WO2024022147A1

WO2024022147A1 - Baev membrane glycoprotein and use thereof

Info

Publication number: WO2024022147A1
Application number: PCT/CN2023/107696
Authority: WO
Inventors: 黄宇康; 陈运凡; 沈俊杰; 徐艳敏; 洪娟
Original assignee: 重庆精准生物技术有限公司; 重庆精准生物产业技术研究院有限公司
Priority date: 2022-07-28
Filing date: 2023-07-17
Publication date: 2024-02-01

Abstract

Provided are a virus membrane glycoprotein or a polypeptide or a composition thereof for packaging viruses and a membrane virus packaged using the membrane glycoprotein or the polypeptide. Also provided is a method for packaging a membrane virus using the virus membrane glycoprotein or the polypeptide or the composition thereof. The method can improve the packaging efficiency of the membrane virus and the transduction efficiency of the packaged virus.

Description

BaEV envelope glycoprotein and its applications

Cross-references to related applications

This application claims the priority rights of Chinese application numbers 2022108997893 and 2022108982703 submitted on July 28, 2022, the entire contents of which are incorporated herein by reference.

Submission of sequence listing

This application contains an electronically submitted sequence listing in XML format, which sequence listing is hereby incorporated by reference in its entirety. The sequence list in XML format was created on July 17, 2023, with the name: TFG00836PCT-seq.xml and a size of 46,261bp.

Technical field

This application relates to lentiviruses or other retroviruses used to transduce NK, αβT, γδT and other cells, plasmids and cell lines used for packaging of said lentiviruses or other retroviruses, and packaging using said plasmids or cell lines Lentiviral or other retroviral methods.

Background technique

In recent years, tumor immunotherapy represented by CAR-T cell therapy has shown good results and huge potential. However, the expression of CAR in T cells is one of the important factors affecting the efficacy of CAR-T cells. With the clinical progress of CAR-T, in order to further optimize the efficacy of CAR-T, improve the immune microenvironment and improve the persistence of CART, there are more and more solutions. In the design of CAR, there are higher requirements for the ability of CAR to transduce T cells. And with the development of technology, there is a demand for universal cell therapy products. There have been studies on using natural killer cells (Nature killer cells, hereinafter abbreviated as "NK" or "NK cells") and γδ T cells for universal immune cell therapy. , but this type of cells is more difficult to transduce or modify and edit using other methods than traditional T cells. In addition, gene editing of innate immune cells and stem cells such as macrophages and DC cells is also attracting more and more attention. These cells are also difficult to genetically manipulate.

Non-viral transduction technology represented by electroporation technology has gradually become widely recognized due to its high safety and convenient process. However, the massive cell death caused by electroporation hinders this further promotion of technology. Although the method of delivering mRNA through electroporation can effectively reduce toxicity and increase cell viability, this transduction is transient and is not conducive to the sustained efficacy of CAR-NK.

The currently used mature VSV-G (pseudotyped lentivirus packaged with VSV-G (vesicular stomatitis virus envelope glycoprotein)) is not enough to solve the above-mentioned transduction and genetic manipulation problems. A large number of studies have shown that VSV-G lentivirus The efficiency of viral transduction of NK cells is extremely low (only 5 to 10%). The reason for this phenomenon is most likely because NK cells have natural antiviral capabilities, and innate immune cells such as γδT also have natural antiviral capabilities. It has also been reported in the literature that the transduction efficiency of cells, DC cells, and macrophages using VSV-G lentivirus is extremely low, and our previous data also confirmed this. Although there are also some reports in the literature on methods to improve the transduction ability of VSV-G pseudotyped lentivirus, the reagents used are difficult to meet the requirements for clinical application. For example, it is reported in the literature that the use of PDK1 inhibitor BX795 can effectively improve the transduction of VSV-G pseudotyped lentivirus. The efficiency of NK cells, but BX795 will have a certain impact on the killing function and proliferation ability of NK cells, which obviously does not meet the requirements for clinical application. Studies have shown that some retroviruses can efficiently transduce NK cells, but there are certain risks in the safety of retrovirus insertion sites. This shortcoming limits their promotion in clinical applications.

Patent WO2013/045639A1 announced that the modified lentivirus (BaEV lentivirus) packaged with Baboon endogenous retrovirus (Baboon endogenous virus, BaEV) envelope glycoprotein can efficiently transduce T cells and B cells. Although BaEV envelope glycoprotein (BaEV-G) has extremely high application value, it is difficult to produce high-titer pseudovirions with BaEV envelope glycoprotein. Currently, there are two main forms of BaEV envelope glycoprotein used for lentivirus packaging: 1. BaEV-Rless, which is the form of BaEV envelope glycoprotein without Fusion restrictive R peptide; 2. BaEV/ TR is the form of BaEV envelope glycoprotein in which the tail domain is replaced by the tail domain of MLV envelope glycoprotein. Expression of BaEV-Rless in 293T will cause 293T to form a large number of syncytia, leading to massive cell death and seriously affecting lentivirus packaging. By optimizing the BaEV-Rless lentivirus packaging process with 293F, BaEV-Rless lentivirus of up to approximately 1E9TU (p24 protein measured by ELISA) can be produced in a 1L system, but the yield of this process is unstable and the titer is still difficult to meet demand (Bauler , M., et al., Production of lentiviral vectors using suspension cells grown in serum-free media. Molecular Therapy-Methods Clinical Development, 2020.17: p.58-68). Compared with BaEV-Rless, the BaEV/TR form has greatly reduced cytotoxicity and greatly reduces the appearance of syncytia during the virus packaging process, but the virus titer is lower than the BaEV-Rless form. Therefore, optimizing the structure of BaEV envelope glycoprotein and improving the packaged virus titer are the keys to whether BaEV envelope glycoprotein can be used in the production and preparation of engineered immune cells and stem cells that are difficult to transduce.

Summary of the invention

In order to improve the packaging titer of enveloped viruses while maintaining or further improving its transduction efficiency for immune cells or stem cells that are difficult to transduce, this application provides a packaging method for pseudoviruses, in which the envelope glycoprotein is used, and Pseudoviruses or virus particles packaged using the method described.

Specifically, this application relates to:

1. A chimeric viral envelope glycoprotein or polypeptide. The chimeric viral envelope glycoprotein or polypeptide includes the extracellular region, the transmembrane region of the BaEV envelope glycoprotein (BaEV-G), and the tail domain of the MoRV envelope glycoprotein. In some embodiments, the chimeric viral envelope glycoprotein or polypeptide is compared to wild-type BaEV-G only in that the chimeric viral envelope glycoprotein or polypeptide has different characteristics compared to wild-type BaEV-G. The tail domain is derived from the tail domain of MoRV envelope glycoprotein, that is, the tail domain is wild-type MoRV envelope glycoprotein or a functional derivative thereof. In some embodiments, the extracellular region, the transmembrane region, and the tail domain of the MoRV envelope glycoprotein of the BaEV envelope glycoprotein (BaEV-G) in the chimeric viral envelope glycoprotein or polypeptide Connect via connectors or directly. In some embodiments, the pseudovirus is a lentivirus or other retrovirus. In some embodiments, the lentivirus or other retrovirus is derived from HIV.

2. The chimeric viral envelope glycoprotein or polypeptide according to item 1, wherein the extracellular region sequence of the BaEV envelope glycoprotein includes the amino acid sequence shown in SEQ ID NO: 1 or a functional derivative thereof, or Contains about 70% or more (such as 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more) identity to the amino acid sequence shown in SEQ ID NO: 1 the sequence of. In some embodiments, the extracellular region sequence of the BaEV envelope glycoprotein is the amino acid sequence shown in SEQ ID NO: 1.

3. The chimeric viral envelope glycoprotein or polypeptide according to item 1 or 2, wherein the transmembrane region sequence of the BaEV envelope glycoprotein includes the amino acid sequence shown in SEQ ID NO: 2 or 19 or its function. Derivatives, or containing about 70% or more (such as 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more) identity with SEQ ID NO: 2 or 19 amino acid sequence. In some embodiments, the amino acid sequence of the transmembrane region of the BaEV envelope glycoprotein is the amino acid sequence shown in SEQ ID NO: 1 or 19.

4. The chimeric viral envelope glycoprotein or polypeptide according to any one of items 1-3, wherein the tail domain of the MoRV envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 3 Or its functional derivative, or has about 70% or more (such as 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more) identical amino acid sequences. In some embodiments, the tail domain of the MoRV envelope glycoprotein is the amino acid sequence set forth in SEQ ID NO: 3.

5. The chimeric viral envelope glycoprotein or polypeptide according to item 1, which contains the amino acid sequence as in SEQ ID NO: 4 or its functional derivatives, or has more than about 70% similarity with SEQ ID NO: 4 (for example 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more) identical amino acid sequences.

In some embodiments, the chimeric viral envelope glycoprotein or polypeptide according to any one of the above, wherein the wild-type BaEV-G and the chimeric viral envelope glycoprotein or polypeptide both comprise SEQ ID NO : The amino acid sequence shown in 13 or its functional derivative, or has more than 70% (such as 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%) with SEQ ID NO: 13 or more than 99%) sequence identity of the amino acid sequence. In some embodiments, the sequence of the chimeric viral envelope glycoprotein or polypeptide is set forth in SEQ ID NO: 4.

6. The protein or polypeptide according to any one of the preceding items, wherein the protease cleavage site of the tail domain of the MoRV envelope glycoprotein is replaced by an HIV protease cleavage site.

7. The chimeric viral envelope glycoprotein or polypeptide according to item 6, wherein the HIV protease cleavage site comprises the amino acid sequence shown in SEQ ID NO: 9 or a functional derivative thereof, or is identical to SEQ ID NO :9 An amino acid sequence having about 70% or more (eg, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more) identity. In some embodiments, the HIV protease cleavage site is the amino acid sequence set forth in SEQ ID NO: 9.

In some embodiments, the protease cleavage site sequence in the tail domain of the MoRV envelope glycoprotein that is replaced by the aforementioned amino acid sequence is the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the tail domain of the chimeric viral envelope glycoprotein or polypeptide comprises an amino acid sequence as in SEQ ID NO: 20 or a functional derivative thereof, or is about 70% identical to SEQ ID NO: 20 Amino acid sequences that are more than (eg, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more) identical. In some embodiments, the tail domain of the chimeric viral envelope glycoprotein or polypeptide is the amino acid sequence in SEQ ID NO: 20. In some embodiments, the chimeric viral envelope glycoprotein or polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 21, or a functional derivative thereof, or is more than 70% identical to SEQ ID NO: 21 (e.g. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more) sequence identity of the amino acid sequence. In some embodiments, the chimeric viral envelope glycoprotein or polypeptide has an amino acid sequence such as SEQ ID NO: shown in 21.

8. Nucleic acid encoding the protein or polypeptide according to any one of items 1-7. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid comprises both deoxyribonucleosides and ribonucleosides. In some embodiments, the nucleic acid contains chemical modifications. In some embodiments, the nucleic acid comprises a promoter for promoting expression of the protein or polypeptide. In some embodiments, the promoter is a eukaryotic promoter. In some embodiments, the promoter is selected from: CAG, miniCMV, SV40.

9. Nucleic acid according to item 8, which contains any one of the polynucleotide sequences shown in SEQ ID NO: 5, 6, 7, 8 and 27, or contains the same polynucleotide sequence as SEQ ID NO: 5, 6, 7, 8 A polynucleoside having about 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more) identity to any of the polynucleotide sequences in 27 acid sequence. In some embodiments, the polynucleotide sequence of the nucleic acid is selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, and 27.

10. Plasmid, virus particle, or artificial nanoparticle containing the nucleic acid according to item 8 or 9. In some embodiments, the plasmid is an envelope plasmid for viral packaging. In some embodiments, the envelope plasmid is any plasmid useful for expressing foreign proteins within eukaryotic cells. In some embodiments, the plasmid is a retroviral envelope plasmid. In some embodiments, the plasmid is a lentiviral envelope plasmid. In some embodiments, the envelope plasmid is the backbone structure of pMD2.G with the nucleic acid sequence of item 8 or item 9 inserted. In some embodiments, the plasmid is a pMD2.G plasmid in which the VSV-G nucleic acid coding sequence is replaced with the nucleic acid sequence of item 8 or item 9. In some embodiments, the envelope plasmid is a pcDNA3.1 plasmid in which the nucleic acid sequence of item 8 or item 9 is inserted.

11. Comprising the chimeric viral envelope glycoprotein or polypeptide according to any one of items 1 to 7, and/or the nucleic acid according to item 8 or 9, and/or the plasmid or virus according to item 10 particles, or cells of artificial nanoparticles.

12. The cell according to item 11, wherein the cell is a 293T cell or a derivative thereof. In some embodiments, the cells are selected from 293T cells, 293F cells, HEK293 cells, 293T/17SF cells. In some embodiments, the cells are immune effector cells or stem cells. In some embodiments, the cells are T cells, B cells, or NK cells.

13. A composition or complex comprising:

VSV envelope glycoprotein or nucleic acid, plasmid, virus particle or artificial nanoparticle encoding VSV envelope glycoprotein; and

The chimeric viral envelope glycoprotein or polypeptide according to any one of items 1-7, according to the item 8 or 9 The nucleic acid described above, the plasmid, virus particle, or artificial nanoparticle according to item 10, or the cell according to item 11 or 12.

