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

Abstract

The invention relates to the field of lentivirus delivery, and particularly provides a mutated vesicular virus envelope protein and application thereof in lentiviral vector packaging.

Description

Technical Field

[0001] This invention relates to the field of lentivirus delivery, specifically providing a mutated vesicular virus envelope protein and its application in lentivirus vector packaging. Background Technology

[0002] In the field of drug delivery, traditional targeted delivery vectors include adenovirus liposomes (LNP), adenovirus (Adv) vectors, adeno-associated virus (AAV) vectors, and lentiviral vectors. Among them, lentiviral vectors have the highest delivery efficiency and can integrate the target gene coding sequence into the target cell genome and achieve persistent expression in infected cells and their progeny cells.

[0003] Lentiviral vectors typically use the vesicular stomatitis virus (VSV)-G envelope protein as the viral envelope. Vesicular stomatitis virus (VSV) is a non-pathogenic, enveloped, negative-sense RNA virus belonging to the genus *Vesicularvirae* of the rhabdovirus family. It is an arbovirus that can infect insects, cattle, horses, and pigs. Its glycoprotein G is widely used in gene therapy, including in lentiviral vectors, as the viral envelope protein. The low-density lipoprotein receptor (LDL-R) is the main entry receptor for 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 structures of two different cysteine-rich domains (CR2 and CR3) of VSV G and LDL-R, showing that they have the same binding site on G. They also identified two basic residues on VSV G, lysine K47 and arginine R354, which are crucial for the interaction of VSV G with LDL R via CR2 and CR3, and mutations in these residues result in the loss of VSV infectivity (PCT / EP2018 / 075824). Connor S. Dobson et al. (Dobson, C. Set 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 mutated VSV G with antibodies, receptors or ligands targeting surface antigen proteins of specific cells on the viral membrane surface can achieve cell-specific infection of lentiviral vectors.

[0004] There remains an unmet need in the field for lentiviral vectors with cell-specific infection capabilities. Summary of the Invention

[0005] In this application, the inventors, utilizing structural biology knowledge and through extensive mutation screening, identified novel key amino sites affecting lentiviral infection of host cells based on the VSV G polypeptide sequence, and provided a lentiviral vector with cell-specific infection capability. This provides the following aspects.

[0006] Mutated vesicular virus envelope protein

[0007] In one aspect, the present invention provides a mutated vesiculovirus envelope protein containing an amino acid mutation (e.g., substitution, insertion, or deletion) at at least one of the following amino acid positions (e.g., position 1, position 2, position 3, position 4, or position 5): amino acid positions 50, 331, 347, 184, and 354, as indicated in SEQ ID NO:3 or SEQ ID NO:22.

[0008] In this document, "the amino acid position corresponding to a given position" refers to the equivalent position in the compared sequences when performing optimal sequence alignment, i.e., when the sequences are aligned to obtain the highest percentage of identity. Therefore, the corresponding amino acid position can be identified by sequence alignment (e.g., to obtain the highest percentage of identity) between the target vesicular virus envelope protein sequence and the sequences shown in SEQ ID NO:3 or SEQ ID NO:22. For example, the statement "a substitution is contained at position 50 corresponding to the amino acid residue position shown in reference SEQ ID NO:3" covers the amino acid substitution at position 50 in the amino acid sequence shown in SEQ ID NO:3 or at the corresponding position in another vesicular virus envelope protein sequence. Similarly, the statement "a substitution is contained at position 50 corresponding to the amino acid residue position shown in reference SEQ ID NO:22" covers the amino acid substitution at position 50 in the amino acid sequence shown in SEQ ID NO:22 or at the corresponding position in another vesicular virus envelope protein sequence.

[0009] I. Substitution Mutation

[0010] In some embodiments, the amino acid mutation is an amino acid substitution. In some embodiments, the amino acid mutation is a substitution with an acidic amino acid (e.g., E or D).

[0011] In some embodiments, the mutant vesicular virus envelope protein provided by the present invention contains amino acid substitutions at at least one of the following amino acid positions (e.g., position 1, position 2, position 3 or 4), referring 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.

[0012] In some embodiments, the mutated vesicular virus envelope protein has at least one (e.g., position 1, 2, 3, or 4) amino acid residue at corresponding positions to acidic amino acids (e.g., E or D), with reference 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.

[0013] In some embodiments, the mutated vesicular virus envelope protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the following: an amino acid residue at position 50 replaced by E or D, an amino acid residue at position 331 replaced by E or D, an amino acid residue at position 347 replaced by E or D, and an amino acid residue at position 184 replaced by E or D.

[0014] In some embodiments, the mutated vesicular virus envelope protein comprises at least one (e.g., 1, 2, 3, or 4) mutation selected from the following: an amino acid residue at position 50 replaced with E, an amino acid residue at position 331 replaced with E, an amino acid residue at position 347 replaced with E, and an amino acid residue at position 184 replaced with D.

[0015] II. Insertion mutation

[0016] In some embodiments, the amino acid mutation is an amino acid insertion. In this document, the expression "containing an amino acid insertion at a specified amino acid position" means inserting an additional amino acid before that position (i.e., between that position and the position preceding it).

[0017] In some embodiments, the amino acid insertion is an insertion of one, two, or three consecutive amino acids.

[0018] In some embodiments, the inserted amino acid is selected from E (Glu), I (Ile), A (Ala), AA (Ala-Ala), GAA (Gly-Ala-Ala), or P (Pro).

[0019] In some embodiments, the mutated vesicular virus envelope protein of the present invention includes 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.

[0020] In some embodiments, the mutant vesicular virus envelope protein of the present invention contains an A (Ala) insertion or a P (Pro) insertion between positions 353 and 354 corresponding to SEQ ID NO:3 or SEQ ID NO:22.

[0021] In some embodiments, the mutated vesicular virus envelope protein contains an I (Ile) insertion between positions 183 and 184 corresponding to SEQ ID NO:3 or SEQ ID NO:22, or contains an A (Ala) insertion or a P (Pro) insertion between positions 353 and 354 corresponding to SEQ ID NO:3 or SEQ ID NO:22.

[0022] In some embodiments, the vesicular viral envelope protein described in any of the above embodiments is selected from 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.

[0023] Mutant VSV G protein

[0024] In some embodiments, the mutated vesicular virus envelope protein is a mutated VSV G protein. In some embodiments, the mutated vesicular virus envelope protein is derived from the wild-type vesicular virus envelope protein, which is the VSV G protein.

[0025] In some embodiments, the mutant VSV G protein, compared to the corresponding wild-type VSV G protein, contains amino acid substitutions at at least one (e.g., position 1, position 2, position 3, or position 4) at amino acid positions corresponding to the positions shown in SEQ ID NO:3 or SEQ ID NO:22: position 50, position 331, position 347, and position 184.

[0026] In some embodiments, positions 50, 331, 347, and 184 of the amino acid positions indicated by reference SEQ ID NO:3 or SEQ ID NO:22 correspond to positions 50, 331, 347, and 184 of the wild-type VSV Indiana strain G protein (e.g., SEQ ID NO:34), respectively.

[0027] In some embodiments, the 50th, 331st, 347th, and 184th amino acid positions indicated by reference SEQ ID NO:3 or SEQ ID NO:22 correspond to the 50th, 335th, 351st, and 184th positions of the wild-type VSV New Jersey strain G protein (e.g., SEQ ID NO:38), respectively.

[0028] In some embodiments, the mutant VSV G protein, compared to the corresponding wild-type VSV 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.

[0029] In some embodiments, the mutated VSV G protein, compared to the corresponding wild-type VSV 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.

[0030] In some embodiments, the mutated VSV 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.

[0031] In some embodiments, the wild-type VSV G protein has a sequence shown in any one of SEQ ID NOs:3, 34, 38 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.

[0032] In some embodiments, the mutant VSV G protein, compared to the wild-type VSV G protein sequence shown in SEQ ID NO:3, contains at least one (e.g., 1, 2, 3, or 4) substitutions at positions selected from the following: K50, I331, I347, M184.

[0033] In some embodiments, the mutated VSV G protein, compared to the wild-type VSV G protein sequence shown in SEQ ID NO:3, contains at least one (e.g., 1, 2, 3, or 4) mutations selected from the following: K50E, I331E, I347E, M184D.

[0034] In some embodiments, the mutant VSV G protein, compared to the wild-type VSV G protein sequence shown in SEQ ID NO:34, contains at least one (e.g., 1, 2, 3, or 4) substitutions at positions selected from the following: K50, I331, I347, M184.

[0035] In some embodiments, the mutated VSV G protein, compared to the wild-type VSV G protein sequence shown in SEQ ID NO:34, contains at least one (e.g., 1, 2, 3, or 4) mutations selected from the following: K50E, I331E, I347E, M184D.

[0036] In some embodiments, the mutant VSV G protein, compared to the wild-type VSV G protein sequence shown in SEQ ID NO:38, contains at least one (e.g., 1, 2, 3, or 4) substitutions at positions selected from the following: T50, L335, V351, V184.

[0037] In some embodiments, the mutated VSV G protein, compared to the wild-type VSV G protein sequence shown in SEQ ID NO:38, contains at least one (e.g., 1, 2, 3, or 4) mutations selected from the following: T50E, L335E, V351E, V184D.

