insertion between E353 and R354, compared to the sequence set forth in SEQ ID NO: 34; or, comprises an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g. an I insertion) between L183 and V184, or an A insertion or a P insertion between V357 and R358, compared to the sequence set forth in SEQ ID NO: 38; (iii) comprises an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g. an I insertion) between S183 and V184, or an A insertion or a P insertion between E353 and R354, compared to the sequence set forth in SEQ ID NO: 35; (iv) comprises an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g. an I insertion) between S183 and M184, or an A insertion or a P insertion between E353 and R354, compared to the sequence set forth in SEQ ID NO: 36; (v) comprises an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g. an I insertion) between S183 and T184, or an A insertion or a P insertion between K353 and R354, compared to the sequence set forth in SEQ ID NO: 37; (vi) comprises an E insertion, an I insertion, an A insertion, an AA insertion, or a GAA insertion (e.g. an I insertion) between A183 and M184, or an A insertion or a P insertion between S357 and R358, compared to the sequence set forth in SEQ ID NO:

39.

9. An isolated nucleic acid molecule encoding the mutated VSV envelope protein of any one of claims 1-8.

10. A vector comprising the isolated nucleic acid molecule of claim 9.

11. A lentiviral vector comprising an envelope and a lentiviral vector genome; wherein, the envelope comprises the mutated VSV envelope protein of any one of claims 1-8, the lentiviral vector genome comprises a nucleic acid of interest; Preferably, the lentiviral vector is pseudotyped by the mutated VSV envelope protein.

12. The lentiviral vector of claim 11, further comprising a non-viral membrane-bound protein, the non-viral membrane-bound protein comprising an extracellular targeting domain and a membrane-binding domain; Preferably, the extracellular targeting domain comprises a specific binding domain for a target cell; Preferably, the extracellular targeting domain is an antibody or a ligand for a target cell surface antigen; Preferably, the extracellular targeting domain is an antibody, such as a scFv or a VHH, for a target cell surface antigen; Preferably, the target cell surface antigen is selected from a T cell marker, a gd T cell marker, a NK T cell marker, a MAIT cell marker, a tumor cell or other cell marker; Preferably, the extracellular targeting domain is an anti-CD3 antibody.

13. The lentivirus vector of claim 12, wherein, The non-viral membrane binding protein further comprises a hinge region between the extracellular targeting domain and the membrane binding domain; Preferably, the hinge region comprises the sequence set forth in SEQ ID NO:

11.

14. The lentiviral vector of claim 12 or 13, wherein, The membrane binding domain comprises a transmembrane region of a transmembrane protein, such as a CD8 transmembrane region, a CD4 transmembrane region, or a NKG2D transmembrane region; Preferably, the membrane binding domain comprises the sequence set forth in SEQ ID NO:

12.

15. The lentiviral vector of any one of claims 12-14, wherein, The non-viral membrane binding protein further comprises a signal peptide at its N-terminus; Preferably, the signal peptide is a CD8 signal peptide, a GM-CSF receptor signal peptide, or an IgG signal peptide; Preferably, the signal peptide comprises the sequence set forth in SEQ ID NO:

10.

16. The lentiviral vector of any one of claims 12-15, wherein, The non-viral membrane binding protein comprises the amino acid sequence set forth in SEQ ID NO: 40 or 49.

17. The lentiviral vector of any one of claims 11-16, wherein, The lentiviral vector genome further comprises a cis-acting element, such as a LTR.

18. One or more vectors comprising a first nucleotide sequence encoding the mutated vesiculovirus envelope protein of any one of claims 1-8 and a second nucleotide sequence encoding a non-viral membrane-bound protein; wherein, The non-viral membrane binding protein is as defined in any one of claims 12-16; Preferably, the first and second nucleotide sequences are on the same vector; preferably, the first and second nucleotide sequences are linked by a 2A peptide (such as T2A, P2A, E2A or F2A) coding sequence or an IRES sequence; Preferably, the first and second nucleotide sequences are on different vectors; Preferably, the vector is an expression vector; Preferably, the vector is a packaging plasmid for lentiviral vector packaging.