In some embodiments, the compositions or complexes are suitable for use in first, second and third generation lentiviral packaging systems.

In some embodiments, the composition further comprises nucleic acids encoding Gag, Pol, Rev, and Tat, or nucleic acids encoding Gag, Pol, and Rev but not Tat. In some embodiments, the coding nucleic acids comprising Gag, Pol, Rev and Tat are present in one or two or more plasmids respectively. In some embodiments, the plasmid comprising the nucleic acid encoding Gag and Pol is pMDlg/pRRE. In some embodiments, the plasmid comprising a Rev-encoding nucleic acid is a pRSV-Rev plasmid.

In some embodiments, nucleic acids encoding Gag, Pol, Rev and Tat are present in the cell according to item 11 or 12. In some embodiments, the composition further comprises nucleic acid encoding a gene sequence of interest, a promoter that drives expression of the gene of interest, an LTR, and a psi packaging signal. In some embodiments, the plasmid comprising a nucleic acid encoding a gene sequence of interest, a promoter for initiating expression of the gene of interest, an LTR, and a psi packaging signal is a transfer plasmid.

In some embodiments, the present application also provides a kit for pseudovirus packaging, which includes the composition or complex in item 13. In some embodiments, the pseudovirus is a retrovirus. In some embodiments, the virus is a lentivirus.

14. The chimeric viral envelope glycoprotein or polypeptide according to any one of items 1 to 7, the nucleic acid according to item 8 or 9, the plasmid, virus particle, or artificial nanoparticle according to item 10 , the cell according to item 11 or 12, or the use of the composition according to item 13 for pseudovirus packaging. In some embodiments, the pseudovirus is a lentivirus or other retrovirus. In some embodiments, the lentivirus or other retrovirus is derived from HIV.

15. A pseudoviral particle for transducing cells, which is packaged by the chimeric viral envelope glycoprotein or polypeptide according to any one of items 1-7. In some embodiments, the envelope glycoprotein of the pseudovirion comprises the chimeric viral envelope glycoprotein or polypeptide according to any one of items 1-7. In some embodiments, the envelope glycoprotein of the pseudovirion comprises a portion of the chimeric viral envelope glycoprotein or polypeptide according to any one of items 1-7 other than the R peptide. In some embodiments, the cells are selected from: NK cells, αβ T cells, γδ T cells, DC cells, and stem cells. In some embodiments, the pseudovirus is a lentivirus or other retrovirus. In some embodiments, the lentivirus or other retrovirus is derived from HIV. In some embodiments, the The pseudoviral particles are packaged with a chimeric antigen receptor (CAR) or its coding sequence.

16. The pseudoviral particle according to item 15, further comprising the envelope glycoprotein of VSV in its envelope. In some embodiments, the protein component in the envelope consists of VSV-G and the chimeric viral envelope glycoprotein of any one of items 1-7.

17. The pseudoviral particle according to item 15 or 16, which is a pseudoviral particle of a lentivirus or other retrovirus.

18. The pseudoviral particle according to item 17, wherein the lentivirus or other retrovirus is derived from HIV virus.

19. The pseudoviral particle according to any one of items 15 to 18, which contains a chimeric antigen receptor or a component thereof, or a coding sequence of the chimeric antigen receptor or a component thereof.

20. The pseudoviral particle according to item 19, wherein the chimeric antigen receptor specifically binds CD19 or CD123.

21. Cells obtained by transducing the pseudoviral particles according to any one of items 15-20.

22. The chimeric viral envelope glycoprotein or polypeptide according to any one of items 1 to 7, the nucleic acid according to item 8 or 9, the plasmid, virus particle, or artificial nanoparticle according to item 10, Use of the cell according to item 11 or 12, the composition according to item 13, the pseudoviral particle according to any one of items 15-20, or the cell according to item 21 in the preparation of a cell therapy drug.

In addition, this application relates to:

1. A method of packaging fake viruses, including:

Introduce BaEV envelope glycoprotein or a vector containing BaEV envelope glycoprotein encoding nucleic acid, target gene encoding nucleic acid and viral packaging elements into target cells; or

Construct a cell line that stably expresses BaEV envelope glycoprotein, and introduce the nucleic acid encoding the target gene and viral packaging elements into the cell line. The cell line that stably expresses BaEV envelope glycoprotein can be a mixed clonal cell line or a screened monoclonal cell line.

2. The method according to item 1, wherein the pseudovirus is a lentivirus or other retrovirus. In some embodiments, the lentivirus is derived from HIV.

3. The method according to item 2, wherein the nucleic acid encoding the target gene is a transfer plasmid of a lentivirus or other retroviral packaging system, and the viral packaging element is a packaging plasmid of a lentivirus or other retroviral packaging system.

4. The method according to any one of items 1-3, wherein constructing a cell line stably expressing BaEV envelope glycoprotein is to insert the nucleic acid encoding BaEV envelope glycoprotein into the target through lentiviral transduction or transposition. realized by the cellular genome.

5. The method according to item 4, wherein the transposon system used for transposition is selected from the group consisting of: PB transposon system, SB transposon system, and ΦC31 integrase system.

6. The method according to item 4 or 5, wherein the lentivirus or other retroviral transduction adds the sensitizing agent DEAE or polybrene, or a reagent having the same active ingredient as the DEAE or polybrene.

7. The method according to any one of items 1 to 6, further comprising introducing VSV envelope glycoprotein or its coding sequence into the target cell or cell line, or the stably expressing BaEV envelope glycoprotein. The cell line also stably expresses VSV envelope glycoprotein. In some embodiments, the VSV envelope glycoprotein is wild-type VSV envelope glycoprotein or a variant thereof. In some embodiments, the VSV envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 18, or a functional derivative thereof, or has a sequence that is more than 70% identical to the amino acid sequence shown in SEQ ID NO: 18 Identity of the amino acid sequence.

8. The method according to any one of items 1 to 7, wherein the protease cleavage site in the tail domain of the BaEV envelope glycoprotein is replaced by an HIV protease cleavage site.

9. The HIV protease cleavage site according to item 8 has an amino acid sequence as shown in SEQ ID NO: 9.

10. The method according to any one of items 1-9, wherein the BaEV envelope glycoprotein comprises the extracellular region, the transmembrane region of the BaEV envelope glycoprotein, and the MoRV viral envelope glycoprotein tail domain . In some embodiments, the BaEV envelope glycoprotein includes the extracellular region, the transmembrane region, the intracellular juxtamembrane region of the BaEV envelope glycoprotein, and the MoRV viral envelope glycoprotein tail domain. In some embodiments, the BaEV envelope glycoprotein includes the signal peptide, extracellular region, transmembrane region, intracellular segment juxtamembrane region of the BaEV envelope glycoprotein, and the MoRV viral envelope glycoprotein tail domain. In some embodiments, the BaEV envelope glycoprotein is different from wild-type BaEV-G only in that it has a different tail domain relative to wild-type BaEV-G, and the tail domain is derived from From the tail domain of MoRV envelope glycoprotein, that is, the tail domain is wild-type MoRV envelope glycoprotein or a functional derivative thereof. In some embodiments, the signal peptide, extracellular region, transmembrane region, intracellular segment juxtamembrane region of the BaEV envelope glycoprotein (BaEV-G), and/or the tail domain of the MoRV envelope glycoprotein The linkage is via a linker or directly via valency (e.g. peptide bond).

11. The method according to item 10, wherein the extracellular region sequence of the BaEV envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 1 or a functional derivative thereof, or contains the same amino acid sequence as SEQ ID NO: 1 The amino acid sequences shown are sequences with more than 70% identity.

12. The method according to item 10 or 11, wherein the transmembrane region sequence of the BaEV envelope glycoprotein includes the amino acid sequence shown in SEQ ID NO: 2 or 19 or a functional derivative thereof, or includes the same amino acid sequence as SEQ ID NO: 2 or 19. The amino acid sequence represented by NO: 2 or 19 is an amino acid sequence having at least 70% identity.

13. The method according to any one of items 10 to 12, wherein the MoRV viral envelope glycoprotein tail domain comprises the amino acid sequence shown in SEQ ID NO: 3 or 20 or a functional derivative thereof, or Contains an amino acid sequence having more than 70% identity with the amino acid sequence shown in SEQ ID NO: 3 or 20. In some embodiments, the amino acid sequence of the protease cleavage site in the tail domain of the MoRV viral envelope glycoprotein is as shown in SEQ ID NO: 14.

14. The method according to item 10, wherein the BaEV envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 4 or a functional derivative thereof, or has the same amino acid sequence as SEQ ID NO: 4. Sequences with more than 70% identity.

15. The method according to item 10, wherein the BaEV envelope glycoprotein encoding nucleic acid is codon-optimized according to different target cells. In some embodiments, the BaEV envelope glycoprotein encoding nucleic acid comprises a polynucleotide sequence selected from any one of SEQ ID NO: 5, 6, 7, 8, 27, or a polynucleotide sequence similar to SEQ ID NO: 5, Any one of the polynucleotide sequences in 6, 7, 8 and 27 has a polynucleotide sequence with more than 70% identity.

16. The method according to any one of items 1-15, wherein the promoter of the BaEV envelope glycoprotein encoding nucleic acid is CAG, miniCMV, or SV40.

17. The method according to any one of items 1 to 16, wherein the target cell is a 293T cell or a derivative thereof. In some embodiments, the target cells are selected from the group consisting of 293T cells, 293T/17 cells, 293F cells, HEK293 cells, and 293T/17SF cells.

18. A chimeric BaEV envelope glycoprotein or polypeptide for pseudovirus packaging, the tail domain of which is the BaEV envelope glycoprotein or the envelope glycoprotein tail domain of a non-BaEV envelope virus, and whose The protease cleavage site in the tail domain is replaced by the HIV protease cleavage site.

19. According to the BaEV chimeric envelope glycoprotein or polypeptide described in item 18, the amino acid sequence of the HIV protease cleavage site is shown in SEQ ID NO: 9.

20. The BaEV chimeric envelope glycoprotein or polypeptide according to item 18 or 19, wherein the pseudovirus is a retrovirus.

21. The BaEV chimeric envelope glycoprotein or polypeptide according to item 20, wherein the pseudovirus is a lentivirus, preferably HIV virus.

22. The BaEV chimeric envelope glycoprotein or polypeptide according to any one of items 18-21, wherein The BaEV chimeric envelope glycoprotein contains R peptide.

23. The BaEV chimeric envelope glycoprotein or polypeptide according to any one of items 18-22, wherein the BaEV chimeric envelope glycoprotein includes the extracellular region and the transmembrane region of the wild-type BaEV envelope glycoprotein. , and the tail domain of wild-type MoRV viral envelope glycoprotein.

24. The BaEV chimeric envelope glycoprotein or polypeptide according to any one of items 18-23, wherein the extracellular region sequence of the BaEV chimeric envelope glycoprotein includes the amino acid shown in SEQ ID NO: 1 The sequence or its functional derivative, or an amino acid sequence containing more than 70% identity with the amino acid sequence shown in SEQ ID NO:1.

25. The BaEV chimeric envelope glycoprotein or polypeptide according to any one of items 18-24, wherein the transmembrane region sequence of the BaEV chimeric envelope glycoprotein includes such as SEQ ID NO: 2 or SEQ ID NO : The amino acid sequence shown in SEQ ID NO: 19 or a functional derivative thereof, or an amino acid sequence having more than 70% identity with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 19.

26. The BaEV chimeric envelope glycoprotein or polypeptide according to any one of items 18-25, wherein the MoRV viral envelope glycoprotein tail domain comprises the amino acid shown in SEQ ID NO: 3 or 20 The sequence or its functional derivative, or an amino acid sequence containing more than 70% identity with the amino acid sequence shown in SEQ ID NO: 3 or 20.

27. The BaEV chimeric envelope glycoprotein or polypeptide according to any one of items 18-26, wherein the BaEV chimeric envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 4 or 21 or its Functional derivatives, or amino acid sequences with more than 70% identity to the amino acid sequence shown in SEQ ID NO: 4 or 21.

28. The BaEV chimeric envelope glycoprotein or polypeptide according to any one of items 18-23, wherein the encoding nucleic acid of the BaEV chimeric envelope glycoprotein includes SEQ ID NO: 5, 6, 7, 8 , the polynucleotide sequence shown in any one of SEQ ID NO: 5, 6, 7, 8 and 27, or a polynucleotide having more than 70% identity with the nucleotide sequence shown in any one of SEQ ID NO: 5, 6, 7, 8 and 27 acid sequence.

29. The BaEV chimeric envelope glycoprotein or polypeptide according to item 28, wherein the promoter of the BaEV chimeric envelope glycoprotein encoding nucleic acid is CAG, miniCMV, or SV40.

30. The BaEV chimeric envelope glycoprotein or polypeptide according to any one of items 18-22, wherein the non-BaEV enveloped virus is selected from: FLV, KoRV, GaLV, MoRV and MLV.

31. The BaEV chimeric envelope glycoprotein or polypeptide according to item 30, wherein the BaEV chimeric envelope glycoprotein includes the extracellular region, the transmembrane region of the wild-type BaEV envelope glycoprotein, and the wild-type FLV , KoRV, GaLV, or MLV viral envelope glycoprotein tail domain.