[0038] In some embodiments, the mutated VSV G protein has a sequence selected from the following: the sequence shown in any one of SEQ ID NOs:5-8, a sequence containing a mutation selected from K50E, I331E, I347E, M184D compared to the sequence shown in SEQ ID NO:34, or a sequence containing a mutation selected from T50E, L335E, V351E, V184D compared to the sequence shown in SEQ ID NO:38.

[0039] In some embodiments, the mutant VSV G protein, compared to the corresponding wild-type VSV G protein, contains an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between positions 183 and 184 corresponding to SEQ ID NO:3 or SEQ ID NO:22, or an A insertion or P insertion between positions 353 and 354 corresponding to SEQ ID NO:3 or SEQ ID NO:22.

[0040] In some embodiments, positions 183, 184, 353, and 354 of the amino acid positions indicated by reference SEQ ID NO:3 or SEQ ID NO:22 correspond to positions 183, 184, 353, and 354 of the wild-type VSV Indiana strain G protein (e.g., SEQ ID NO:34), respectively.

[0041] In some embodiments, positions 183, 184, 353, and 354 of the amino acid positions indicated by reference SEQ ID NO:3 or SEQ ID NO:22 correspond to positions 183, 184, 353, and 354 of the wild-type VSV New Jersey strain G protein (e.g., SEQ ID NO:38), respectively.

[0042] In some embodiments, the mutated VSV G protein has a sequence selected from the following: containing an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between S183 and M184 compared to the sequence shown in SEQ ID NO:3, or containing an A insertion or P insertion between E353 and R354.

[0043] In some embodiments, the mutated VSV G protein has a sequence selected from the following: containing an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between S183 and M184 compared to the sequence shown in SEQ ID NO:34, or containing an A insertion or P insertion between E353 and R354.

[0044] In some embodiments, the mutated VSV G protein has a sequence selected from the following: containing an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between L183 and V184 compared to the sequence shown in SEQ ID NO:38, or containing an A insertion or P insertion between V357 and R358.

[0045] Mutant CocalG protein

[0046] In some embodiments, the mutated vesicular virus envelope protein is a mutated CocalVirus G protein. In some embodiments, the mutated vesicular virus envelope protein is derived from the wild-type vesicular virus envelope protein, which is the CocalG protein.

[0047] In some embodiments, the mutant Cocal G protein, compared to the corresponding wild-type Cocal G protein, contains amino acid substitutions at at least one (e.g., position 1, 2, 3, or 4) of the amino acid positions corresponding to the positions shown in SEQ ID NO:3 or SEQ ID NO:22: position 50, position 331, position 347, and position 184.

[0048] In some embodiments, the 50th, 331st, 347th, and 184th amino acid positions indicated by reference SEQ ID NO:3 or SEQ ID NO:22 correspond to the 50th, 331st, 347th, and 184th positions of the wild-type CocalG protein (e.g., SEQ ID NO:22), respectively.

[0049] In some embodiments, the mutant Cocal G protein, compared to the corresponding wild-type Cocal 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.

[0050] In some embodiments, the mutated Cocal G protein, compared to the corresponding wild-type Cocal 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.

[0051] In some embodiments, the mutated Cocal 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.

[0052] In some embodiments, the wild-type CocalG protein has the sequence shown 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 with it.

[0053] In some embodiments, the mutant CocalG protein, compared to the wild-type CocalG protein sequence shown in SEQ ID NO:22, contains at least one (e.g., 1, 2, 3, or 4) substitutions at positions selected from the following: K50, I331, I347, T184.

[0054] In some embodiments, the mutated CocalG protein, compared to the wild-type CocalG protein sequence shown in SEQ ID NO:22, contains at least one (e.g., 1, 2, 3, or 4) mutations selected from the following: K50E, I331E, I347E, T184D.

[0055] In some embodiments, the mutated Cocal G protein has a sequence selected from any of the following: SEQ ID NOs:24-27.

[0056] In some embodiments, the mutant Cocal G protein, compared to the corresponding wild-type Cocal G protein, contains an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between positions 183 and 184 of SEQ ID NO:3 or SEQ ID NO:22, or an A insertion or P insertion between positions 353 and 354 of SEQ ID NO:3 or SEQ ID NO:22.

[0057] In some embodiments, positions 183, 184, 353, and 354 of the amino acid positions indicated by reference SEQ ID NO:3 or SEQ ID NO:22 correspond to positions 183, 184, 353, and 354 of the wild-type Cocal G protein (e.g., SEQ ID NO:22), respectively.

[0058] In some embodiments, the mutated Cocal G protein has the sequence shown in any one of SEQ ID NOs:41-47. In some embodiments, the mutated Cocal G protein has the sequence shown in any one of SEQ ID NOs:42, 46, and 47.

[0059] Mutant Maraba G protein

[0060] In some embodiments, the mutated vesicular virus envelope protein is a mutated Maraba virus G protein. In some embodiments, the mutated vesicular virus envelope protein is derived from the wild-type vesicular virus envelope protein, which is the Maraba G protein.

[0061] In some embodiments, the mutant Maraba G protein, compared to the corresponding wild-type Maraba G protein, contains amino acid substitutions at at least one (e.g., position 1, position 2, position 3, or position 4) at amino acid positions corresponding to the positions shown in SEQ ID NO:3 or SEQ ID NO:22: position 50, position 331, position 347, and position 184.

[0062] In some embodiments, positions 50, 331, 347, and 184 of the amino acid positions indicated by reference SEQ ID NO:3 or SEQ ID NO:22 correspond to positions 50, 331, 347, and 184 of the wild-type Maraba G protein (e.g., SEQ ID NO:35), respectively.

[0063] In some embodiments, the mutant Maraba G protein, compared to the corresponding wild-type Maraba 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.

[0064] In some embodiments, the mutated Maraba G protein, compared to the corresponding wild-type Maraba 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.

[0065] In some embodiments, the mutated Maraba 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.

[0066] In some embodiments, the wild-type Maraba G protein has the sequence shown in SEQ ID NO:35 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.

[0067] In some embodiments, the mutated Maraba G protein, compared to the wild-type Maraba G protein sequence shown in SEQ ID NO:35, contains at least one (e.g., 1, 2, 3, or 4) substitutions at positions selected from the following: K50, I331, M347, V184.

[0068] In some embodiments, the mutated Maraba G protein, compared to the wild-type Maraba G protein sequence shown in SEQ ID NO:35, contains at least one (e.g., 1, 2, 3, or 4) mutations selected from the following: K50E, I331E, M347E, V184D.

[0069] In some embodiments, the mutated Maraba G protein has a sequence selected from the following: a sequence containing a mutation selected from K50E, I331E, M347E, and V184D compared to the sequence shown in SEQ ID NO:35.

[0070] In some embodiments, the mutant Maraba G protein, compared to the corresponding wild-type Maraba G protein, contains an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between positions 183 and 184 corresponding to SEQ ID NO:3 or SEQ ID NO:22, or an A insertion or P insertion between positions 353 and 354 corresponding to SEQ ID NO:3 or SEQ ID NO:22.

[0071] In some embodiments, positions 183, 184, 353, and 354 of the amino acid positions indicated by reference SEQ ID NO:3 or SEQ ID NO:22 correspond to positions 183, 184, 353, and 354 of the wild-type Maraba G protein (e.g., SEQ ID NO:35), respectively.

[0072] In some embodiments, the mutated Maraba G protein has a sequence selected from the following: containing an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between S183 and V184 compared to the sequence shown in SEQ ID NO:35, or containing an A insertion or P insertion between E353 and R354.

[0073] Mutant Morreton G protein

[0074] In some embodiments, the mutated vesicular virus envelope protein is a mutated Morreton virus G protein. In some embodiments, the mutated vesicular virus envelope protein is derived from the wild-type vesicular virus envelope protein, which is the Morreton G protein.

[0075] In some embodiments, the mutant Morreton G protein, compared to the corresponding wild-type Morreton G protein, contains amino acid substitutions at at least one (e.g., position 1, position 2, position 3, or position 4) at amino acid positions corresponding to the positions shown in SEQ ID NO:3 or SEQ ID NO:22: position 50, position 331, position 347, and position 184.

[0076] In some embodiments, positions 50, 331, 347, and 184 of the amino acid positions indicated by reference SEQ ID NO:3 or SEQ ID NO:22 correspond to positions 50, 331, 347, and 184 of the wild-type Morreton G protein (e.g., SEQ ID NO:36), respectively.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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) mutations selected from the following: K50E, I331E, I347E, M184D.

[0083] In some embodiments, the mutated Morreton G protein has a sequence selected from the following: a sequence containing a mutation selected from K50E, I331E, I347E, or M184D compared to the sequence shown in SEQ ID NO:36.

[0084] In some embodiments, the mutated Morreton G protein, compared to the corresponding wild-type Morreton G protein, contains an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between positions 183 and 184 corresponding to SEQ ID NO:3 or SEQ ID NO:22, or an A insertion or P insertion between positions 353 and 354 corresponding to SEQ ID NO:3 or SEQ ID NO:22.

[0085] In some embodiments, positions 183, 184, 353, and 354 of the amino acid positions indicated by reference SEQ ID NO:3 or SEQ ID NO:22 correspond to positions 183, 184, 353, and 354 of the wild-type Morreton G protein (e.g., SEQ ID NO:36), respectively.