19. A lentiviral vector packaging system, comprising: a first nucleic acid molecule encoding the mutated vesicular virus envelope protein of any one of claims 1-8; a second nucleic acid molecule encoding a non-viral membrane binding protein, the non-viral membrane binding protein being as defined in any one of claims 12-16; a third nucleic acid molecule encoding gag and pol; a fourth nucleic acid molecule encoding rev; and a fifth nucleic acid molecule comprising a nucleic acid of interest; Preferably, the first to fifth nucleic acid molecules are present on one or more expression vectors.

20. The lentiviral vector packaging system of claim 19, comprising: a packaging plasmid comprising the first and second nucleic acid molecules; a packaging plasmid comprising the third and fourth nucleic acid molecules; and a transfer plasmid comprising the fifth nucleic acid molecule.

21. A method of producing a lentiviral vector, comprising expressing the lentiviral vector packaging system of claim 19 in a host cell; Preferably, the method comprises: introducing into the host cell the packaging plasmid, the packaging plasmid, and the transfer plasmid as described in claim 20; Preferably, the method further comprises recovering viral particles from the culture of the host cell.

22. A method of delivering a nucleic acid of interest to a target cell, comprising: (i) providing the lentiviral vector of any one of claims 11-17; (ii) contacting the lentiviral vector with a target cell, thereby delivering the nucleic acid of interest to the cell; Preferably, the target nucleic acid comprises an exogenous gene, for example encoding a therapeutic protein or an antigen of a pathogen (e.g. a viral antigen), a gene editing tool (e.g. a Cas protein and / or a gRNA of a CRISPR / Cas system), or a gene silencing tool (e.g. an shRNA); Preferably, the method is performed in vitro; Preferably, the method is performed in vivo, the method comprising administering the lentiviral vector to a subject, thereby delivering the target nucleic acid to a target cell in vivo; Preferably, the method is for transgenesis of the target cell; Preferably, the method is for gene editing of the target cell.

23. A pharmaceutical composition comprising the lentiviral vector of any one of claims 11-17 or the target cell obtained by the method of claim 22, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the target nucleic acid carried by the lentiviral vector comprises an exogenous gene; Preferably, the exogenous gene encodes a therapeutic protein or an antigen (e.g. an antigen of a pathogen); Preferably, the exogenous gene encodes a gene editing tool (e.g. a Cas protein and / or a gRNA of a CRISPR / Cas system) or a gene silencing tool (e.g. an shRNA).

24. Use of the lentiviral vector of any one of claims 11-17, the lentiviral vector packaging system of claim 19 or 20, the target cell obtained by the method of claim 22, or the pharmaceutical composition of claim 23, in the manufacture of a medicament for, or in the use of, gene editing or gene therapy; Preferably, the target nucleic acid carried by the lentiviral vector comprises an exogenous gene; Preferably, the exogenous gene encodes a therapeutic protein or an antigen (e.g. an antigen of a pathogen); Preferably, the exogenous gene encodes a gene editing tool (e.g. a Cas protein and / or a gRNA of a CRISPR / Cas system) or a gene silencing tool (e.g. an shRNA).

25. Use of the lentiviral vector of any one of claims 11-17, the lentiviral vector packaging system of claim 19 or 20, or the pharmaceutical composition of claim 23, in the manufacture of, or as, a nucleic acid vaccine (e.g. an mRNA vaccine); Preferably, the target nucleic acid carried by the lentiviral vector encodes an antigen, e.g. an antigen of a pathogen.

26. A method for gene editing or gene therapy, comprising administering to a subject in need thereof an effective amount of the lentiviral vector of any one of claims 11-17, the lentiviral vector packaging system of claim 19 or 20, the target cell obtained by the method of claim 22, or the pharmaceutical composition of claim 23; Preferably, the subject is a mammal, e.g. a human.

27. A method for inducing an immune response in a subject, comprising administering to a subject in need thereof an effective amount of the lentiviral vector of any one of claims 11-17, the lentiviral vector packaging system of claim 19 or 20, or the pharmaceutical composition of claim 23; Preferably, the lentiviral vector carries a nucleic acid of interest that encodes an antigen, such as a pathogen antigen; Preferably, the subject is a mammal, such as a human; Preferably, the subject is administered the pharmaceutical composition of claim 23, which is a nucleic acid vaccine (e.g., an mRNA vaccine).

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