32. Pseudovirus packaged according to the method described in any one of items 1-17. In some embodiments, the wild-type virus of the pseudovirus is itself an enveloped virus. In some embodiments, the wild-type virus of the pseudovirus is not itself an enveloped virus. In some embodiments, the pseudovirus is a lentivirus or other retrovirus. In some embodiments, the pseudovirus is a retroviral or lentiviral vector.

Description of drawings

Figure 1. Schematic diagram of the structure of wild-type BaEV envelope glycoprotein.

Figure 2. Schematic diagram of the structure of the BaEV chimeric envelope glycoprotein with the tail replaced.

Figure 3. Flow cytometric detection results of positive markers (GFP or CD19-CAR) after infection with 293T using SERV-Rless envelope glycoprotein for lentiviral packaging.

Figure 4. Titer detection results of enveloped lentivirus formed using different envelope glycoprotein packaging.

Figure 5. The transduction efficiency measured by flow cytometry after transducing PBNK (PBMC-derived NK cells) with HERV-wt or HERV-Rless packaged lentivirus at MOI=5.

Figure 6. Lentivirus packaged with BaEVRless, BaEVTR, BaEV-MoRV tail, BaEV-FLV tail, BaEV-KLV tail, BaEV-GaVL tail envelope glycoprotein was used to transduce PBNK at MOI=1, 3 days after transduction Detection, flow cytometric detection results of its transduction efficiency (expressed as GFP positive rate).

Figure 7 uses lentivirus packaged with BaEV-MoRV tail, BaEV-GaVL tail envelope glycoprotein and BaEV-HIV protease cleavage site to transduce PBNK at MOI=3 or MOI=5, and perform detection 3 days after transduction. Flow cytometry test results of transduction efficiency (expressed as CAR-NK positive rate).

Figure 8. Titer comparison of enveloped lentivirus packaged with different envelope glycoproteins and the preferred solutions (BaEVRless and BaEVTR) in comparative literature.

Figure 9. Shows that the BaEV-MoRV tail envelope glycoprotein encoding nucleic acid was transposed into the 293T cell genome, and 293T cells stably expressing the BaEV-MoRV tail envelope glycoprotein were successfully constructed.

Figure 10. Flow cytometry test results of the transduction efficiency of BaEV-MoRV-tail::VSV-G chimeric lentivirus and BaEV-MoRV-tail chimeric lentivirus on PBNK cells.

Figure 11. Comparison of transduction efficiency between enveloped lentivirus with protease cleavage site replaced and enveloped lentivirus without protease cleavage site replaced.

Figure 12. Shows that lentivirus containing CD123-CAR or mbIL15 packaged in the 293T-BaEV-MoRV Tail (LV) cell line was used to transduce PBNK cells activated for 2 days at MOI=3, and flow cytometry was used three days after transduction. NK cell transduction efficiency detected by instrument.

Figure 13. For difficult-to-transfect cells such as immune cells, test results of CAR delivery and transduction efficiency of enveloped lentivirus produced by different packaging methods.

Figure 14. The expression abundance and abundance levels of BaEV in different clones detected by flow cytometry after the positive monoclonal expression BaEV-G constructed by transposition was expanded and cultured.

Figure 15. Comparison of packaging efficiency of enveloped lentivirus constructed through different methods. PB means transposition method, LV means lentiviral transduction method.

Figure 16. 293T-BaEV-Rless (PB), a lentivirus packaged using a cell line constructed by PB transposon transposition (left) and 293T-BaEV-Rless (LV), a lentivirus constructed using lentiviral transposition Efficiency of transducing PBNK cells with cell line packaged lentivirus (right).

Figure 17. Titer detection results after lentiviral packaging of a variety of different target genes.

Figure 18. Transduction efficiency of PBNK using lentivirus packaged with different target genes using the 293T-BaEV-Rless cell line.

Figure 19. Comparison of the efficiency of BaEV-Rless:VSV-G chimeric envelope lentivirus and VSV-G envelope lentivirus in transducing γδ T cells.

Figure 20. Efficiency of transducing mouse B cells using 293T-BaEV-Rless packaged BaEV-Rless enveloped virus.

Figure 21. Comparison of the effects of BaEV-Rless and BaEV-Rless::VSV-G chimeric chronic disease transduction of PBNK

Application details

The present application provides a chimeric envelope glycoprotein/polypeptide, an engineered cell expressing the polypeptide, a method for packaging a pseudovirus with high transfection/transduction efficiency using the engineered cell expressing the polypeptide, and a method packaged by the method. Fake virus. The chimeric envelope glycoprotein/polypeptide retains the R peptide required to inhibit the toxicity of the envelope glycoprotein, reducing its toxicity, and the conditionally sheared form ensures its activity when forming a virus; the method It saves the steps of fake virus packaging and improves packaging efficiency. Viruses are available for cells that are difficult to transduce, such as immune cells, stem cells, primary cells, etc. The virus can be concentrated and purified by conventional methods, and stored at -80°C for a long time.

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following references provide the skilled person with information for use in this application General definitions of many terms: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R .Rieger et al (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings assigned to them unless otherwise stated.

As used herein, the term "transduction" refers to the process by which natural or artificially engineered viral particles enter a cell and bring the genetic material contained therein into said cell.

As used herein, the term "wild-type" has the meaning commonly understood by those skilled in the art to mean the typical form of an organism, strain, gene or characteristic found in nature, without deliberate modification by man. Unless otherwise stated, "envelope glycoprotein" as used herein includes "wild-type" envelope glycoprotein and engineered envelope glycoprotein, such as chimeric envelope glycoprotein or wild-type envelope glycoprotein with the envelope glycoprotein removed. A capsule glycoprotein formed by adding a portion of a domain or adding a portion of a domain derived from other capsule glycoproteins. But when referring to a part of the "envelope glycoprotein" of a particular virus, we are referring to that part of the "wild-type" envelope glycoprotein of that particular virus. For example, the tail domain of MoRV envelope glycoprotein refers to the tail domain of wild-type MoRV envelope glycoprotein. "Wild-type" proteins include any reference protein mentioned herein and naturally occurring variants thereof. In this application, any reference to a certain part of the envelope glycoprotein of a specific virus or a certain amino acid position thereof refers to its corresponding part or corresponding amino acid position relative to the wild-type protein. For convenience of description, this application uses one of the wild-type proteins as a reference protein. For the specific reference proteins listed in this application, their specific sequences and the division of functional segments of the sequences can be obtained by those skilled in the art through known databases. In this application, the reference protein of human endogenous viral envelope glycoprotein (HERV-G) is as shown in NCBI GeneBank No. AAM68163.1, and the reference protein of koala retrovirus envelope glycoprotein (KLV-G) is as shown in NCBI GeneBank. Reference number ALX81658.1, reference protein of gibbon leukemia virus envelope glycoprotein (GaLV-G) is shown as NCBI GeneBank number AAC96085.1, reference protein of murine endogenous retrovirus envelope glycoprotein (MoRV-G) The reference protein is shown in NCBI GeneBank number AAC42271.1, the reference protein of feline leukemia virus envelope glycoprotein (FLV-G) is shown in NCBI GeneBank number ACB05740.1, and the reference protein of feline endogenous virus envelope glycoprotein (RD114- The reference protein of G) is shown as NCBI GeneBank No. CAA61093.1, the reference protein of simian endogenous retroviral envelope glycoprotein (SERV-G) is shown as NCBI GeneBank No. AEJ22866.1, and the reference protein of murine leukemia virus vesicle The reference protein of membrane glycoprotein (MLV-G) is shown as NCBI GeneBank number AAP13891.1. in this application , unless otherwise stated, when referring to, for example, the tail domain of MoRV-G, it refers to the part of the MoRV-G corresponding to the tail domain of NCBI GeneBank No. AAC42271.1. Those skilled in the art can understand that, in addition to the specific reference proteins exemplified in this application, other naturally occurring wild-type variant proteins are also within the protection scope of this application, and their corresponding structural domains and amino acid positions can also be determined by those skilled in the art. .

As used herein, "vector" refers to a vector through which a polynucleotide sequence (eg, a gene sequence of interest) can be introduced into a host cell to transform the host and facilitate expression (eg, transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, artificial nanoparticles, etc.

As used herein, the term "artificial nanoparticle" refers to artificially synthesized or artificially engineered particles with a diameter of less than 1000 nm, which are suitable for delivering the nucleic acids and/or proteins of the present application into cells. Exemplary artificial nanoparticles include, but are not limited to: lipid nanoparticles, exosomes, and the like. The "lipid nanoparticles" are typically spherical vesicle structures composed of a single or multilamellar lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. The nanoparticles can be made from several different types of lipids; however, phospholipids are most commonly used to generate lipid nanoparticles. Although lipid nanoparticle formation is spontaneous when lipid films are mixed with aqueous solutions, formation of lipid nanoparticles can also be accelerated by applying force in the form of shaking using a homogenizer, sonicator, or extrusion device . Several other additives can be added to lipid nanoparticles in order to modify their structure and properties. For example, cholesterol or sphingomyelin can be added to the lipid nanoparticle mixture to help stabilize the lipid nanoparticle structure and prevent leakage of the lipid nanoparticle inner cargo. Lipid nanoparticle formulations may consist essentially of natural phospholipids and lipids such as 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine (DSPC), sphingomyelin, lecithin and monosialoganglioside. May be provided as solid nanoparticles (eg metals such as silver, gold, iron, titanium), non-metals, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof.

As used herein, a "gene of interest" refers to a gene or its coding sequence contained in a vector, such as a viral vector, intended to initiate expression in a target cell by introducing the vector into the target cell.

Unless otherwise specified, the terms "lentiviral vector" and "lentiviral particle" in this application can be used interchangeably, and both refer to pseudotyped lentiviral particles packaging the target gene sequence. The construction method of lentiviral vectors is known in the art and is specifically described in documents such as Naldini et al. (2000) Adv.Virus.Res.55:599 609 and Negre et al. (2002) Biochimie84:1161-1171. Typically, lentiviral vector particles according to the present invention comprise at least the following components: (i) an envelope component ("envelope" and "envelope" are used interchangeably in this application), which consist of binding to an envelope protein; consists of a phospholipid bilayer, in which the envelope The protein comprises at least a chimeric or modified glycoprotein as defined above, said envelope surrounding (ii) a core component consisting of a binding of gag proteins, which itself surrounds (iii) a genome usually composed of ribonucleic acid (RNA) component and (iv) enzyme component (pol). The biological material may be present within the envelope, within the core and/or within the genomic component. Lentiviral vectors can be readily prepared by those skilled in the art, for example, by following the general guidance provided by Sandrin et al. (2002) Biood 100:823 832. Briefly, lentiviral vector particles can be generated by co-expressing packaging elements (i.e., core and enzyme components), genomic components, and envelope components in producer cells (eg, 293T human embryonic kidney cells or cells derived therefrom). Typically 3 to 4 plasmids can be used, but the number of plasmids can be higher depending on the extent to which the lentiviral components are broken down into individual elements. In some embodiments, the partial components, such as envelope components, enzyme components, etc., can be inserted into the genome of a production cell, and the production cell is used for packaging of viral vectors. In some embodiments, the packaging elements and envelope components may be present in plasmids, a plasmid comprising viral genome components, a plasmid comprising the envelope components, and a plasmid comprising enzyme components and/or core components. The components divided into protein coding sequences are called transfer plasmids, packaging plasmids and envelope plasmids respectively. Commonly used lentiviral packaging plasmids include psPAX2, as well as pMDlg/pRRE and pRSV-Rev, which are components of the second- and third-generation lentiviral packaging systems, respectively. The psPAX2 plasmid also contains the coding sequences for gag, pol, rev and tat, the pMDlg/pRRE plasmid contains the coding sequences for gag and pol, and pRSV-Rev contains the coding sequence for rev.

In this application, "pseudovirus" and "pseudovirion" are used interchangeably and refer to viral vectors containing foreign viral envelope glycoproteins. For example, a viral vector according to the present application may be pseudotyped using a chimeric envelope glycoprotein as defined below or a variant of said envelope glycoprotein. "Pseudoviruses" include "pseudotyped lentiviruses" and other pseudotyped retroviruses. Among them, "lentivirus" is the collective name for "pseudotype lentivirus", wild-type lentivirus and other engineered lentivirus. "Retrovirus" is a collective term for "pseudotyped retroviruses", wild-type retroviruses and other engineered retroviruses. Those skilled in the art will know that lentivirus is a type of retrovirus.

In this application, "viral vector" is a type of "viral particle". "Viral vector" emphasizes that the virus particles are engineered viruses that contain artificially introduced or modified proteins or nucleic acid fragments.