[0086] In some embodiments, the mutated Morreton G protein has a sequence selected from the following: containing an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between S183 and M184 compared to the sequence shown in SEQ ID NO:36, or containing an A insertion or P insertion between E353 and R354.

[0087] Mutant AlagoaG protein

[0088] In some embodiments, the mutated vesicular virus envelope protein is a mutated Alagoa virus G protein. In some embodiments, the mutated vesicular virus envelope protein is derived from the wild-type vesicular virus envelope protein, which is the Alagoa G protein.

[0089] In some embodiments, the mutant Alagoa G protein, compared to the corresponding wild-type Alagoa G protein, contains amino acid substitutions at at least one (e.g., position 1, position 2, position 3, or position 4) at amino acid positions corresponding to the positions shown in SEQ ID NO:3 or SEQ ID NO:22: position 50, position 331, position 347, and position 184.

[0090] In some embodiments, the 50th, 331st, 347th, and 184th amino acid positions indicated by reference SEQ ID NO:3 or SEQ ID NO:22 correspond to the 50th, 331st, 347th, and 184th positions of the wild-type Alagoa G protein (e.g., SEQ ID NO:37), respectively.

[0091] In some embodiments, the mutant Alagoa G protein, compared to the corresponding wild-type Alagoa 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.

[0092] In some embodiments, the mutated Alagoa G protein, compared to the corresponding wild-type Alagoa 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.

[0093] In some embodiments, the mutated Alagoa 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.

[0094] In some embodiments, the wild-type Alagoa G protein has the sequence shown in SEQ ID NO:37 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.

[0095] In some embodiments, the mutated Alagoa G protein, compared to the wild-type Alagoa G protein sequence shown in SEQ ID NO:37, contains at least one (e.g., 1, 2, 3, or 4) substitutions at positions selected from the following: K50, L331, V347, T184.

[0096] In some embodiments, the mutated Alagoa G protein, compared to the wild-type Alagoa G protein sequence shown in SEQ ID NO:37, contains at least one (e.g., 1, 2, 3, or 4) mutations selected from the following: K50E, L331E, V347E, T184D.

[0097] In some embodiments, the mutated Alagoa G protein has a sequence selected from the following: a sequence containing a mutation selected from K50E, L331E, V347E, and T184D compared to the sequence shown in SEQ ID NO:37.

[0098] In some embodiments, the mutated Alagoa G protein, compared to the corresponding wild-type Alagoa G protein, contains an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between positions 183 and 184 corresponding to SEQ ID NO:3 or SEQ ID NO:22, or an A insertion or P insertion between positions 353 and 354 corresponding to SEQ ID NO:3.

[0099] In some embodiments, positions 183, 184, 353, and 354 of the amino acid positions indicated by reference SEQ ID NO:3 or SEQ ID NO:22 correspond to positions 183, 184, 353, and 354 of the wild-type Alagoa G protein (e.g., SEQ ID NO:37), respectively.

[0100] In some embodiments, the mutated Alagoa G protein has a sequence selected from the following: containing an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between S183 and T184 compared to the sequence shown in SEQ ID NO:37, or containing an A insertion or P insertion between K353 and R354.

[0101] Mutant CarajasG protein

[0102] In some embodiments, the mutated vesicular virus envelope protein is a mutated Carajas virus G protein. In some embodiments, the mutated vesicular virus envelope protein is derived from the wild-type vesicular virus envelope protein, which is the Carajas G protein.

[0103] In some embodiments, the mutant Carajas G protein, compared to the corresponding wild-type Carajas G protein, contains amino acid substitutions at at least one (e.g., position 1, position 2, position 3, or position 4) at amino acid positions corresponding to the positions shown in SEQ ID NO:3 or SEQ ID NO:22: position 50, position 331, position 347, and position 184.

[0104] In some embodiments, positions 50, 331, 347, and 184 of the amino acid positions indicated by reference SEQ ID NO:3 or SEQ ID NO:22 correspond to positions 50, 335, 351, and 184 of the wild-type Carajas G protein (e.g., SEQ ID NO:39), respectively.

[0105] In some embodiments, the mutant Carajas G protein, compared to the corresponding wild-type Carajas 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.

[0106] In some embodiments, the mutated Carajas G protein, compared to the corresponding wild-type Carajas 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.

[0107] In some embodiments, the mutated Carajas 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.

[0108] In some embodiments, the wild-type Carajas G protein has the sequence shown in SEQ ID NO:39 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.

[0109] In some embodiments, the mutated Carajas G protein, compared to the wild-type Carajas G protein sequence shown in SEQ ID NO:39, contains at least one (e.g., 1, 2, 3, or 4) substitutions at positions selected from the following: K50, I335, V351, M184.

[0110] In some embodiments, the mutated Carajas G protein, compared to the wild-type Carajas G protein sequence shown in SEQ ID NO:39, contains at least one (e.g., 1, 2, 3, or 4) mutations selected from the following: K50E, I335E, V351E, M184D.

[0111] In some embodiments, the mutated Carajas G protein has a sequence selected from the following: a sequence containing a mutation selected from K50E, I335E, V351E, and M184D compared to the sequence shown in SEQ ID NO:39.

[0112] In some embodiments, the mutated Carajas G protein, compared to the corresponding wild-type Carajas G protein, contains an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between positions 183 and 184 corresponding to SEQ ID NO:3 or SEQ ID NO:22, or an A insertion or P insertion between positions 353 and 354 corresponding to SEQ ID NO:3 or SEQ ID NO:22.

[0113] In some embodiments, positions 183, 184, 353, and 354 of the amino acid positions indicated by reference SEQ ID NO:3 or SEQ ID NO:22 correspond to positions 183, 184, 357, and 358 of the wild-type Carajas G protein (e.g., SEQ ID NO:39), respectively.

[0114] In some embodiments, the mutated Carajas G protein has a sequence selected from the following: containing an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between A183 and M184 compared to the sequence shown in SEQ ID NO:39, or containing an A insertion or P insertion between S357 and R358.

[0115] In another aspect, the present invention provides isolated nucleic acid molecules encoding the vesicular virus envelope protein described herein.

[0116] In another aspect, the present invention provides a vector (e.g., an expression vector) comprising the isolated nucleic acid molecules described above.

[0117] Lentiviral vector

[0118] In another aspect, the present invention provides a lentiviral vector comprising an envelope and a viral genome, wherein the envelope comprises the mutated vesicular virus envelope protein described herein. In some embodiments, the lentiviral vector of the present invention is pseudotyped by the mutated vesicular virus envelope protein described herein.

[0119] Encapsulation

[0120] The lentiviral vector defined in this invention is a pseudo-packaged lentiviral vector composed of vector particles carrying envelope proteins, and is therefore also referred to as a lentiviral vector particle. The envelope protein is derived from a virus different from the specific lentivirus providing the lentiviral vector genome.

[0121] In some embodiments, the lentiviral vector further comprises a non-viral membrane-binding protein. In some embodiments, the envelope of the lentiviral vector further comprises a non-viral membrane-binding protein.

[0122] In some embodiments, the non-viral membrane-binding protein includes an extracellular targeting domain and a membrane-binding domain.

[0123] In some embodiments, the extracellular targeting domain includes a target cell-specific binding domain. In some embodiments, the extracellular targeting domain specifically binds to an antigen or receptor on the surface of the target cell. In some embodiments, the binding interaction between the extracellular targeting domain and the antigen or receptor on the cell surface enables the lentivirus to enter the cell (e.g., antigen-specific cells, such as T cells).

[0124] In some implementations, the extracellular targeting domain binds to antigens or receptors present on the cell surface of a single T cell or a subset of a T cell population.

[0125] In some embodiments, the target cell surface antigen is selected from:

[0126] (1) T cell markers, such as those 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;

[0127] (2) γδT cell markers, such as those selected from γδTCR, Vδ1, Vδ2 and NKG2D (KLRK1, CD314);

[0128] (3) NKT cell markers, such as those selected from constant TCR (Va24-Ja18), CD185 (CXCR5), CXCR6 and IL-21R, or any combination thereof;

[0129] (4) MAIT cell markers, such as those selected from Va7.2, Ja33, CXCR6, IL-18R, KLRB1 (CD161) and VLA4 (α4β1 integrin) or any combination thereof;

[0130] (5) Tumor cells or other cell markers, such as those selected from TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, TnAg, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, liver glycoprotein B2, IGF-I receptor, CAIX, LMP2, gp100. bcr-abl, tyrosinase, EphA2, fucose GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, 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 A1, ETV6-AML, Sperminin 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostein, survivability protein and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS 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 carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, DLL3 or combinations thereof.

[0131] In some implementations, the extracellular targeting domain is an antibody or ligand targeting a target cell surface antigen.

[0132] In some embodiments, the extracellular targeting domain is an antibody against a target cell surface antigen, such as scFv or VHH.

[0133] In some embodiments, the extracellular targeting domain is an anti-CD3 antibody. In some exemplary embodiments, the anti-CD3 antibody comprises the VH and VL sequences of the scFv shown in SEQ ID NO:9, for example, the scFv shown in SEQ ID NO:9. In some exemplary embodiments, the anti-CD3 antibody comprises the VH and VL sequences of the scFv shown in SEQ ID NO:48, for example, the scFv shown in SEQ ID NO:48.