As used herein, "baboon endogenous retrovirus" or "BaEV" is a type C retrovirus present as multiple proviral copies in baboon DNA. The term "BaEV envelope glycoprotein" (BaEV-G) is also known in the art as "BaEV envelope glycoprotein". BaEV envelope glycoprotein is specifically described in Benveniste et al. (1974) Nature 248:17-20 and Todaro et al. (1974) Cell 2:55-61. The BaEV envelope glycoprotein described in the present application contains the amino acid sequence shown in SEQ ID NO: 13 sequence, or an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 95% identical to the sequence set forth in SEQ ID NO: 13, provided that the amino acid sequence remains as determined by SEQ ID NO: 13 The basic function of the protein or polypeptide, the difference relative to SEQ ID NO: 13 does not result in the ability of the glycoprotein to adsorb the cell membrane of the host cell, fuse with the host cell membrane, and assist in injecting the genomic nucleic acid or the nucleic acid encoding the target gene into the host cell. loss. In this application, when a chimeric envelope glycoprotein is referred to, it is named after the envelope glycoprotein from which its extracellular domain is derived. For example, the BaEV chimeric envelope glycoprotein is a chimeric protein form in which certain parts of the BaEV envelope glycoprotein except the extracellular region are replaced with domains of other viral envelope glycoproteins. For example, "BaEV/TR" contains or consists of a chimeric envelope glycoprotein consisting of a fusion of the transmembrane and extracellular regions of the BaEV envelope glycoprotein and the tail domain of the MLV (murine leukemia virus) envelope glycoprotein. "BaEVRLess" refers to the modified BaEV envelope glycoprotein lacking the fusion inhibitory R peptide in the tail domain. The specific forms of "BaEVRLess" and "BaEV/TR" are described in detail in Chinese patent CN104080917B. .

In this application, the term "fusion-inhibitory R peptide" or "R peptide" refers to the C-terminal portion of the tail domain of the envelope glycoprotein, which carries the tyrosine endocytosis signal-YXXL and matures during viral particle It is cleaved by viral protease during the process, thereby enhancing the membrane fusion ability of envelope glycoprotein. The fusion-inhibitory R peptide of BaEV envelope glycoprotein is usually located between amino acid sequence 547 and 564 of the wild-type BaEV envelope glycoprotein. Therefore, when referring to a "tail domain", unless otherwise specified, the "tail domain" includes the R peptide.

From the amino terminus to the carboxyl terminus, the envelope glycoprotein usually contains an extracellular region, a transmembrane region, an intracellular segment, a juxtamembrane region, and a cytoplasmic tail domain (Cytoplasmic tail domain, sometimes represented by "tail" in this application). In enveloped viruses, the transmembrane region passes through the viral envelope and is connected to the extracellular region located outside the viral envelope and the tail domain located inside the viral envelope. In this application, the "extracellular region" is the part corresponding to amino acid positions 1-503 (including the endpoints) of the reference BaEV envelope glycoprotein (NCBI sequence registration number: YP_009109691.1), and the "transmembrane region" is the part corresponding to The reference BaEV envelope glycoprotein amino acid position 504 to 524 (including the endpoint) or the part of the amino acid 504 to 532 (including the endpoint), the "intracellular segment juxtamembrane region" corresponds to the reference BaEV capsule. The portion of the membrane glycoprotein at amino acid positions 525 to 532 (inclusive), and the intracellular domain is the portion corresponding to amino acid positions 534 to 563 (inclusive) of the reference BaEV envelope glycoprotein. Unless otherwise specified, the "transmembrane region" in this application may or may not include the intracellular segment juxtamembrane region.

As used herein, "functional derivatives" of a protein include various variants or functional domains of the protein as long as the variants or functional domains retain the properties of a functional domain of the protein. The function (whether it is an enhanced function or a weakened function) can be called a functional derivative of the protein.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to a group of engineered polypeptides or proteins that, when in immune effector cells, bind to a specific antigen contained on a target cell and upon recognition of said The specific antigen generates an intracellular signal and activates the downstream pathway of the cell where the receptor is located to initiate the killing effect of the immune effector cells on the target cells. The immune effector cells include but are not limited to NK cells, macrophages, neutrophils, T cells, etc. CARs generally include at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain. The extracellular antigen-binding domain can specifically recognize an antigen. Non-limiting examples include single-chain variable fragments (scFv) derived from antibodies, fragmented antigen-binding regions (Fab) selected from libraries, single-domain fragments, or combinations thereof. A natural ligand that engages its cognate receptor. In some embodiments, the extracellular antigen binding region may comprise scFv, Fab or natural ligands, as well as any derivatives thereof. An extracellular antigen-binding region may refer to a molecule other than an intact antibody, which may comprise a portion of an intact antibody and which may bind the antigen to which the intact antibody binds. Examples of antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, linear antibodies; single chain antibody molecules (eg, scFv); and those formed from antibody fragments Multispecific antibodies. The "signal transduction domain" usually includes immune-receptor tyrosine-based activation motifs (ITAM), whose basic composition is: YXXL/V. Among them, Y is tyrosine, L/V refers to leucine or valine, and X can be any amino acid. When the receptor binds to the corresponding ligand, the tyrosine in the ITMA linked to it can be phosphorylated by PTK, a type of protein tyrosine kinase associated with the cell membrane, thereby recruiting other free protein kinases in the cell. or adapter proteins that transmit activation signals into cells. In some embodiments, the "signal transduction domain" is selected from the intracellular signal transduction domain of TCRζ (CD3ζ) or FcεRIγ. As used herein, the "costimulatory domain" is also called a "costimulatory signal domain" and is mainly used to provide a costimulatory signal to enhance the ability of immune cells, including, for example, enhancing the proliferation, survival and/or development of memory cells. In some embodiments, the "costimulatory domain" is selected from CD28, 4-1BB (CD137), OX40 (CD134), and the like. As used herein, the "transmembrane domain", also known as the "transmembrane region", refers to a thermodynamically stable protein structural region anchored in the cell membrane. Transmembrane domains can be obtained from natural proteins, such as those derived from T cell receptors (TCR). In some embodiments, the transmembrane domain is selected from the group consisting of CD4, CD8α, CD28, and CD3ζ.

As used herein, the term "linker" is a short peptide used to connect multiple domains or components in a protein or polypeptide. For example, the BaEV-MoRV-tail envelope glycoprotein in this application contains BaEV The extracellular region, transmembrane region, and tail domain of envelope glycoproteins can be connected through linkers or other amino acid chains with certain functions, or directly. The term "directly connected" means that the domains or components do not contain any other amino acid residues between them.

In this application, "293T cells", namely HEK 293T cells, are an immortalized cell line derived from human embryonic kidneys. HEK 293T cells are a cell line derived from HEK 293 cells through genetic technology. HEK 293 cells are transfected with the adenovirus E1A gene and can stably express the SV40 large T antigen and contain the SV40 replication origin and promoter region. HEK 293 cells and all other derivative cells of HEK293 cells are classified as "derivative cells of 293T cells" in this application, including but not limited to 293F cells and 293T/17SF cells.

As used herein, the term "protease cleavage site" refers to a stretch of amino acid sequence contained in the tail domain of a retroviral envelope glycoprotein that is recognized for cleavage by the protease expressing it. When the protease recognizes the "protease cleavage site" and completes the cleavage, the tail domain of the viral envelope glycoprotein will lose the R peptide. The protease cleavage sites contained in various envelope glycoproteins used in the present application are known in the art. For example, the BaEV envelope glycoprotein includes the amino acid sequence shown in SEQ ID NO: 14. The amino acid sequence of the HIV protease cleavage site is shown in SEQ ID NO: 9. In this article, whenever a protease cleavage site of a specific envelope glycoprotein is mentioned, it refers to the protease cleavage site contained in the tail domain of the wild-type protein of the specific envelope glycoprotein.

chimeric viral envelope glycoprotein

On the one hand, the present application provides a chimeric viral envelope glycoprotein (also referred to as "chimeric envelope glycoprotein") or polypeptide for pseudovirus packaging. "Envelope glycoprotein", also known as "envelope glycoprotein (Glycoprotein, GP)". For wild-type enveloped viruses, the envelope glycoprotein is encoded by the viral genome and is coated in the outer layer of the virus. GP is a multifunctional protein that plays a crucial role in virus adsorption, penetration into host cells, pathogenicity, downregulation of host cell surface protein expression, and increased virus assembly and budding. Therefore, the choice of envelope glycoproteins plays a critical role in both viral packaging titers and transduction of host cells.

The chimeric viral envelope glycoprotein or polypeptide provided by the present application is composed of multiple segments derived from different envelope glycoproteins, that is, the "chimeric viral envelope glycoprotein" includes at least two viral envelope glycoproteins. Domain or peptide segment of a membrane glycoprotein. In some embodiments, the chimeric viral envelope glycoprotein or polypeptide comprises the extracellular region of BaEV envelope glycoprotein (BaEV-G), the transmembrane region, and the MoRV (murine endogenous retrovirus) envelope glycoprotein The tail domain of the protein. In some embodiments, the chimerism Compared with wild-type BaEV-G, the only difference between the vesicle glycoprotein or polypeptide and the chimeric virus envelope glycoprotein or polypeptide is that the chimeric virus envelope glycoprotein or polypeptide has a different cytological tail domain than the wild-type BaEV-G, and the cytotoxicity The tail domain is derived from the tail domain of MoRV envelope glycoprotein, that is, the tail domain is wild-type MoRV envelope glycoprotein or a functional derivative thereof. In some embodiments, the extracellular region, the transmembrane region, and the tail domain of the MoRV envelope glycoprotein of the BaEV envelope glycoprotein (BaEV-G) in the chimeric viral envelope glycoprotein or polypeptide Connect via connectors or directly. The embodiments of this application take lentivirus as an example to test the impact of the chimeric virus envelope glycoprotein on virus particle packaging and transduction, and confirm that the virus packaged using the chimeric virus envelope glycoprotein is more effective than the current There are technologies that use VSV-G or other variants of BaEV-G to package viruses with higher packaging efficiency and packaging stability. And for cells that are difficult to transduce, such as NK cells, T cells and other immune cells, it has a transduction efficiency that is not inferior to other existing variants of BaEV-G, or has better transduction efficiency. Therefore, those skilled in the art should know that the chimeric viral envelope glycoprotein or polypeptide provided in this application for packaging pseudoviruses can not only be used for packaging lentiviruses or retroviruses, but can also be applied to other enveloped viruses. packaging, and have similar effects as described above on the viruses packaged by it.

The extracellular region sequence of the exemplary BaEV envelope glycoprotein is the amino acid sequence shown in SEQ ID NO: 1 or a functional derivative thereof, or has about 70% or more identity with the amino acid sequence shown in SEQ ID NO: 1 amino acid sequence. The transmembrane region of the exemplary BaEV envelope glycoprotein is the sequence shown in SEQ ID NO: 2 or 19 or a functional derivative thereof, or an amino acid sequence having about 70% or more identity with SEQ ID NO: 2 or 19. The tail domain of an exemplary MoRV envelope glycoprotein includes the amino acid sequence shown in SEQ ID NO: 3 or a functional derivative thereof, or an amino acid sequence having about 70% or more identity with SEQ ID NO: 3. Those skilled in the art should know that although in the scheme provided in this application, in the process of using the chimeric virus envelope glycoprotein to package virus particles, the tail domain contains an R peptide segment, and the packaging is completed by the R peptide segment. In viruses, the chimeric viral envelope glycoprotein may not include the R peptide segment of the tail. The location and sequence of the R peptide segment of MoRV are known in the art. In some embodiments, the chimeric viral envelope glycoprotein or polypeptide is BaEV-MoRV tail envelope glycoprotein, and an exemplary amino acid sequence is the amino acid sequence of SEQ ID NO: 4 or a functional derivative thereof, or with SEQ ID NO: 4 is an amino acid sequence with about 70% or more identity.

In some embodiments, the chimeric viral envelope glycoprotein or polypeptide comprises the entire sequence of wild-type BaEV-G except for the tail domain. Exemplarily, the wild-type BaEV-G and the chimeric viral envelope glycoprotein or polypeptide both comprise the amino acid sequence shown in SEQ ID NO: 13 or a functional derivative thereof, or are identical to SEQ ID NO: 13 Amino acid sequences with 70% sequence identity. During the packaging process, even if the chimeric envelope glycoprotein or polypeptide provided by this solution contains a signal peptide, the chimeric envelope glycoprotein or polypeptide in the virus particles formed through its packaging may not contain the signal peptide. Because, those skilled in the art will know that the signal peptide molecule can be cleaved by protease during the packaging process. As used herein, the term "signal peptide" refers to a short peptide chain, typically 5-30 amino acids in length, that directs the transfer of newly synthesized proteins to the secretory pathway. In some embodiments, the signal peptide is an amino acid sequence used to direct the transmembrane transfer (localization) of a protein. In most cases, the signal peptide is located at the N-terminus of the amino acid sequence. In mRNA, the coding sequence of the signal peptide is usually located after the start codon and is an RNA region encoding a hydrophobic amino acid sequence. After the signal peptide guides the protein to complete its positioning, it is usually cleaved by the action of signal peptidase. Modification or modification of signal peptide molecules can alter or improve the transfer, localization or assembly properties of the protein, which is well known in the art. Therefore, the chimeric viral envelope glycoprotein or polypeptide provided in this application may not include a signal peptide, or the signal peptide may be replaced or modified. The signal peptide used does not need to be replaced by the exemplary signal peptide in SEQ ID NO: 11. limit.