[0134] In some 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 some embodiments, the membrane-binding domain includes at least a transmembrane domain. In some embodiments, the membrane-binding domain includes both an intracellular domain and a transmembrane domain.

[0135] In some embodiments, the membrane-binding domain comprises a transmembrane region of a transmembrane protein, such as a CD8 transmembrane region, a CD4 transmembrane region, a CD28 transmembrane region, or an NKG2D transmembrane region. In some embodiments, the membrane-binding domain comprises a human CD8 transmembrane region. In some embodiments, the membrane-binding domain comprises the sequence shown in SEQ ID NO:12.

[0136] In some embodiments, the nonviral membrane-binding protein further includes a hinge region between the extracellular targeting domain and the membrane-binding domain. In some embodiments, the hinge region may 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 consecutive amino acids). In some embodiments, the hinge region is the hinge region of CD8 or CD28 or a portion thereof. In some embodiments, the hinge region comprises the sequence shown in SEQ ID NO:11.

[0137] In some embodiments, the nonviral membrane-binding protein further includes a signal peptide at its N-terminus. In some embodiments, the signal peptide functions to transfer the nonviral membrane-binding protein to the membrane (or envelope) of a lentivirus. In some embodiments, the signal peptide is a CD8 signal peptide, a GM-CSF receptor signal peptide, an IgG signal peptide, etc. In some embodiments, the signal peptide is a human CD8 signal peptide. In some embodiments, the signal peptide comprises the sequence shown in SEQ ID NO:10.

[0138] In some exemplary embodiments, the non-viral membrane-binding protein has the following structure: [signal peptide]-[extracellular targeting domain]-[hinge region]-[membrane-binding domain].

[0139] In some exemplary embodiments, the non-viral membrane-binding protein comprises the amino acid sequence shown in SEQ ID NO:40. In some exemplary embodiments, the non-viral membrane-binding protein comprises the amino acid sequence shown in SEQ ID NO:49.

[0140] In some exemplary embodiments, the C-terminus of the nonviral membrane-binding protein may further include an additional peptide sequence (e.g., a peptide linker), such as a peptide linker containing one or more Gs and / or Ss, for example, GSG.

[0141] In some 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 virus envelope protein coding sequence], wherein the linker is selected from cleavable linkers, such as 2A peptide (e.g., T2A, P2A, E2A, or F2A) coding sequences or IRES sequences. In some exemplary embodiments, a nucleotide sequence encoding an additional peptide sequence (e.g., a peptide linker) is further included between the [non-viral membrane-binding protein coding sequence] and the [linker], such as a peptide linker containing one or more Gs and / or Ss, for example, GSG. In some exemplary embodiments, the nucleic acid molecule comprises a nucleotide sequence selected from the following: a nucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NOs:17-20, 30-33, or a degenerate sequence thereof. In some exemplary embodiments, the nucleic acid molecule comprises a nucleotide sequence selected from the following: a nucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NOs:52-58 (preferably SEQ ID NOs:53, 57, 58), or a degenerate sequence thereof.

[0142] Vector genome

[0143] The lentiviral vector contains a vector genome, which is either substantially devoid of the original lentiviral sequence encoding the lentiviral protein, or modified, if present, to specifically prevent the expression of biologically active pol antigens and optionally further lentiviral structural and / or accessory and / or regulatory proteins. The biologically active pol antigens comprise viral enzymes (RT), reverse transcriptases (RT and RNase H), and integrase (IN), generated via gag-pol polyprotein cleavage. The pol antigen is not biologically active when the biological activity of at least one of these enzymes is not activated. The vector genome also contains the polynucleotide or transgene of interest (i.e., the target nucleic acid).

[0144] In some implementations, the polynucleotides or transgenes in the vector genome lack the functional pol gene, and in particular, do not contain the complete pol gene.

[0145] In addition to the heterologous polynucleotides of interest placed under appropriate regulatory sequence control, the vector genome defined herein contains lentiviral genomic sequences that are non-coding regions of the genome and are essential for providing 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 vectors of this invention are based on principles in the prior art.

[0146] In some embodiments, the vector genome contains the polynucleotide of interest located between two long terminal repeats (LTRs). In some embodiments, the vector genome may be a substitution vector, wherein the viral protein-coding sequence between the two long terminal repeats (LTRs) is replaced by the polynucleotide of interest.

[0147] In some embodiments, the lentiviral vector genome contains the target nucleic acid as well as flanking LTRs (5' LTR and 3' LTR).

[0148] In some embodiments, the lentiviral vector is a non-replicating lentiviral vector, which is a result of the fact that the gag and pol functional genes are provided in absolute trans form and therefore not present in the vector genome. In this case, when the lentiviral vector is given to the host, it cannot replicate in the host cell. Therefore, it provides therapeutically interested polynucleotides into the host cell for expression but does not form further lentiviral vector particles. This non-replicating state of the lentiviral vector is achieved particularly when the lentiviral gag, pol, and env genes are not provided in the vector genome or are not provided as functional genes. "Functional" means that the gene is correctly transcribed and / or correctly expressed. Thus, the lentiviral vector genome of the present invention in this embodiment contains at least one of the gag, pol, and env genes that are not transcribed or are incompletely transcribed; the term "incomplete transcription" refers to alterations in the transcripts gag, gag-pro, or gag-pro-pol, one or more of these being not transcribed. Other sequences involved in lentiviral replication may also be mutated in the vector genome to achieve this state.

[0149] In some embodiments, the 3'LTR sequence of the lentiviral vector genome lacks at least the promoter of the activator (enhancer) and possibly the U3 region. In some embodiments, the 3'LTR region lacks the U3 region (delta U3).

[0150] In some embodiments, the lentiviral vector of the present invention is derived from HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Visina-medie virus (VMV); Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); Simian immunodeficiency virus (SIV), etc.

[0151] In some implementations, an HIV-based vector backbone (i.e., HIV cis-acting sequence element) is used.

[0152] In some implementations, the lentiviral vector particle is human immunodeficiency virus-1 (HIV-1).

[0153] Lentiviral packaging envelope plasmid

[0154] In another aspect, the present invention provides one or more vectors comprising a first nucleotide sequence encoding a mutated vesicular virus envelope protein described herein and a second nucleotide sequence encoding a non-viral membrane-binding protein, wherein the non-viral membrane-binding protein is as defined in the foregoing aspects.

[0155] In some implementations, the first nucleotide sequence and the second nucleotide sequence are located on the same vector.

[0156] In some embodiments, the first and second nucleotide sequences are linked by a 2A peptide (e.g., T2A, P2A, E2A, or F2A) coding sequence or an IRES sequence. When multiple ORFs are expressed using a single promoter, each ORF separated by an internal ribosome entry site (IRES) or a 2A peptide in a single mRNA transcript will produce multiple proteins. Furthermore, those skilled in the art will understand that additional 2A peptide residues may be added to the C-terminus of the upstream protein separated by the 2A peptide, and additional residues may be added to the N-terminus of the downstream protein, without affecting the respective functions of the upstream and downstream proteins.

[0157] In some embodiments, the first and second nucleotide sequences are linked by a 2A peptide (e.g., T2A, P2A, E2A, or F2A) coding sequence. In some exemplary embodiments, the first and second nucleotide sequences are linked by a T2A peptide (e.g., SEQ ID NO:14) coding sequence.

[0158] In some exemplary embodiments, when the first nucleotide sequence and the second nucleotide sequence are located on the same vector, it has the following structure: [second nucleotide sequence]-[linker]-[first nucleotide sequence], wherein the linker is selected from cleavable linkers, such as 2A peptide (e.g., T2A, P2A, E2A, or F2A) coding sequences or IRES sequences. In some exemplary embodiments, the vector comprises a nucleotide sequence selected from the following: a nucleotide sequence encoding an amino acid sequence shown in any one of SEQ ID NOs:17-20, 30-33, or a degenerate sequence thereof. In some exemplary embodiments, the vector comprises a nucleotide sequence selected from the following: a nucleotide sequence encoding an amino acid sequence shown in any one of SEQ ID NOs:52-58 (preferably SEQ ID NOs:53, 57, 58), or a degenerate sequence thereof. In some exemplary embodiments, a nucleotide sequence encoding an additional peptide sequence (e.g., a peptide linker) is further included between the [second nucleotide sequence] and the [linker], the additional peptide sequence being, for example, a peptide linker containing one or more Gs and / or S, such as GSG.

[0159] In some implementations, the first nucleotide sequence and the second nucleotide sequence are located on different vectors.

[0160] In some implementations, the vector is an expression vector.

[0161] In some implementations, the vector is used as an envelope plasmid in a lentiviral vector package.

[0162] Packaging of lentiviruses

[0163] Methods for lentiviral packaging are known in the prior art. Typically, the lentiviral vector of the present invention is a non-replicating lentiviral vector; therefore, in order to obtain the lentiviral vector of the present invention, the vector genome must be packaged into particles or pseudo-particles. Thus, lentiviral proteins, except for envelope proteins, must be trans-provided to the vector genome in the production system, particularly in the production cells.

[0164] In another aspect, the present invention provides a lentivirus vector packaging system comprising:

[0165] The first nucleic acid molecule encodes the mutant vesicular virus envelope protein described herein;

[0166] The second nucleic acid molecule encodes the nonviral membrane-bound protein described herein;

[0167] The third nucleic acid molecule encodes gag and pol;

[0168] The fourth nucleic acid molecule, encoding rev; and

[0169] The fifth nucleic acid molecule, namely the lentiviral vector genome, contains the target nucleic acid.