In some embodiments, the protease cleavage site of the tail domain of the MoRV envelope glycoprotein is replaced by an HIV protease cleavage site. Taking the tail domain of the exemplary wild-type MoRV envelope glycoprotein as an example, the amino acid sequence shown in SEQ ID NO: 14 is replaced by the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, the tail domain of the chimeric enveloped virus glycoprotein or polypeptide comprises the amino acid sequence of SEQ ID NO: 20 or a functional derivative thereof, or is identical to the amino acid sequence set forth in SEQ ID NO: 20 Amino acid sequences with about 70% or more identity. In some embodiments, the chimeric viral envelope glycoprotein or polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 21, or a functional derivative thereof, or has 70% sequence identity with SEQ ID NO: 21 amino acid sequence. In some embodiments, the amino acid sequence of the chimeric viral envelope glycoprotein or polypeptide is set forth in SEQ ID NO: 21.

In addition, the present application also provides a BaEV chimeric envelope glycoprotein or polypeptide in which the protease cleavage site of the tail domain is replaced by the HIV protease cleavage site. In some embodiments, the sequence of the HIV protease cleavage site is shown in SEQ ID NO: 9. In some embodiments, the tail domain of the BaEV chimeric envelope glycoprotein or polypeptide is the tail domain of a wild-type BaEV envelope glycoprotein. In some embodiments, the tail domain of the BaEV chimeric envelope glycoprotein or polypeptide is not the tail domain of the wild-type BaEV envelope glycoprotein, but is replaced with the envelope of an envelope virus other than BaEV. Glycoprotein tail domain. In some embodiments, the tail domain of the BaEV chimeric envelope glycoprotein or polypeptide is replaced with the envelope glycoprotein tail domain of FLV, KoRV, GaLV, MoRV or MLV, and the MLV or MoRV The tail domain of the envelope glycoprotein contains or does not contain R Peptides. In some embodiments, the BaEV chimeric envelope glycoprotein or polypeptide used for pseudovirus packaging is selected from: BaEV-MoRV-tail, BaEVRless, BaEV/TR, BaEV-FLV-tail, BaEV-KoRV-tail, BaEV-GaLV-tail. Among them, the exemplary structures of BaEV-MoRV-tail, BaEVRless, BaEV/TR, BaEV-FLV-tail, BaEV-KoRV-tail, and BaEV-GaLV-tail are specifically described in Example 1.

nucleic acid

In a second aspect, this application also discloses a nucleic acid encoding the aforementioned chimeric virus envelope glycoprotein. In some embodiments the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid comprises both deoxyribonucleosides and ribonucleosides. In some embodiments, the nucleic acid contains chemical modifications. In some embodiments, the nucleic acid comprises a promoter for promoting expression of the protein or polypeptide. In some embodiments, the promoter is a eukaryotic promoter. In some embodiments, the promoter is selected from: CAG, miniCMV, SV40. In some embodiments, the nucleic acids are codon-optimized for different host cells. The nucleic acid of the present application can be circular, linear, single-stranded or double-stranded.

In some embodiments the nucleic acid comprises a polynucleotide sequence as set forth in SEQ ID NO: 5, or is about 70% identical to any of the polynucleotide sequences of SEQ ID NO: 5, 6, 7, 8 and 27. Polynucleotide sequences with more than % identity. In some embodiments the nucleic acid comprises a polynucleotide sequence as set forth in SEQ ID NO: 6, or is about 70% identical to any of the polynucleotide sequences in SEQ ID NO: 5, 6, 7, 8 and 27 Sequences with the above identity. In some embodiments the nucleic acid comprises a polynucleotide sequence as set forth in SEQ ID NO: 7, or is about 70% identical to any of the polynucleotide sequences of SEQ ID NO: 5, 6, 7, 8 and 27. Sequences with more than % identity. In some embodiments the nucleic acid comprises a polynucleotide sequence as set forth in SEQ ID NO: 5, 6 and 7, or a polynucleotide sequence corresponding to any one of SEQ ID NO: 5, 6, 7, 8 and 27 Sequences with about 70% or more identity. In some embodiments the nucleic acid comprises a polynucleotide sequence as set forth in SEQ ID NO: 8, or is about 70% identical to any of the polynucleotide sequences of SEQ ID NO: 5, 6, 7, 8 and 27. Polynucleotide sequences with more than % identity. In some embodiments the nucleic acid comprises a polynucleotide sequence as set forth in SEQ ID NO: 27, or is about 70% identical to any of the polynucleotide sequences of SEQ ID NO: 5, 6, 7, 8 and 27. Polynucleotide sequences with more than % identity.

Another aspect of the application also provides plasmids, viral particles, or artificial nanoparticles comprising any of the nucleic acids described above. In some embodiments, the plasmid, viral particle, or artificial The nanoparticle further contains a promoter that can initiate the expression of the aforementioned chimeric virus envelope glycoprotein or polypeptide in the host cell. In some embodiments, the plasmid is an envelope plasmid for viral packaging. In some embodiments, the envelope plasmid is any plasmid useful for expressing foreign proteins within eukaryotic cells. In some embodiments, the plasmid is a retroviral envelope plasmid. In some embodiments, the plasmid is a lentiviral envelope plasmid. In some embodiments, the envelope plasmid is the backbone structure of pMD2.G with the nucleic acid sequence of item 8 or item 9 inserted. In some embodiments, the plasmid is a pMD2.G plasmid in which the VSV-G nucleic acid coding sequence is replaced with the nucleic acid sequence of item 8 or item 9. In some embodiments, the envelope plasmid is a pcDNA3.1 plasmid in which the nucleic acid sequence of item 8 or item 9 is inserted. The viral particles are any viral particles or viral vectors that can infect or transduce the target cells of interest and express therein, introduce into the cytoplasm of the target cells, or insert the aforementioned nucleic acid into the genome of the target cells. For example, in some embodiments, the target cells are eukaryotic cells, and common viral particles include, but are not limited to: lentiviral vectors (LV), adenoviral vectors (ADV), adeno-associated virus vectors (AAV), Mouse leukemia virus (MLV), etc. Exemplary artificial nanoparticles include, for example, lipid nanoparticles, quantum dots, and the like.

The present application also provides engineered cells comprising the aforementioned chimeric viral envelope glycoprotein or polypeptide, or the aforementioned nucleic acid, plasmid, virus particle, or artificial nanoparticle. In some embodiments, the cells can express the aforementioned embedded cells transiently or over a period of time simply due to the introduction of the viral particles, plasmids, or artificial nanoparticles containing the previously described nucleic acids, or any of the aforementioned nucleic acids. Synthesized viral envelope glycoprotein or polypeptide. In some embodiments, the nucleic acid, plasmid, viral particle, or artificial nanoparticle further comprises a transposable element, which inserts the aforementioned nucleic acid into the engineered cell, and the engineered cell is constructed to stably express the aforementioned chimeric Cells of viral envelope glycoproteins or polypeptides.

composition or complex

In a third aspect, the present application also provides a composition or complex comprising the aforementioned chimeric viral envelope glycoprotein or polypeptide, the aforementioned nucleic acid, plasmid, virus particle, artificial nanoparticle, or cell. In some embodiments the composition or complex further comprises a VSV envelope glycoprotein or a nucleic acid, plasmid, viral particle or artificial nanoparticle encoding a VSV envelope glycoprotein. The composition or complex can be used for packaging of pseudoviruses, and provides the aforementioned chimeric virus envelope glycoprotein or polypeptide for packaging of pseudoviruses.

In some embodiments, the compositions or complexes are suitable for use in first, second and third generation lentiviral packaging systems. In some embodiments, the composition further comprises nucleic acids encoding Gag, Pol, Rev, and Tat, or nucleic acids encoding Gag, Pol, and Rev but not Tat. In some embodiments, the coding nucleic acids comprising Gag, Pol, Rev and Tat are present in one or two plasmids respectively. In some embodiments, the plasmid comprising the nucleic acid encoding Gag and Pol is pMDlg/pRRE. In some embodiments, the plasmid comprising a Rev-encoding nucleic acid is a pRSV-Rev plasmid. In some embodiments, nucleic acids encoding Gag, Pol, Rev and Tat are present in the aforementioned cells. In some embodiments, the composition further comprises nucleic acid encoding a gene sequence of interest, a promoter that drives expression of the gene of interest, an LTR, and a psi packaging signal. In some embodiments, the nucleic acid comprising a sequence encoding a gene of interest, a promoter for initiating expression of the gene of interest, an LTR and a psi packaging signal is a transfer plasmid.

In some embodiments, the composition or complex includes a cell with a nucleic acid capable of stably expressing the aforementioned chimeric viral envelope glycoprotein or polypeptide inserted into the genome, which can be used to package the gene sequence of interest into pseudoviral particles. Therefore, the composition or complex further includes nucleic acid encoding a gene sequence of interest, a promoter for initiating expression of the gene of interest, LTR and psi packaging signals, and other auxiliary components for virus packaging. For example, in some embodiments, the composition or complex further comprises a nucleic acid encoding Gag, Pol, Rev and/or Tat. The nucleic acid encoding the target gene sequence, the promoter for initiating the expression of the target gene, the LTR and the psi packaging signal, and the coding nucleic acid containing Gag, Pol, Rev and/or Tat can be plasmids or genome components that are convenient for use in the cells. in the form of expression or packaging. In some embodiments, the nucleic acid expressing the aforementioned chimeric viral envelope glycoprotein or polypeptide is inserted into the genome of the cell through viral transduction or transposition.

In some embodiments, the complex or composition includes the aforementioned chimeric viral envelope glycoprotein or polypeptide, which may be complexed or complexed with other nucleic acids or proteins that package the required elements. The complex may be a nucleoprotein formed by the complex of protein and nucleic acid.

In some embodiments, the present application also provides a kit for pseudovirus packaging, which includes the aforementioned composition or complex. In some embodiments, the pseudovirus is a retrovirus. In some embodiments, the virus is a lentivirus.

viral vector

In a fourth aspect, the present application also provides a pseudoviral particle for transducing target cells, which contains the aforementioned chimeric viral envelope glycoprotein or polypeptide in the envelope. In some embodiments, the target cells are immune effector cells or hematopoietic stem/progenitor cells. In some embodiments, the target cells are selected from: NK cells, αβ T cells, γδ T cells, DC cells, and stem cells. In some embodiments, the pseudovirus is a lentivirus or other retrovirus. In some embodiments, the lentivirus or other Retroviruses originate from HIV. In some embodiments, the pseudoviral particle is packaged with a chimeric antigen receptor (CAR) or its coding sequence. In some embodiments, after transduction using the viral vector, the immune effector cells are modified into engineered immune effector cells that express foreign proteins, such as CAR-T cells, CAR-NK cells, etc. In some embodiments, the pseudoviral particles comprise a chimeric antigen receptor or a component thereof, or a coding sequence for the chimeric antigen receptor or a component thereof. In some embodiments, the chimeric antigen receptor specifically binds CD19 or CD123.

In some embodiments, the envelope of the pseudoviral particle further contains the envelope glycoprotein of VSV. In some embodiments, the protein component in the envelope consists of VSV-G and the aforementioned chimeric viral envelope glycoprotein. In some embodiments the VSV-G comprises an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence having more than 70% identity to the amino acid sequence set forth in SEQ ID NO: 18.

On the other hand, the present application also provides virus particles or pseudovirions in which the target gene itself contains a nucleic acid sequence encoding the aforementioned chimeric virus envelope glycoprotein or polypeptide. In some embodiments, the viral particles are selected from engineered viral vectors such as lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated virus vectors, and the like. In some embodiments, the envelope glycoprotein of the pseudoviral particle may or may not include the aforementioned chimeric viral envelope glycoprotein or polypeptide. In some embodiments, the target cells of the pseudoviral particles whose gene of interest itself contains the nucleic acid sequence encoding the aforementioned chimeric virus envelope glycoprotein or polypeptide are 293T cells or cells derived from them, for example, selected from: 293T/17, 293F , HEK293, 293T/17SF target cells.

In some embodiments, the pseudoviral particles are lentiviral or other retroviral pseudoviral particles. In some embodiments, the lentivirus or other retrovirus is derived from the HIV virus.

cell

In one aspect, the present application also provides cells that express or contain the aforementioned chimeric viral envelope glycoproteins or polypeptides. The cells can be used for packaging of enveloped lentiviruses and provide envelope glycoproteins for the packaging. In some embodiments, the cells are 293T cells or derivatives thereof. In some embodiments, the cells are selected from 293T cells, 293F cells, HEK293 cells, 293T/17SF cells.

On the other hand, the present application also provides engineered cells transduced by viral vectors packaged by the aforementioned chimeric viral envelope glycoprotein or polypeptide. The engineered cells express exogenous genes of interest, or overexpress certain endogenous genes, or regulate the expression of endogenous genes of the cells through the expression of the genes of interest, or modify the cells. Examples of the target gene sequence include, but are not limited to: chimeric Antigen receptor (CAR) coding sequence, Ig gene coding sequence, cytokine gene coding sequence, shRNA, CRISPR gene editing system and other gene editing systems. In some embodiments, the cells are immune effector cells or stem cells. In some embodiments, the cell is a T cell, B cell, NK cell, DC cell, αβ T cell, or γδ T cell, for example. In some embodiments, the cells are transduced by the viral vector to form, for example, CAR-NK cells or CAR-T cells.

use

In one aspect, the present application also provides the use of the aforementioned chimeric virus envelope glycoprotein or polypeptide, the aforementioned nucleic acid, the aforementioned plasmid, virus particles, artificial nanoparticles, and the aforementioned cells, especially for packaging pseudoviruses. In some embodiments, the viral packaging is a lentiviral packaging or other retroviral packaging. In some embodiments, the lentivirus or other retrovirus is derived from HIV.