[0170] In some embodiments, the first, second, third, fourth, and fifth nucleic acid molecules are present on one or more expression vectors. In some embodiments, the first and second nucleic acid molecules are present on one expression vector. In some embodiments, the third and fourth nucleic acid molecules are present on one expression vector, or on two different expression vectors respectively. In some embodiments, the fifth nucleic acid molecule is present on one expression vector.

[0171] In some embodiments, the lentiviral vector packaging system comprises an envelope plasmid, a packaging plasmid, and a packaging plasmid, wherein:

[0172] The enveloped plasmid contains the first nucleic acid molecule and the second nucleic acid molecule;

[0173] The packaging plasmid contains the third nucleic acid molecule and the fourth nucleic acid molecule; and

[0174] The transfer plasmid contains the fifth nucleic acid molecule.

[0175] In some implementations, the envelope plasmid encodes only viral envelope proteins.

[0176] In some implementations, the packaging plasmid encodes lentiviral proteins essential only for viral particle synthesis. Accessory genes present in the plasmid that could raise safety concerns are therefore removed. Trans-packaged viral proteins, exemplified in HIV-1, include: 1. Gag proteins, used to construct the matrix (MA, with an epigenetic molecular weight of p17), capsid (CA, p24), and nucleocapsid (NC, p6); 2. Pol-encoded enzymes: integrase, protease, and reverse transcriptase; 3. Tat and Rev-encoded regulatory proteins, with Tat required to initiate LTR-mediated transcription; it can be omitted if the U3 region of the 5′ LTR is replaced by a promoter driving Tat-independent transcription.

[0177] In some embodiments, the transfer plasmid contains a vector genome, wherein the vector genome contains the target nucleic acid but lacks the viral coding sequence and / or cis-acting genetic elements required for particle formation.

[0178] In some implementations, the vector genome contains a 5'LTR and a 3'LTR, wherein the 3'LTR is optionally deleted in the U3 region without interfering with the function required for gene transfer.

[0179] In some embodiments, the vector genome contains one or more (e.g., all) of the following: a 5' promoter (e.g., for controlling the expression of the entire packaged RNA), a 5' LTR (e.g., which includes R (polyadenylation tail signal) and / or U5 including primer activation signal), a primer binding site, a psi packaging signal, an RRE element for nuclear output, a promoter located directly upstream of the target nucleic acid to control the expression of the target nucleic acid, the target nucleic acid, a polypurine region, and a 3' LTR (e.g., which includes mutated U3, R, and U5). In some embodiments, the vector genome also contains one or more of cPPT, WPRE, and / or insulator elements.

[0180] In some embodiments, the lentiviral vector of the present invention is derived from HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Visina-medie virus (VMV); Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); Simian immunodeficiency virus (SIV), etc.

[0181] In some implementations, an HIV-based vector backbone (i.e., HIV cis-acting sequence element) is used.

[0182] In some implementations, the lentiviral vector is human immunodeficiency virus-1 (HIV-1).

[0183] In another aspect, the present invention provides a method for preparing a lentiviral vector, comprising expressing the lentiviral vector packaging system described herein in a host cell.

[0184] In some embodiments, the method includes introducing the envelope plasmid, packaging plasmid, and transfer plasmid contained in the lentiviral vector packaging system into the host cell. Methods for introducing the vector into the host cell are well known to those skilled in the art, such as transfection, including chemical transfection (calcium phosphate, liposomes, or cationic polymers), electroporation, photoporation, etc. The transfection can be transient or stable.

[0185] In some embodiments, the host cell can be any prokaryotic (bacterial), eukaryotic (yeast, insect, or animal, including mammals, especially humans) cell. In some embodiments, the host cell is a mammalian cell, such as 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, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In some embodiments, the host cell is a 293 cell, a 293T cell, or an A549 cell.

[0186] In some embodiments, the method further includes recovering viral particles from the cell culture.

[0187] Delivery of nucleic acids

[0188] In another aspect, the present invention provides a method for delivering a target nucleic acid to a target cell, comprising:

[0189] (i) Provide the lentiviral vectors described herein;

[0190] (ii) The lentiviral vector is brought into contact with the target cell to deliver the target nucleic acid to the target cell.

[0191] In some embodiments, nucleic acids are delivered to the cells when the lentivirus enters or infects the cells during step (ii). In some embodiments, the method does not require a transfection agent (e.g., a lipophilic transfection agent, such as Lipofectin).

[0192] In some implementations, the method is performed in vitro.

[0193] In some implementations, the method is carried out in vivo and includes administering the lentiviral vector described herein to a subject, thereby delivering the target nucleic acid to target cells in the body.

[0194] In some embodiments, the method is used to genetically modify the target cells.

[0195] In some embodiments, the method is used to perform gene editing on the target cells.

[0196] In some embodiments, the target nucleic acid comprises a foreign gene. The foreign gene encodes, for example, a therapeutic protein (e.g., a protein that compensates for a subject's disease condition) or an antigen (such as a pathogen antigen), a gene editing tool (e.g., the Cas protein and / or gRNA of a CRISPR / Cas system), or a gene silencing tool (e.g., shRNA).

[0197] In some embodiments, the target nucleic acid encodes an mRNA molecule, optionally wherein the mRNA is the aforementioned exogenous gene.

[0198] In some implementations, the target nucleic acid encodes double-stranded RNA, antisense RNA, microRNA, or any other RNA molecule.

[0199] In some embodiments, the target cell surface contains antigens or receptors that can be targeted by non-viral membrane-binding proteins contained in the lentiviral vector described herein.

[0200] In some embodiments, the target cell can be any prokaryotic (bacterial), eukaryotic (yeast, insect, or animal, including mammals, especially humans) cell. In some embodiments, the target cell is a human, mouse, rat, or non-human primate cell. In some embodiments, the target cell is a somatic cell or germ cell. In some embodiments, the target cell is an epithelial cell, nerve cell, hormone-secreting cell, immune cell, secretory cell, blood cell, interstitial cell, or germ cell. In some embodiments, the target cell is an antigen-specific cell (e.g., a cell that binds to a specific antigen). In some embodiments, the antigen-specific cell is an immune cell. In some embodiments, the antigen-specific cell is a B cell or a T cell.

[0201] In some implementations, the target cells are cells used for cell therapy.

[0202] Therapeutic applications

[0203] In another aspect, the present invention provides a pharmaceutical composition comprising a lentiviral vector as described herein, or target cells obtained by the method described herein for delivering target nucleic acids to target cells, and a pharmaceutically acceptable carrier and / or excipient.

[0204] In some embodiments, the pharmaceutical composition comprises an effective amount (e.g., a therapeutic or preventative effective amount) of the lentiviral vector or target cells.

[0205] In some implementations, the target nucleic acid includes a foreign gene.

[0206] In some implementations, the exogenous gene encodes, for example, a therapeutic protein (e.g., a protein that compensates for a subject’s disease condition) or an antigen (such as a pathogen antigen).

[0207] In some embodiments, the exogenous gene encodes a gene editing tool (e.g., the Cas protein and / or gRNA of the CRISPR / Cas system) or a gene silencing tool (e.g., shRNA).

[0208] In some embodiments, the pharmaceutical composition is a nucleic acid vaccine (e.g., an mRNA vaccine), and the target nucleic acid carried by the lentiviral vector is an antigen, such as a pathogen antigen.

[0209] In another aspect, the present invention provides a method for gene editing or gene therapy, comprising administering to a subject in need an effective amount of the lentiviral vector described herein or target cells obtained by the method for delivering the target nucleic acid described herein.

[0210] In some implementations, the target nucleic acid includes a foreign gene.

[0211] In some implementations, the exogenous gene encodes, for example, a therapeutic protein (e.g., a protein that compensates for a subject’s disease condition) or an antigen (such as a pathogen antigen).

[0212] In some embodiments, the exogenous gene encodes a gene editing tool (e.g., the Cas protein and / or gRNA of the CRISPR / Cas system) or a gene silencing tool (e.g., shRNA).

[0213] In another aspect, the present invention provides the use of the lentiviral vectors, lentiviral vector packaging systems, or target cells obtained by the methods for delivering target nucleic acids described herein in the preparation of medicaments for gene editing or gene therapy.

[0214] In another aspect, the present invention provides a method for inducing an immune response in a subject (e.g., a human), comprising administering an effective amount of the lentiviral vector or pharmaceutical composition described herein to a subject in need of doing so. In some embodiments, the target nucleic acid carried by the lentiviral vector is an antigen, such as a pathogen antigen. In some embodiments, the pharmaceutical composition is a nucleic acid vaccine (e.g., an mRNA vaccine).

[0215] In another aspect, the present invention provides the use of the lentiviral vector or lentiviral vector packaging system described herein in the preparation of nucleic acid vaccines (e.g., mRNA vaccines). In some embodiments, the target nucleic acid carried by the lentiviral vector is an antigen, such as a pathogen antigen.

[0216] Terminology Definition

[0217] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0218] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0219] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0220] The term "corresponding to" regarding the position of a protein / peptide, such as the statement that a nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in a disclosed sequence (such as that shown in the sequence listing), refers to the nucleotide or amino acid position identified based on structural sequence alignment or by aligning the sequence to the disclosed sequence using a standard alignment algorithm (such as the GAP algorithm). For example, corresponding residues in similar sequences (e.g., fragments or species variants) can be determined by aligning a structural sequence to a reference sequence. By aligning the sequences, those skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guidance.

[0221] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap may be introduced in the first amino acid sequence or nucleic acid sequence to best align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid 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, the molecule is identical at that position. The percentage identity between two sequences is a function of the number of identity positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions / total number of positions × 100%). In some embodiments, the two sequences are of the same length.

[0222] The determination of percentage identity between two sequences can also be achieved using mathematical algorithms. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as an improvement upon that in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are integrated into the NBLAST and XBLAST procedures of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0223] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0224] 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 Escherichia 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 fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0225] As used herein, the term “vector genome” refers to nucleic acids, specifically lentiviral nucleic acids, that make up the genome of the lentiviral vector particle. Therefore, the term encompasses any suitable nucleic acid, i.e., DNA or RNA, double-stranded or single-stranded, including forms containing DNA flaps, as a triple-stranded sequence. The properties and structure of the nucleic acids (DNA, RNA) depend on the stage of the particle cycle, including transfer plasmids (used for co-transfection of cells with packaging and envelope plasmids to express viral particles), or the RNA genome of the viral particle, or the nucleic acids of this genome in various forms in the transduced cells of the host given the viral particle (including genomic mRNA transcripts, linear unintegrated DNA reverse transcripts, or unintegrated one or two LTR DNA circular forms or integrated proviruses), including the vector pre-integration complex.

[0226] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delay agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic 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. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Absorption delay agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol).

[0227] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to the expected survival (if no treatment was received).

[0228] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for disease prevention is an amount sufficient to prevent, stop, or delay the onset of disease; an effective amount for disease treatment is an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0229] Beneficial effects of the invention

[0230] This invention identifies key amino acid sites affecting lentiviral targeted infection. Mutations at these sites can significantly inhibit lentiviral infection of target cells. Simultaneously, targeted infection of cells by lentiviruses carrying mutated membrane proteins is achieved using redirected targeting molecules (e.g., antibodies). Therefore, this invention provides a lentiviral vector with cell-specific infection capabilities, possessing broad application prospects and significant clinical importance. Attached Figure Description

[0231] Figure 1 Three lentiviral structures used for infectivity analysis.

[0232] Figure 2 Lentiviral viruses with different VSV G envelope protein mutations affect CD3 + Tests of the infectivity of Jurkat cells.

[0233] Figure 3 Lentiviral viruses with different VSV G envelope protein mutations affect CD3 - Test of the infectivity of Raji cells.

[0234] Figures 4A-4CInfectivity assays of lentiviruses with different VSV G envelope protein mutations against T cells, NK cells, and B cells in PBMCs. A: anti-CD3 retargeted lentivirus; B: lentivirus without anti-CD3 retargeting; C: Statistical results of infectivity assays.

[0235] Figure 5 Lentiviral viruses with different mutations in the Cocal G envelope protein affect CD3. + Tests on the infectivity of Jurkat cells.

[0236] Figure 6 Lentivirals with different mutations in the CocalG envelope protein affect CD3. - Test of the infectivity of Raji cells.

[0237] Figure 7 Lentivirals carrying different insertion mutations in the CocalG envelope protein respectively target CD3 + Human primary cells, CD3 + Jurkat cells, and CD3 - Infectivity testing of Raji cells. A: Infectivity test of lentivirus packaged with a mutant envelope protein inserted at the Cocal GT T184 site on the above cells; B: Infectivity test of lentivirus packaged with a mutant envelope protein inserted at the Cocal GT R354 site on the above cells.

[0238] Explanation of sequence information

[0239] Information about the sequence involved in this invention is provided in Table 1.

[0240] Table 1: Sequence Information

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252] Example

[0253] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0254] Those skilled in the art will understand that the embodiments are described by way of example only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0255] Example 1: Packaging of Lentiviral Vectors

[0256] 1. G protein mutant

[0257] Using structural biology knowledge and through extensive mutation screening, four novel key amino sites affecting lentiviral infection of host cells were identified in the VSV G polypeptide sequence. For the wild-type VSV G sequence shown in SEQ ID NO:3, these are lysine at position 50 (K50), isoleucine at position 331 (I331), isoleucine at position 347 (I347), and methionine at position 184 (M184). 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 mutants VSV G-K50E, VSV G-I331E, VSV G-I347E, and VSV G-M184D are shown in SEQ ID NO:5-8, respectively.

[0258] The Cocal virus G protein, structurally similar to VSV G, shares 72.93% sequence homology, differing only in the flexible region of the proximal membrane domain (MP) and effectively resisting neutralization in serum. Therefore, the aforementioned mutation sites were introduced into the wild-type Cocal G protein (SEQ ID NO:22). The specific mutations in CocalG are: lysine at position 50 to glutamic acid (K50E), isoleucine at position 331 to glutamic acid (I331E), isoleucine at position 347 to glutamic acid (I347E), and threonine at position 184 to aspartic acid (T184D). The amino acid sequences of the four mutants, CocalG-K50E, CocalG-I331E, CocalG-I347E, and CocalG-T184D, are shown in SEQ ID NO:24-27, respectively.

[0259] In addition, the above mutation sites were 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), and Carajas virus G protein (SEQ ID NO:39), with the mutation forms shown below:

[0260] The specific mutations in Indiana G are: lysine at position 50 is mutated to glutamic acid (K50E), isoleucine at position 331 is mutated to glutamic acid (I331E), isoleucine at position 347 is mutated to glutamic acid (I347E), and methionine at position 184 is mutated to aspartic acid (M184D).

[0261] The specific mutations in New Jersey G are: threonine at position 50 is mutated to glutamic acid (T50E), leucine at position 335 is mutated to glutamic acid (L335E), valine at position 351 is mutated to glutamic acid (V351E), and valine at position 184 is mutated to aspartic acid (V184D).

[0262] The specific mutations in Maraba G are: lysine at position 50 is mutated to glutamic acid (K50E), isoleucine at position 331 is mutated to glutamic acid (I331E), methionine at position 347 is mutated to glutamic acid (M347E), and valine at position 184 is mutated to aspartic acid (V184D).

[0263] The specific mutations in Morreton G are: lysine at position 50 is mutated to glutamic acid (K50E), isoleucine at position 331 is mutated to glutamic acid (I331E), isoleucine at position 347 is mutated to glutamic acid (I347E), and methionine at position 184 is mutated to aspartic acid (M184D).

[0264] The specific mutations in Alagoa G are: lysine at position 50 is mutated to glutamic acid (K50E), leucine at position 331 is mutated to glutamic acid (L331E), valine at position 347 is mutated to glutamic acid (V347E), and threonine at position 184 is mutated to aspartic acid (T184D).

[0265] The specific mutations in Carajas G are: lysine at position 50 is mutated to glutamic acid (K50E), isoleucine at position 335 is mutated to glutamic acid (I335E), valine at position 351 is mutated to glutamic acid (V351E), and methionine at position 184 is mutated to aspartic acid (M184D).

[0266] 2. Plasmid / Sequence

[0267] Lentiviral packaging systems include envelope plasmids, packaging plasmids, and transfer plasmids.

[0268] The envelope plasmid carries either (i) a G protein coding sequence or (ii) a construct of the G protein and target molecule, wherein the construct of the G protein and target molecule has the following structure: [signal peptide]-[CD3scFv(αCD3)]-[hinge region]-[transmembrane region]-[connector]-[T2A]-[G protein], and the full-length amino acid sequence corresponding to this nucleic acid construct is shown in SEQ ID NOs:15-20, 28-33. Those skilled in the art will understand that self-cleaving peptides (such as T2A) are used to cleave multiple proteins in a single transcript. Two proteins connected by a self-cleaving peptide (such as T2A) will be cleaved during translation to form two independent proteins, thereby generating an independent G protein and target molecule (αCD3).

[0269] The construction of the above-mentioned envelope plasmids was commissioned to Qingke Biotechnology Co., Ltd. The lentiviral transfer plasmid Len-E carrying EGFP and the endotoxin-free plasmid pSPAX2 encoding gag-pol-rev were both prepared by Qingke Biotechnology.

[0270] 3. Cells

[0271] HEK 293T17 (Nanjing Kebai) cells were maintained in DMEM containing 10% FBS, 1% Pen / Strep, and 1% L-glutamine.

[0272] 4. Packaging and concentration preparation of lentiviruses

[0273] Packaging plasmids were introduced into HEK 293T17 cells using PEI (Polysciences) to prepare... Figure 1 The three recombinant lentiviral particles with the shown structures were transfected with three plasmids: (1) a plasmid expressing VSV-G glycoprotein and / or targeting molecules (αCD3), (2) a plasmid encoding gag-pol-rev (pSPAX2), and (3) a lentiviral transfer plasmid carrying EGFP. 30-48 h after transfection, the culture supernatant was collected and refilled with fresh DMEM medium preheated with 5% FBS, and stored temporarily at 4°C. 72 h after transfection, the culture supernatant was collected and mixed with the supernatant collected 30-48 h after transfection. The virus in the cell supernatant was concentrated using PEG8000 and resuspended in serum-free RPMI1640. The lentiviral particle titer was determined using the Lenti-X p24 rapid titer kit (Takara Bio). The specific experimental steps are as follows:

[0274] 1) Place 1.7×10⁻⁶ plants in a 15cm culture plate. 7 HEK 293T cells were added to 20 mL of DMEM (Hyclone, SH30243.01) medium containing 10% FBS (Gibco, 10099-141C), and the cells were thoroughly mixed and cultured overnight at 37°C.