In some embodiments, the aforementioned chimeric viral envelope glycoprotein or polypeptide includes a nucleic acid, plasmid, virus particle, or artificial nanoparticle that contains the encoding sequence of the aforementioned chimeric viral envelope glycoprotein or polypeptide and a nucleic acid encoding a gene of interest, and other elements required for virus packaging or their expression vectors, such as plasmids, together constitute a packaging system. The packaging system is introduced into the target cells and uses relevant organelles, functional proteins, etc. in the target cells to package the pseudovirus.

In some embodiments, the nucleic acid, plasmid, virus particle, or artificial nanoparticle containing the aforementioned chimeric viral envelope glycoprotein or polypeptide coding sequence is first introduced into the target cell, and the chimeric viral envelope glycoprotein or polypeptide is performed. Transient expression of polypeptides, or insertion into the target cell genome to construct cells that stably express chimeric viral envelope glycoproteins or polypeptides; subsequently, other nucleic acids encoding the target genes, as well as other elements required for virus packaging or their expression vectors are introduced into the cells, Or similarly, it can be inserted into the cell genome, and then the pseudovirus can be packaged using relevant organelles, functional proteins, etc. in the target cell.

Exemplary specific usage methods include but are not limited to the following steps:

(1)Vector construction

The BaEV-MoRV tail is integrated into the genome of the cell line used for virus packaging using the aforementioned lentiviral vector or non-viral vector (including but not limited to artificial nanoparticles and plasmids) inserted with the BaEV-MoRV tail coding sequence. The integration methods include but are not limited to lentiviral system, PB transposon system, SB transposon system, ΦC31 integrase system, etc. The lentivirus can be selected from the aforementioned lentiviral vectors, and the PB transposon system can be selected from the aforementioned plasmids. The promoter driving BaEV expression can be a promoter of different strengths, including CAG, miniCMV, SV40, etc. In the plasmid structure of the PB transposon system, WPRE or bGH poly A can be added to the 3' end of the ORF to improve the stability of the transcript. Qualitative. Resistance genes including but not limited to puromycin, neomycin, etc. are also added to the plasmid to facilitate the subsequent screening of cell lines.

(2) Construction of cells

Introduce the vector constructed in (1) into cells. When the vector is a lentiviral vector, the introduction can introduce the coding sequence of BaEV-MoRV-tail into the target cell genome under the action of sensitizing reagents including DEAE, polybrene, etc. The target cells are selected from 293T cells or cells derived therefrom.

When the vector is a non-viral vector, the introduction method includes but is not limited to electroporation, lipofection, calcium transfer, PEI and other methods, and the plasmid containing the transposon and transposase is introduced into the target cell. , different forms of BaEV coding sequences are inserted into the genome of target cells under the action of transposase.

After the introduction of the target cells, cells expressing BaEV-MoRV tail can be sorted out through cell line screening methods (including but not limited to flow sorting, drug screening, etc.). On this basis, in order to further optimize the efficiency of virus packaging, the above cells can be monocloned through methods such as flow sorting and limiting dilution. It has been identified that, in some embodiments, BaEV expression abundance among different clones of the 293T-BaEV cell line is different, and clones with higher expression abundance are more advantageous in virus packaging efficiency.

(3)Virus packaging

The above-mentioned 293T cell line stably expressing BaEV-MoRV-Tail was transduced with the plasmid containing the target gene, RRE, and Rev at a certain ratio, and then the virus was packaged. The culture supernatant was harvested 48 hours after transfection, and the virus was concentrated by PEG6000 for flow cytometry. The titer can reach above 1e8TU/ml. On this basis, VSV-G encoding plasmid can be added during the packaging process to further increase the lentivirus titer by up to 5-8 times.

(4) Testing of virus transduction effect

The lentivirus in (3) was slowly pressed at MOI=1-5 to transduce activated PBMC-derived NK cells (PBNK). Cationic polymers such as polybrene and DEAE can be added before adding viruses to improve transduction efficiency. Three days after transduction, the positive rate of target gene expression was detected by flow cytometry, and the results showed that the positive rate could reach 50-80%.

On the other hand, the present application also provides the use of the aforementioned chimeric virus envelope glycoprotein or polypeptide, the aforementioned nucleic acid, the aforementioned plasmid, viral particles, artificial nanoparticles, the aforementioned cells, the aforementioned compositions or complexes, and the aforementioned pseudoviral particles in the preparation of cells. Use in therapeutic medicines. Exemplary methods of use include, for example, using the lentivirus packaged with the chimeric viral envelope glycoprotein or polypeptide to transduce immune effector cells to prepare engineered immune effector cells with precise targeting, such as CAR-T cells, CAR-NK cells, etc.; prepare pseudoviral particles that express the aforementioned chimeric virus envelope glycoprotein or polypeptide in the envelope. The pseudoviral particles contain the target gene and can carry the target gene for transduction in vivo or in vitro. from subjects To make the cells express therapeutic exogenous proteins, to regulate the expression of endogenous genes of the cells, or to modify the cells, examples of the target gene sequences include but are not limited to: embedded Synthetic antigen receptor (CAR) coding sequence, Ig gene coding sequence, cytokine gene coding sequence, shRNA, CRISPR gene editing system and other gene editing system components.

method of packing

The seventh aspect of the present application provides a packaging method for a pseudovirus with improved packaging efficiency. The pseudovirus has high transduction efficiency for target cells that are difficult to transduce, especially immune cells. As used herein, the term "target cell" refers to a cell into which exogenous nucleic acid or protein is introduced for protein expression or viral packaging.

The packaging method of the pseudovirus includes: introducing the aforementioned BaEV envelope glycoprotein or a vector containing the aforementioned BaEV envelope glycoprotein encoding nucleic acid, the nucleic acid encoding the target gene and the viral packaging element into the target cell; or constructing a stable expression of the BaEV envelope glycoprotein. Protein-producing cell lines, and the nucleic acid encoding the target gene and viral packaging elements are introduced into the cell lines. The vector may be selected from: plasmids, phages, viruses, artificial nanoparticles, etc. In some embodiments, in addition to the nucleic acid encoding the BaEV envelope glycoprotein, the vector further includes a promoter that can initiate expression of the BaEV envelope glycoprotein. In this application, "virus packaging elements" refer to regulatory elements, structural proteins (other than envelope glycoproteins) and related enzymes required for virus packaging, or nucleic acids encoding the regulatory elements, structural proteins and related enzymes. For a specific virus, its packaging components are recorded in relevant published academic documents in the field, which can be obtained by those skilled in the art. In some embodiments, the pseudovirus is a lentivirus or other retrovirus. Therefore, the BaEV envelope glycoprotein or a vector comprising a BaEV envelope glycoprotein encoding nucleic acid, a target gene encoding nucleic acid and a viral packaging element, Or a cell line stably expressing BaEV envelope glycoprotein, nucleic acid encoding the target gene and viral packaging components form a lentivirus or other retrovirus packaging system. The lentivirus or other retrovirus packaging system can be selected from the group consisting of first-generation, second-generation, and third-generation lentivirus packaging systems. In some embodiments, the nucleic acid encoding the gene of interest is a transfer plasmid of a lentivirus or other retroviral packaging system, which contains the LTRs and psi packaging signal of the lentivirus or other retrovirus. In some embodiments, the nucleic acid encoding the gene of interest can also refer to any nucleic acid or vector containing the nucleic acid encoding the gene of interest and LTRs and psi packaging signals of lentivirus or other retrovirus, for example, it can be a linear nucleic acid, Viral vectors, artificial nanoparticles, etc. In some embodiments, the pseudovirus is a lentivirus or other retrovirus, the packaging element of which includes the coding sequences of Gag, Pol, Rev and Tat genes. In some embodiments, the pseudovirus is slow Viruses or other retroviruses, the packaging element only contains the coding sequences of Gag, Pol and Rev genes, and the nucleic acid encoding the target gene also contains a specialized promoter. In some embodiments, the pseudovirus is a lentivirus and the "viral packaging element" is selected from the psPAX2 plasmid, or a combination of pMDlg/pRRE and pRSV-Rev plasmids. Among them, psPAX2, pMDlg/pRRE and pRSV-Rev are common plasmids in the art, and the necessary functional elements are known in the art. Specifically, the psPAX2 plasmid contains the coding sequences of gag, pol, rev and tat, the pMDlg/pRRE plasmid contains the coding sequences of gag and pol, and pRSV-Rev contains the coding sequence of rev. The method described in this application is applicable to a variety of target genes, including but not limited to chimeric antigen receptors (such as CAR targeting CD19, CD123) and genes of various cytokines.

In some embodiments, the pseudovirus is a lentivirus or other retrovirus, and the lentivirus or other retrovirus is selected from: Rous sarcoma virus, Rous-related virus, chicken tumor virus, avian leukosis virus ( ALV), murine sarcoma virus (MSV), murine leukemia virus (MLV), murine endogenous viruses, pork tumour virus, bovine leukemia virus, porcine leukemia virus, murine mammary tumor virus, primate sarcoma virus, simian leukemia virus, Baboon tumor virus type C, Mason-Pfizer monkey virus (MPMV), human T-cell virus types I, II, V (HTLV-I, II, V), HIV (human immunodeficiency virus), ovine demyelinating Leukoencephalitis virus, sheep lung adenoma virus, equine infectious anemia virus (EIAV), primate foamy virus, feline foamy virus, bovine foamy virus, human foamy virus, etc. In some embodiments, the lentivirus or other retrovirus is derived from HIV.

As used herein, the term "nucleic acid" may refer to any form of nucleic acid, including, but not limited to, linear nucleic acid, circular nucleic acid (e.g., plasmid), genomic nucleic acid, artificially modified nucleic acid, DNA, RNA, or a nucleic acid composed of DNA and RNA. nucleic acids. In some embodiments, the nucleic acid encoding the target gene described in this application is a transfer plasmid of a lentivirus or other retroviral packaging system.

Any method for constructing a cell line that stably expresses a foreign protein can be used to construct a cell line that stably expresses the BaEV envelope glycoprotein, for example, using simple homologous recombination. In some embodiments, constructing a cell line stably expressing the BaEV envelope glycoprotein is achieved by inserting the nucleic acid encoding the BaEV envelope glycoprotein into the genome of the target cell through lentiviral transduction or transposition. In some embodiments, the lentivirus can be lentivirus packaged using the method provided in this application or can be lentivirus packaged using traditional methods. In some embodiments, the transposition can be performed using any common transposon system, for example, the transposon system is selected from: PB transposon system, SB transposon system, ΦC31 integrase system. In some embodiments, the method for constructing a cell line that stably expresses foreign proteins is a gene editing method, such as CRISPR gene editing method, ZFN gene editing method, TALEN gene editing method, Mega nuclease gene editing method, etc.

In some embodiments, the method for constructing a cell line stably expressing a foreign protein is a method of lentiviral transduction, which includes contacting a lentivirus comprising the BaEV envelope glycoprotein encoding nucleic acid with a target cell, or Before or after contact with the encapsulated cells, add a sensitizing reagent (or transfer-assisting reagent) DEAE or polybrene, or a reagent with the same active ingredient as DEAE or polybrene. The active ingredients of DEAE and polybrene reagents are known in the art, and there is no difference in the active ingredients of the reagents provided by various manufacturers, and the reagents from different manufacturers will not cause significant differences in viral transduction efficiency. For the active ingredients of DEAE, please refer to the product information on the official website of Sangon Bioengineering (Shanghai) Co., Ltd. For the active ingredients of Polybrene, please refer to the corresponding product information on the official website of Santa Cruz Animal Health.

In some embodiments, the packaging method of the pseudovirus further includes introducing VSV envelope glycoprotein or its coding sequence into the target cell or cell line, or making the cell line stably expressing the BaEV envelope glycoprotein simultaneously VSV envelope glycoprotein is also stably expressed. In some embodiments, the VSV envelope glycoprotein is wild-type VSV envelope glycoprotein or a variant thereof. In some embodiments, the VSV envelope glycoprotein has the amino acid sequence set forth in SEQ ID NO: 18, or a functional derivative thereof, or has at least 70% sequence identity with the sequence set forth in SEQ ID NO: 18 amino acid sequence. In some embodiments, the amino acid sequence of the VSV envelope glycoprotein is set forth in SEQ ID NO: 18.

In some embodiments, the BaEV envelope glycoprotein used in the present methods is a chimeric envelope glycoprotein in which the protease cleavage site in the tail domain of the envelope glycoprotein is determined by the HIV protease cleavage site. replace. In some embodiments, the amino acid sequence of the HIV protease cleavage site is as shown in SEQ ID NO: 9. It should be noted that the aforementioned BaEV envelope glycoprotein may be a wild-type BaEV envelope glycoprotein or a modified BaEV envelope glycoprotein. The modified BaEV envelope glycoprotein includes a chimeric protein whose tail domain is replaced with the tail domain of a non-BaEV envelope glycoprotein. The "non-BaEV envelope glycoprotein" may refer to any other envelope glycoprotein except wild-type BaEV envelope glycoprotein, including but not limited to: FLV, KoRV, GaLV, MoRV and MLV.