[0275] 2) On the second day, when the HEK 293T (ATCC, CRL-3216) cells reach about 90% confluence, replace the medium with DMEM containing 5% FBS.

[0276] 3) Prepare plasmid complexes with the following amounts: 21 μg plasmid DNA, 14 μg psPAX2 and 7 μg pMD2. Dissolve them in 1 mL opti-MEM (Gibco, 31985-070) and add 126 μL PEI (Polysciences, 24765-2). Vortex for 20 s, let stand at room temperature for 15 minutes, and then gently add the mixture along the edge to HEK293T medium. Continue culturing at 37°C.

[0277] 4) 30-48 h after transfection, collect the culture supernatant, add fresh DMEM medium preheated with 5% FBS, and store temporarily at 4°C.

[0278] 5) 72 h after transfection, collect the culture supernatant and mix it with the supernatant from 30-48 h. Centrifuge at 1000 g for 10 min. Filter the supernatant through a 0.45 μm filter and add PEG8000 (Sigma, 89510-1KG-F) to a final concentration of 5.3% and NaCl (Sigma, S5150-1L) to a final concentration of 0.27 M. Mix thoroughly and incubate overnight at 4 °C.

[0279] 6) Centrifuge the viral supernatant at 4000g for 40 minutes at 4℃, discard the supernatant, dissolve the precipitate with 400μL of serum-free DMEM, aliquot and store in a -80℃ freezer.

[0280] 7) Lentivirus particle titers were determined using the Lenti-X p24 rapid titer kit (Takara Bio).

[0281] Example 2: Lentiviral infection of Jurkat and Raji cells based on VSV G envelope protein mutation

[0282] 1. Cells

[0283] Jurkat cells were maintained in PRMI 1640 medium containing 10% FBS, 1% Pen / Strep, and 1% L-glutamine. Raji cells were maintained in PRMI 1640 medium containing 10% FBS, 1% Pen / Strep, and 1% L-glutamine.

[0284] 2. The experimental steps for lentiviral infection of Jurkat and Raji cells are as follows:

[0285] 1) Take Jurkat tumor cell line (T cell-derived) or Raji cell line (B cell-derived) into 96-well U-plates, with a cell count of 3 × 10⁶ cells / well. 4 Each sample was resuspended in PRMI 1640 medium containing 10% FBS, 1% Pen / Strep, and 1% L-glutamine, at a volume of 50 μl / well.

[0286] 2) Based on the p24 quantification results of concentrated lentivirus titers, lentivirus (50 μL / well) and 12 μg / mL protamine (Sigma, P4005) were added in 2-fold serially diluted lentivirus to 96-well U-diban containing Jurkat or Raji cells to complete the target cell infection. After 8 h, PRMI 1640 medium containing 10% FBS, 1% Pen / Strep, and 1% L-glutamine was added to 100 μL / well.

[0287] 3) 48 hours after lentiviral infection, the infected target cells were centrifuged at 300g for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS solution. The proportion of EGFP-positive cells was then analyzed on a CytoFLEX flow cytometer (Beckman).

[0288] The results of this experiment are as follows: Figure 2 and Figure 3 The results showed that, compared with wild-type VSV G envelope proteins, lentiviruses packaged with VSV G envelope proteins containing K50E, I331E, I347E, or M184D mutants exhibited significantly reduced infectivity against target cells. However, when the infectivity of lentiviruses packaged with various mutant envelope proteins was redirected using anti-CD3 antibodies (constructs introducing G proteins and targeting molecules), all redirected lentiviruses regained infectivity against CD3-positive Jurkat cells, while their infectivity against CD3-negative Raji cells was weak. In summary, the four sites of VSV G—K50, I331, I347, and M184—play similarly crucial roles in the targeted infection of VSV G.

[0289] Example 3: Lentiviral infection of PBMC cells based on VSV G envelope protein mutation

[0290] 1) Commercially available PBMCs (Shanghai Heyousheng Biotechnology) cells were cultured in X-Vivo 15 (Lonza) medium containing 1% human AB serum, 1% HSA, 1% Pen / Strep, and 1% L-glutamine, with an initial cell density of 1×10⁻⁶ cells / year. 6 / mL;

[0291] 2) Add T cellTransact (Miltenyibiotec, 130-111-160) at a ratio of 10ul / ml, and add 1000IU / mL of IL-2 (Tetracycline Biotech, National Drug Approval Number S10970016) to activate T cell proliferation.

[0292] 3) After 48 hours of cell activation, PMBCs were transferred to 96-well U-plates at a cell count of 3 × 10⁶ cells / well. 4 Inoculate with 50 μl of concentrated lentivirus (500 ng P24 concentration) and 12 μg / mL protamine (Sigma, P4005) per well. After 12 hours of infection, add 100 μl of fresh culture medium per well.

[0293] 4) On day 4 of lentiviral infection, flow cytometry was used to analyze the transduced cells to determine the proportion of GFP-positive cells. Specifically, anti-CD3 antibody was used to stain cells and detect CD3-positive cells (BV421 mouse anti-human CD3, clone UCHT1, BD) and GFP expression; anti-CD19 antibody was used to stain cells and detect CD19-positive cells (PE mouse anti-human CD19, clone 4G7, Abcam) and GFP expression; anti-CD56 antibody was used to stain cells and detect CD56-positive cells (APC mouse anti-human CD56, clone TULY56, eBioscience) and GFP expression.

[0294] The results of this experiment are shown in Figure 4. VSV-G pseudotyped lentivirus carrying the target molecule CD3, when transduced into human PBMCs, was able to transduce CD3-positive T cells, almost no CD19-positive B cells, and a small number of CD56-positive NK cells. This may be due to the expression of CD3e on the surface of NK cells. Lentiviral viruses that did not express anti-CD3 redirection molecules did not show transduction ability in any of the three cell populations.

[0295] Example 4: Lentiviral infection of Jurkat and Raji cells based on mutation of the CocalG envelope protein

[0296] The VSV G protein used in this study is highly homologous to the Indiana VSV strain G protein, differing by only two amino acids. Furthermore, the Cocal virus G protein is also highly similar in structure to the VSV G protein used in this study, with a sequence homology of 72.93%, differing only in the flexible region of the proximal membrane domain (MP), and effectively resists neutralization in serum. Therefore, we also tested the effect of the aforementioned mutation sites in the Cocal G protein on the specificity of lentiviral infection. The results of this experiment are as follows: Figure 5 and Figure 6 The results showed that the corresponding Cocal G mutant protein could also block the ability of lentiviruses to infect target cells.

[0297] The above embodiments have verified that the four sites K50, I331, I347, and M184 on VSV G and their corresponding sites on the Cocal G protein can block the target cell infectivity of lentiviruses. Therefore, those skilled in the art will understand that the above-mentioned mutation sites are also applicable to the G proteins of other VSV strains (e.g., Indiana and New Jersey strains), as well as the G proteins of other vesicular viruses such as Maraba, Morreton, Alagoa, and Carajas, all of which have high homology with the VSV G protein in this study. The corresponding mutation sites of these VSV strains' G proteins, or their combined mutations, should also be within the scope of protection of this invention.

[0298] Example 5: Lentiviral infection of Jurkat and Raji cells based on insertional mutation of Cocal G envelope protein

[0299] In addition to the four new key amino sites and corresponding substitution mutations identified in the above embodiments, it was also found that insertion mutations at specific amino acid sites can also weaken or eliminate the target cell infectivity of lentiviruses.

[0300] In this study, E (Glu), I (Ile), A (Ala), AA (Ala-Ala), or GAA (Gly-Ala-Ala) were inserted before the T184 site of the Cocal viral G protein (i.e., between D183 and T184), or P (Pro) or A (Ala) was inserted before the R354 site (i.e., between E353 and R354) to form the corresponding insertion mutant proteins of the Cocal viral G protein, the sequences of which are shown in SEQ ID NO:41-47. Following the method described in Example 1, constructs of the G protein and target molecules were constructed and packaged lentiviruses. The full-length amino acid sequences of these nucleic acid constructs are shown in SEQ ID NOs:52-58. Subsequently, the lentiviral infectivity was tested according to the method described in Example 2.

[0301] The results are as follows Figure 7 As shown, inserting Ile before the T184 site or Pro or Ala amino acids before the R354 site of the CocalG protein can weaken or eliminate the infectivity of the corresponding mutant protein-packaged lentivirus.

[0302] Those skilled in the art will understand that the above-mentioned insertion mutations also apply to the G proteins of VSV strains, VSV Indiana strains, and VSV New Jersey strains, as well as to the G proteins of other vesicular viruses such as Maraba, Morreton, Alagoa, and Carajas, all of which have a high degree of homology with the Cocal G protein in this study.