In some embodiments, the BaEV envelope glycoprotein comprises the extracellular region, the transmembrane region of the BaEV envelope glycoprotein, and the MoRV viral envelope glycoprotein tail domain. In some embodiments, the BaEV envelope glycoprotein includes the extracellular region, the transmembrane region, the intracellular juxtamembrane region of the BaEV envelope glycoprotein, and the MoRV viral envelope glycoprotein tail domain. In some embodiments, the BaEV envelope glycoprotein includes a signal peptide, an extracellular region, a transmembrane region, an intracellular segment and a juxtamembrane region of the BaEV envelope glycoprotein. and the tail domain of MoRV viral envelope glycoprotein. In some embodiments, the BaEV envelope glycoprotein is different from wild-type BaEV-G only in that it has a different tail domain relative to wild-type BaEV-G, and the tail domain is derived from From the tail domain of MoRV envelope glycoprotein, that is, the tail domain is wild-type MoRV envelope glycoprotein or a functional derivative thereof. In some embodiments, the signal peptide, extracellular region, transmembrane region, intracellular juxtamembrane region of the BaEV envelope glycoprotein (BaEV-G), and/or the tail domain of the MoRV envelope glycoprotein Connect via connectors or directly. In some embodiments, the BaEV envelope glycoprotein is BaEV-MoRV-tail, that is, a BaEV envelope glycoprotein in which the tail domain is replaced with the tail domain of the MoRV envelope glycoprotein. In some embodiments, the BaEV envelope glycoprotein is BaEVRless, that is, the BaEV envelope glycoprotein with the R peptide in the tail domain removed. In some embodiments, the BaEV envelope glycoprotein is BaEV/TR, that is, a BaEV envelope glycoprotein in which the tail domain is replaced with the tail domain of the MLV envelope glycoprotein.

In some embodiments, the extracellular region sequence of the BaEV envelope glycoprotein comprises the sequence shown in SEQ ID NO: 1 or a functional derivative thereof, or a sequence having more than 70% identity thereto. In some embodiments, the extracellular region sequence of the BaEV envelope glycoprotein is shown in SEQ ID NO: 1. In some embodiments, the transmembrane region sequence of the BaEV envelope glycoprotein comprises a sequence as shown in SEQ ID NO: 2 or 19 or a functional derivative thereof, or a sequence having more than 70% identity thereto. In some embodiments, the sequence of the transmembrane region of the BaEV envelope glycoprotein is as shown in SEQ ID NO: 2 or 19. In some embodiments, the MoRV viral envelope glycoprotein tail domain comprises the sequence shown in SEQ ID NO: 3 or 20 or a functional derivative thereof, or a sequence having more than 70% identity thereto. In some embodiments, the MoRV viral envelope glycoprotein tail domain sequence is shown in SEQ ID NO: 3 or 20. In some embodiments, the MoRV viral envelope glycoprotein comprises SEQ ID NO: 1-3, or SEQ ID NO: 1, 3, 19, or SEQ ID NO: 1, 2, 20, Or the sequences shown in SEQ ID NO: 1, 19, 20 or functional derivatives thereof, or sequences having more than 70% identity with them. In some embodiments, the MoRV viral envelope glycoprotein sequence consists of SEQ ID NO: 1-3, or consists of SEQ ID NO: 1, 3, 19, or consists of SEQ ID NO: 1, 2, 20, or consists of The sequences of SEQ ID NO: 1, 19, and 20, or the sequences of SEQ ID NO: 1, 3, and 19 are sequentially connected. In some embodiments, the order of the following domains in the BaEV envelope glycoprotein from N-terminus to C-terminus is: BaEV-G extracellular region, BaEV-G extracellular region transmembrane region, and MoRV virus vesicle Membrane glycoprotein tail domain. In some embodiments, the extracellular region, transmembrane region, and MoRV viral envelope glycoprotein tail domain of the BaEV envelope glycoprotein are directly connected or connected through a linker. In some embodiments, the BaEV envelope glycoprotein comprises SEQ ID NO: The sequence shown in 4 or its functional derivative, or a sequence having more than 70% identity with it. In some embodiments, the BaEV envelope glycoprotein sequence is set forth in SEQ ID NO: 4. In some embodiments, the protease cleavage site shown in SEQ ID NO: 14 in the MoRV viral envelope glycoprotein tail domain is replaced with an HIV protease cleavage site. In some embodiments, the MoRV viral envelope glycoprotein comprises the sequence shown in SEQ ID NO: 21 or a functional derivative thereof, or a sequence having more than 70% identity thereto. In some embodiments, the BaEV envelope glycoprotein sequence is set forth in SEQ ID NO: 21.

In some embodiments, the BaEV envelope glycoprotein-encoding nucleic acid is codon-optimized for different target cells. In some embodiments, the BaEV envelope glycoprotein encoding nucleic acid comprises a nucleotide sequence selected from any one of SEQ ID NO: 5, 6, 7, 8, 27, or is identical to SEQ ID NO: 5, 6 , 7, 8 and 27 any one of the nucleotide sequences has more than 70% identity. The promoter used to initiate the expression of BaEV envelope glycoprotein can be any promoter suitable for target cells, preferably a promoter that facilitates expression in target cells. Preferred promoters for specific target cells are known in the art. For example, in some embodiments, the target cell is a 293T cell or a derivative cell thereof, then the promoter of the BaEV envelope glycoprotein encoding nucleic acid is CAG, miniCMV, or SV40. Those skilled in the art should know that 293T cells or derivative cells thereof include, but are not limited to: 293T cells, 293T/17 cells, 293F cells, HEK293 cells, and 293T/17SF cells.

Specific exemplary embodiments include:

(1) Select transgenic system

Methods to integrate BaEV of different structures into the genome of cell lines used for virus packaging include but are not limited to lentiviral systems, PB transposon systems, SB transposon systems, ΦC31 integrase systems, etc. Specifically, lentiviral systems and PB transposon system.

(2) Vector construction

Insert the coding sequences of BaEV of different structures (including BaEV-Rless, BaEV-MoRV, BaEV-HIV cleavage site (that is, BaEV-G that replaces the BaEV protease cleavage site with the HIV protease cleavage site)) into lentiviral system vectors or non-virus in the system carrier. The promoter driving BaEV expression can be promoters of different strengths, including CAG, miniCMV, SV40, etc. In the plasmid structure of the PB transposon system, WPRE or bGH poly A can be added to the 3' end of the ORF to improve the stability of the transcript. Resistance genes including but not limited to puromycin, neomycin, etc. are also added to the plasmid to facilitate the subsequent screening of cell lines.

(3) Cell transduction

The lentiviral system can insert the BaEV-MoRV coding sequence into the 293T genome under the action of sensitizing reagents including DEAE, polybrene, etc. For non-viral systems, the transposon and transposase plasmids need to be introduced into 293T through methods including but not limited to electroporation, lipofection, calcium transfection, PEI, etc., and different forms will be transformed under the action of transposase. The BaEV coding sequence was inserted into the 293T genome.

After transduction, 293T can be screened through cell line screening methods including but not limited to flow cytometry sorting, drug screening, etc. On this basis, in order to further optimize the efficiency of virus packaging, the above cells can be monocloned through methods such as flow sorting and limiting dilution. It was identified that the BaEV expression abundance of different clones of the 293T-BaEV cell line was different, and clones with medium expression abundance had more advantages in virus packaging efficiency.

(4)Virus packaging

The transfer plasmid (such as a plasmid encoding a chimeric antigen receptor), pMDLg/pRRE and pRSV-Rev are transfected into the above-mentioned 293T-BaEV cell line at a certain ratio and then the virus is packaged. The culture and supernatant are harvested 48 hours after transfection. After concentration with PEG6000, the viral flow titer can reach over 1e8TU/ml. On this basis, VSV-G encoding plasmid can be added during the packaging process to further increase the lentivirus titer by up to 5-8 times.

(5) Testing of virus transduction effect

The above lentivirus was slowly pressed at MOI=1-5 to transduce activated PBMC-derived NK (PBNK). Cationic polymers such as polybrene and DEAE can be added before adding viruses to improve transduction efficiency. Three days after transduction, the positivity rate was detected by flow cytometry, and the results showed that the positivity rate could reach 50-80%.

fake virus

In the eighth aspect, the present application also provides a pseudovirus packaged using the aforementioned packaging method, and the pseudovirus envelope contains the aforementioned chimeric envelope glycoprotein or polypeptide, and/or VSV envelope glycoprotein. In some embodiments, the wild-type virus of the pseudovirus is itself an enveloped virus. In some embodiments, the wild-type virus of the pseudovirus is not itself an enveloped virus. In some embodiments, the pseudovirus is a lentivirus or other retrovirus. In some embodiments, the pseudovirus is a retroviral or lentiviral vector.

The beneficial effects of the solution provided by this application are:

1. During the packaging process of pseudovirus, the BaEV-MoRV-tail structure can be used without missing the R peptide. A lentivirus that forms a functional BaEV envelope under conditions with lower cytotoxicity than BaEV-Rless, BaEV/RT and other existing technology preferred solutions, and the virus titer is equivalent to or higher than the preferred solutions;

2. Adding VSV-G during the lentivirus packaging process can form BaEV::VSV-G chimeric envelope lentivirus, further improving the titer of the lentivirus and expanding its scope of application;

3. The constructed 293T-BaEV cell line can improve the transduction efficiency of the virus.

4. Effectively improve the safety of packaging cell lines and reduce the risk of autonomous replication of lentivirus.

It is to be understood that the present application encompasses various aspects, embodiments, and combinations of said aspects and/or embodiments described herein. The above description and the examples that follow are intended to illustrate but not to limit the scope of the application. Other aspects, improvements and modifications within the scope of this application will be apparent to those skilled in the art to which this application belongs. Accordingly, those of ordinary skill in the art will recognize that the scope of the present application also includes such improvements and modifications to the described aspects and embodiments.

Exemplary embodiments of the present application are described below in conjunction with the accompanying drawings, which include various details of the embodiments of the present application to facilitate understanding, and should be considered to be merely exemplary. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the application. Also, descriptions of well-known features and structures are omitted from the following description for the sake of clarity and brevity.

Example

Example 1: BaEV structure optimization and screening

Search the NCBI database to search and collect type-C or D endogenous retroviruses, including (AAM68163.1 (human endogenous viral envelope glycoprotein, HERV-G), ALX81658.1 (koala Retroviral envelope glycoprotein, KLV-G), AAC96085.1 (Gibbon leukemia virus envelope glycoprotein, GaLV-G), AAC42271.1 (Murine endogenous retroviral envelope glycoprotein, MoRV-G), ACB05740.1 (feline leukemia virus envelope glycoprotein, FLV-G), CAA61093.1 (feline endogenous viral envelope glycoprotein, RD114-G), AEJ22866.1 (simian endogenous retroviral envelope glycoprotein protein, SERV-G), AAP13891.1 (murine leukemia virus envelope glycoprotein, MLV-G)).

After replacing the tail domain and R peptide of BaEV envelope glycoprotein (BaEV-G) with FLV-G, KLV-G, GaLV-G, or MoRV-G (as shown in Figure 2 (shown) are respectively called: BaEV-FLV tail, BaEV-KLV tail, BaEV-GaVL tail, and BaEV-MoRV tail. SERV-Rless (SERV-G with R peptide removed), HERV-wt (wild type HERV-G), HERV-Rless (R peptide removed) HERV-G), BaEV-FLV tail, BaEV-KLV tail, BaEV-GaVL tail, and BaEV-MoRV tail coding sequences were connected to pMD2 through a series of processes such as codon optimization, sequence synthesis, double enzyme digestion, ligation, and sequencing verification. The following vector plasmids SERV-Rless-pcDNA 3.1, HERV-wt-pcDNA 3.1, HERV-Rless-pcDNA 3.1, BaEV-FLV tail-pcDNA 3.1, and BaEV-KLV tail-pcDNA 3.1, BaEV-GaVL tail-pcDNA 3.1, BaEV-MoRV tail-pcDNA 3.1.

The lentiviral transfer plasmid: a lentiviral transfer plasmid containing a CAR (chimeric antigen receptor) coding sequence (the CAR targeting CD19 is used in this example, and its specific information is detailed in Chinese patent: CN107226867A, whose full text incorporated herein by reference) or pBKL2-GFP (a homemade plasmid, a lentiviral transfer plasmid with inserted GFP green fluorescent protein coding sequence), as well as pMDLg/pRRE and pRSV-Rev; respectively with SERV-Rless-pcDNA 3.1, HERV-wt -pcDNA 3.1, HERV-Rless-pcDNA 3.1, BaEV-FLV tail-pcDNA 3.1, BaEV-KLV tail-pcDNA 3.1, BaEV-GaVL tail-pcDNA 3.1 or BaEV-MoRV tail-pcDNA 3.1 combination, co-transfected 293T cells, Perform lentivirus packaging and concentration.