[0303] The T184 site of the Cocal G protein corresponds to M184 of the VSV G protein (SEQ ID NO:3), M184 of the VSV Indiana strain G protein (SEQ ID NO:34), V184 of the VSV New Jersey strain G protein (SEQ ID NO:38), V184 of the Maraba virus G protein (SEQ ID NO:35), M184 of the Morreton virus G protein (SEQ ID NO:36), T184 of the Alagoa virus G protein (SEQ ID NO:37), and M184 of the Carajas virus G protein (SEQ ID NO:39).

[0304] The R354 site 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), and R358 of the Carajas virus G protein (SEQ ID NO:39).

[0305] The corresponding insertion mutations in the G protein of these VSV strains should also be within the scope of protection of this invention.

Claims

1. A mutated vesiculovirus envelope protein comprising an amino acid mutation (e.g., substitution, insertion, or deletion) at at least one of the following amino acid positions (e.g., position 1, position 2, position 3, position 4, or position 5): amino acid positions 50, 331, 347, 184, and 354, as indicated in SEQ ID NO:3 or SEQ ID NO:

22.

2. The mutated vesicular virus envelope protein of claim 1, wherein, The amino acid mutation is an amino acid substitution; Preferably, the amino acid is mutated and replaced with an acidic amino acid (e.g., E or D).

3. The mutant vesicular virus envelope protein of claim 2, wherein at least one (e.g., position 1, position 2, position 3 or 4) of the amino acid residues at the positions corresponding to the amino acid positions shown in SEQ ID NO:3 or SEQ ID NO:22 is replaced with an acidic amino acid (e.g., E or D).

4. The mutated vesicular virus envelope protein of claim 1, wherein, The amino acid mutation is replaced by an amino acid insertion; Preferably, the amino acid insertion is the insertion of one, two, or three consecutive amino acids; Preferably, the inserted amino acid is selected from E (Glu), I (Ile), A (Ala), AA (Ala-Ala), GAA (Gly-Ala-Ala), or P (Pro).

5. The mutated vesicular virus envelope protein of claim 4, wherein it 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, or, wherein it comprises an A (Ala) insertion or a P (Pro) insertion between positions 353 and 354 corresponding to SEQ ID NO:3 or SEQ ID NO:22; Preferably, the mutated vesicular virus envelope protein contains an I (Ile) insertion between positions 183 and 184 corresponding to SEQ ID NO:3 or SEQ ID NO:22, or contains an A (Ala) insertion or a P (Pro) insertion between positions 353 and 354 corresponding to SEQ ID NO:3 or SEQ ID NO:

22.

6. The mutated vesicular virus envelope protein according to any one of claims 1-5, wherein the protein is selected from 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 vesicular virus envelope protein has a mutation as defined in any one of claims 1-5 compared to the wild-type protein from which it originates; Preferably, wherein: Wild-type VSV G protein has the sequence shown in any one of SEQ ID NOs:3, 34, 38 or a sequence that has at least 80% identity with it; The wild-type CocalG protein has the sequence shown in SEQ ID NO:22 or a sequence that is at least 80% identical to it; The wild-type Maraba G protein has the sequence shown in SEQ ID NO:35 or a sequence that is at least 80% identical to it; The wild-type Morreton G protein has the sequence shown in SEQ ID NO:36 or a sequence that is at least 80% identical to it; Wild-type Alagoa G protein has the sequence shown in SEQ ID NO:37 or a sequence having at least 80% identity with it; and / or, The wild-type Carajas G protein has the sequence shown in SEQ ID NO:39 or a sequence that is at least 80% identical to it.

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

39.

8. The vesicular virus envelope protein according to any one of claims 1-6, having a sequence selected from the following: (i) The sequence shown in any one of SEQ ID NOs:41-47; for example, the sequence shown in any one of SEQ ID NOs:42,46,47; (ii) Compared with the sequence shown in SEQ ID NO:3, it contains an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between S183 and M184, or an A insertion or P insertion between E353 and R354; or, compared with the sequence shown in SEQ ID NO:34, it contains an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between S183 and M184, or an A insertion or P insertion between E353 and R354; or, compared with the sequence shown in SEQ ID NO:38, it contains an E insertion, I insertion, A insertion, AA insertion, or GAA insertion (e.g., I insertion) between L183 and V184, or an A insertion or P insertion between V357 and R358; (iii) Compared with the sequence shown in SEQ ID NO:35, it contains an E insertion, I insertion, A insertion, AA insertion or GAA insertion (e.g. I insertion) between S183 and V184, or contains an A insertion or P insertion between E353 and R354; (iv) Compared with the sequence shown in SEQ ID NO:36, it contains an E insertion, I insertion, A insertion, AA insertion or GAA insertion (e.g. I insertion) between S183 and M184, or contains an A insertion or P insertion between E353 and R354; (v) Compared with the sequence shown in SEQ ID NO:37, it contains an E insertion, I insertion, A insertion, AA insertion or GAA insertion (e.g. I insertion) between S183 and T184, or contains an A insertion or P insertion between K353 and R354; (vi) Compared with the sequence shown in SEQ ID NO:39, it contains an E insertion, I insertion, A insertion, AA insertion or GAA insertion (e.g. I insertion) between A183 and M184, or contains an A insertion or P insertion between S357 and R358.

9. An isolated nucleic acid molecule encoding the mutant vesicular virus envelope protein as described in 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 vesicular virus envelope protein according to any one of claims 1-8, and the lentiviral vector genome comprises the target nucleic acid; Preferably, the lentiviral vector is pseudotyped by the mutated vesicular virus envelope protein.

12. The lentiviral vector of claim 11, further comprising a nonviral membrane-binding protein, said nonviral membrane-binding protein comprising an extracellular targeting domain and a membrane-binding domain. Preferably, the extracellular targeting domain includes a specific binding domain for target cells; Preferably, the extracellular targeting domain is an antibody or ligand targeting a target cell surface antigen; Preferably, the extracellular targeting domain is an antibody against a target cell surface antigen, such as scFv or VHH; Preferably, the target cell surface antigen is selected from T cell markers, γδT cell markers, NK T cell markers, MAIT cell markers, tumor cells, or other cell markers; Preferably, the extracellular targeting domain is an anti-CD3 antibody.

13. The lentiviral vector of claim 12, wherein, The non-viral membrane-bound protein also includes a hinge region between the extracellular targeting domain and the membrane-bound domain. Preferably, the hinge region comprises the sequence shown in SEQ ID NO:

11.

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

12.

15. The lentiviral vector according to any one of claims 12-14, wherein, The non-viral membrane-bound protein also contains 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 shown in SEQ ID NO:

10.

16. The lentiviral vector according to any one of claims 12-15, wherein, The non-viral membrane-bound protein contains the amino acid sequence shown in SEQ ID NO:40 or 49.

17. The lentiviral vector according to any one of claims 11-16, wherein, The lentiviral vector genome further includes cis-acting elements, such as LTR.

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

19. A lentiviral vector packaging system, comprising: A first nucleic acid molecule encoding the mutant vesicular virus envelope protein as described in any one of claims 1-8; A second nucleic acid molecule encoding a non-viral membrane-bound protein, as defined in any one of claims 12-16; The third nucleic acid molecule encodes gag and pol; The fourth nucleic acid molecule, encoding rev; and The fifth nucleic acid molecule contains the target nucleic acid; 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: An enveloped plasmid comprising the first nucleic acid molecule and the second nucleic acid molecule; Packaging plasmid, comprising the third and fourth nucleic acid molecules; and The transfer plasmid contains the fifth nucleic acid molecule.

21. A method for preparing a lentiviral vector, comprising expressing the lentiviral vector packaging system of claim 19 in a host cell; Preferably, the method includes: Introduce the envelope plasmid, packaging plasmid, and transfer plasmid as described in claim 20 into the host cell; Preferably, the method further includes recovering viral particles from the culture of the host cells.

22. A method for delivering a target nucleic acid to a target cell, comprising: (i) providing a lentiviral vector according to any one of claims 11-17; (ii) The lentiviral vector is brought into contact with the target cell to deliver the target nucleic acid into the cell; Preferably, the target nucleic acid includes exogenous genes, such as those encoding therapeutic proteins or pathogen antigens (e.g., viral antigens), gene editing tools (e.g., Cas proteins and / or gRNAs of the CRISPR / Cas system), or gene silencing tools (e.g., shRNA). Preferably, the method is performed in vitro; Preferably, the method is used to genetically modify the target cells; Preferably, the method is used for gene editing of the target cells.

23. A pharmaceutical composition comprising a lentiviral vector as described in any one of claims 11-17 or a 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 includes a foreign gene; Preferably, the exogenous gene encodes a therapeutic protein or antigen (e.g., a pathogen antigen); Preferably, the exogenous gene encodes a gene editing tool (e.g., the Cas protein and / or gRNA of the CRISPR / Cas system) or a gene silencing tool (e.g., 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 cells obtained by the method of claim 22, or the pharmaceutical composition of claim 23 in the preparation of a medicament for gene editing or gene therapy; Preferably, the target nucleic acid carried by the lentiviral vector includes a foreign gene; Preferably, the exogenous gene encodes a therapeutic protein or antigen (e.g., a pathogen antigen); Preferably, the exogenous gene encodes a gene editing tool (e.g., the Cas protein and / or gRNA of the CRISPR / Cas system) or a gene silencing tool (e.g., 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 preparation of a nucleic acid vaccine (e.g., an mRNA vaccine); Preferably, the lentiviral vector carries a target nucleic acid encoding an antigen, such as a pathogen antigen.

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