The concentrated lentivirus was titer tested using 293T. Inoculate 1×10 ⁵ 293T cells into a 24-well plate, dilute the concentrated virus 10 times, and infect 293T cells at 1, 2, and 5 ul/well respectively, and add DEAE transfer agent (Shanghai Sangon, Cat. No. : A600147), 2 days after infection, the infected 293T cells were collected for flow cytometric detection. Among them, the CAR structure positive rate (the proportion of cells containing the CAR structure in the total number of cells, hereinafter referred to as the "positive rate") was detected by flow cytometry using PE-coupled L protein (Sino biological, product number: 11044-H07E-P). The titer calculation method is: Titer (TU/ml) = 1 × 10 ⁵ × positive rate × dilution factor ÷ virus volume × 1000. The results show that the combination containing SERV-Rless cannot form lentivirus (Figure 3), while the combination containing HERV-wt, HERV-Rless, BaEV-FLV tail, BaEV-KLV tail, BaEV-GaVL tail, and BaEV-MoRV tail can form Lentivirus (Figure 4).

Using DEAE as an auxiliary agent, lentivirus packaged with HERV-wt or HERV-Rless was used to transduce PBNK (peripheral blood NK cells) at MOI=5. Flow cytometry was performed 3 days after transduction. The results showed that the transduction efficiency was less than 5. % (Figure 5), that is, effective transduction cannot be performed.

Use lentivirus packaged with BaEV-FLV tail, BaEV-KLV tail, BaEV-GaVL tail, and BaEV-MoRV tail envelope glycoprotein to transduce PBNK at MOI=1, and conduct flow cytometry 3 days after transduction, as shown in Figure 6 shown.

Use lentivirus packaged with BaEV-MoRV tail and BaEV-GaLV tail envelope glycoproteins as follows MOI=3 or MOI=5 was used to transduce PBNK, and flow cytometry was performed 3 days after transduction, as shown in Figure 7

Based on Figure 4, Figure 6 and Figure 7, it can be seen that compared to other lentiviruses mentioned above, the lentivirus packaged with BaEV-MoRV tail has a relatively higher packaging titer and has a relatively higher transduction efficiency for peripheral blood NK cells. , especially when transducing CAR genes, BaEV-MoRV-tail envelope glycoprotein shows more prominent advantages in transduction efficiency.

Lentivirus packaged with BaEV-MoRV tail and lentivirus packaged with BaEV-Rless and BaEV/TR (the specific structure is described in the international application WO2013/045639A1, the full text of which is incorporated herein by reference). Comparing the transfection efficiency (Figure 8) and the transfection efficiency of PBNK cells (Figure 6), the results show that the lentivirus packaged with BaEV-MoRV tail still has a relatively higher packaging titer and has a relatively higher effect on peripheral blood NK cells. High transduction efficiency.

Example 2 PB transposon system construction of 293T-BaEV-MoRV tail cell line and its application

The codon-optimized BaEV-MoRV tail coding sequence was ligated into the PB transposon plasmid (synthesized by Genscript, which contains the necessary functional components of the PiggyBac transposon) through double enzyme digestion. After verification by enzyme digestion and sequencing , amplify and extract the corresponding plasmid pPBK-CAG-BaEVRless-WPRE-bGH, and the plasmid concentration used should not be less than 1000ng/ul. Mix 1×10 ⁷ 293T cells with 5 μg pPBK-CAG-BaEVRless-WPRE-bGH plasmid and 5 μg transposase plasmid 2P (synthesized by Genscript, which can express PB transposase in mammalian cells) in the total volume. Mix well in 100 μl of electroporation solution, and perform electroporation using the Lonza 4D electroporation system. The electroporation program is DG130, and the electroporation solution is OPTI-MEM.

The electroporated 293T cells were expanded and cultured, and positive cells were sorted by flow cytometry. The labeled antibody used was a mouse polyclonal antibody for the extracellular region of BaEV (homemade, which can be obtained by any polyclonal antibody known in the art). Preparation method), the fluorescent secondary antibody is rabbit anti-mouse Alexa 647 (Thermos product number: A21239). The sorted 293T cells expressing BaEV-MoRV tail (293T-BaEV-MoRV tail) were expanded and cultured and the positive rate was retested again (Figure 9). As can be seen from the figure below, the sorted 293T cells expressing BaEV-MoRV tail were expanded and cultured. The 293T cells of MoRV tail still have a high positive rate of expression.

The above-sorted 293T cells expressing BaEV-MoRV tail (293T-BaEV-MoRV tail cell line) were transduced using the composition shown in Table 1, and the titer of the envelope lentivirus was detected. The titer test results are also shown in Table 1. It can be seen that adding VSV-G and BaEV-MoRV-tail envelope glycoprotein at the same time during the packaging process can further improve the quality of lentivirus compared with only adding BaEV-MoRV-tail envelope glycoprotein. Packaging efficiency.

Table 1. Virus titers packaged by 293T-BaEV-MoRV tail (PB)

The above-mentioned lentivirus was used to transduce PBNK cells at MOI=5, and 3 days after transduction, the anti-CD19-scFv-APC monoclonal antibody (homemade, can be prepared using any known monoclonal antibody preparation method in the field) was used for detection. CAR-NK positive rate. The results are shown in Figure 10. It can be seen that lentivirus packaged with BaEV-MoRV-tail envelope glycoprotein, whether or not it further contains VSV-G, can achieve high infection efficiency on PBNK cells.

Example 3 Modification of glycoprotease cleavage sites in BaEV envelope with different structures

Replace the protease cleavage sites of BaEV/TR and BaEV-MoRV tail with HIV protease cleavage site (HIV cleavage site) or synthetic sequence (as shown in Figure 2). pBKL2-GFP, pMDLg/pRRE, pRSV-Rev and candidate envelope glycoproteins BaEV/TR, BaEV-MoRV Tail, BaEV-MoRVT-HIV protease cleavage site (corresponding protease cleavage site of BaEV-MoRV-tail tail domain lentivirus packaging is performed by combining the BaEV-HIV protease cleavage site (replacing its own protease cleavage site with an HIV protease cleavage site or artificially synthesized site) and the BaEV-HIV protease cleavage site (replacing its own protease cleavage site with an HIV protease cleavage site or artificially synthesized site). Same as Example 1; after harvesting the lentivirus crude extract, digest the 293T cells with trypsin and collect them into a centrifuge tube. After labeling 7-AAD, use a flow cytometer to detect the cell viability, as shown in the table below, by As can be seen in Table 2, the packaged lentivirus can maintain a high cell viability rate.

Table 2. Detection of cell viability after transduction with envelope lentivirus containing different envelope glycoproteins

The above lentivirus was used to transduce PBNK at MOI=5. Three days after transduction, flow cytometry was used for detection. The results are shown in Figure 11. It can be seen that the protease cleavage site was changed in the BaEV-MoRV Tail structure. For example, changing its protease cleavage site to an HIV protease cleavage site can enhance the cell transduction efficiency of this structure.

Example 4 The 293T-BaEV-MoRV Tail cell line constructed by the lentiviral method is used for lentiviral packaging of CD123-CAR and mbIL15.

The BaEV-MoRV Tail coding sequence was obtained through codon optimization and sequence synthesis, and was ligated into the lentiviral transfer plasmid pBKL2 using different promoters through enzyme digestion. After enzyme digestion and sequencing verification, the corresponding plasmids pBKL2-MoRV tail (the expression of envelope glycoprotein in this plasmid uses the CAG promoter), pBKL2-miniCMV-MoRV tail and pBKL2-SV40-MoRV tail were extracted.

pBKL2-BaEV-MoRV tail or pBKL2-miniCMV-BaEV-MoRV tail or pBKL2-SV40-BaEV-MoRV tail were transfected into 293T cells with lentiviral packaging plasmids pMDLg/pRRE and pRSV-Rev respectively through calcium transfection. The virus crude extract was harvested at 48 h. After concentrating the crude virus extract with PEG-6000, the virus titer was determined by RT-PCR.

According to MOI=1-2, transduce 293T with the above lentivirus. 3 days after transduction, the transduction efficiency is detected by flow cytometry. If the positive rate is >85%, subsequent verification can be carried out. The BaEV cell line positive rate detection antibody is a mouse polyclonal antibody of the extracellular region of BaEV, and the fluorescent secondary antibody is rabbit anti-mouse Alexa 647. The results show that using lentivirus transduction cells can also successfully construct a cell line expressing BaEV-MoRV tail envelope glycoprotein (293T-BaEV-MoRV Tail (LV)) and be used for subsequent lentivirus packaging.

The lentiviral plasmids of CD123-CAR (derived from patent ZL201810207761.2) and mbIL15 and three lentiviral packaging plasmids pMDLg/pRRE, pRSV-Rev, and pMD2.G were transduced into 293T expressing BaEV-MoRV Tail obtained in the above steps. cell. The virus crude extract was harvested at 48 h. After concentrating the crude virus extract with PEG-6000, the virus titer was detected by flow cytometry. Viruses can be stored at -80°C, as shown in the table 3 It can be seen that lentivirus packaging using CD123-CAR and mbIL15 as target genes can also be successfully carried out using the envelope glycoprotein of the present application.

Table 3. CD123-CAR and mbIL15 packaging titer test

Subsequently, the lentivirus was transduced into PBNK cells activated for 2 days at MOI=3. Three days after transduction, the positive rate of NK cells was detected by flow cytometry. For the detection method, please refer to Example 2. The results are shown in Figure 12. It can be seen that lentiviruses containing CD123-CAR, mbIL15 and other foreign protein coding sequences packaged with the envelope glycoprotein of the present application can also infect PBNK cells with high efficiency.

Example 5. Construction of 293T-BaEV-Rless cell line using lentiviral system and its application

The BaEV-Rless coding sequence was obtained through codon optimization and sequence synthesis, and was ligated into the lentiviral transfer plasmid pBKL2 with different promoters through enzyme digestion. After restriction enzyme digestion and sequencing verification, the corresponding plasmids pBKL2-BaEVRless (CAG promoter), pBKL2-miniCMV-BaEVRless (miniCMV promoter) and pBKL2-SV40-BaEVRless (SV40 promoter) were extracted.

pBKL2-BaEVRless or pBKL2-miniCMV-BaEVRless or pBKL2-SV40-BaEVRless and three lentiviral packaging plasmids pMDLg/pRRE, pRSV-Rev, and pMD2.G were transfected into 293T cells by calcium transfection method. The virus crude extract was harvested at 48 h. After concentrating the crude virus extract with PEG-6000, the virus titer was determined by RT-PCR.

According to MOI=1-2, transduce 293T with the above lentivirus, and detect the transduction efficiency by flow cytometry 3 days after transduction. If the positive rate is >85%, follow-up verification can be carried out. The BaEV cell line positive rate detection antibody is a mouse polyclonal antibody of the extracellular region of BaEV, and the fluorescent secondary antibody is rabbit anti-mouse Alexa 647;

The lentiviral transfer plasmid containing the coding sequence of the CD19-targeting CAR (PCAR-19B, whose specific information is detailed in Chinese patent: CN107226867A, the full text of which is incorporated herein by reference) and two lentiviral packaging plasmids pMDLg/pRRE , pRSV-Rev (the rightmost column of Table 2), or 293T cells transduced with the three lentiviral packaging plasmids pMDLg/pRRE, pRSV-Rev, and pMD2.G respectively (Table 4-5). The virus crude extract was harvested at 48 h. After concentrating the crude virus extract with PEG-6000, the virus titer was detected by flow cytometry. Viruses can be stored at -80°C.

The results show that using BaEVRless and VSV-G at the same time can further improve the packaging efficiency compared to using BaEVRless alone (and BaEV-G with the R peptide removed) for lentivirus packaging. Based on the results in Table 1 and Table 4, the results of lentivirus packaging using BaEV-MoRV-tail and BaEV-Rless envelope glycoproteins together with VSV-G show that on the basis of using BaEV-G, VSV- The conclusion that G can improve the packaging efficiency of enveloped viruses is universal. . In addition, as shown in Table 5, it can be seen that the packaging method using BaEVRless and VSV-G is suitable for a variety of promoters, including but not limited to CAG, miniCMV and SV40.

Table 4. 293T-BaEVRless cell line packaging BaEV lentivirus and BaEV::VSV-G chimeric lentivirus

Table 5. BaEV::VSV-G chimeric lentivirus packaged in the 293T-BaEVRless cell line with different promoters (the envelope contains VSV-G and BaEVRless lentiviral particles)

The PBNK selected from the above-mentioned lentivirus transduction and activation for 2 days were used to detect the positive rate of CAR-NK, in vitro killing and IFN-γ secretion by flow cytometry 7 days after transduction. The results are shown in Figure 13 and Table 6. The group marked with (2G) represents the group that was simultaneously transfected with pMD2.G plasmid, that is, the group of BaEV::VSV-G chimeric envelope lentivirus, while the group marked with (BaEV) represents the group among which The packaged lentiviral envelope glycoprotein is BaEV and does not contain VSV-G. 293T-BaEVmini indicates that the promoter used for expression of BaEV in the cell line is miniCMV, and 293T-BaEV indicates that the promoter used for expression of BaEV in the cell line is CAG. 293T means that the cell line used is unmodified 293T cells, which are modified to package the lentivirus by introducing the BaEV-G envelope plasmid into the 293T cells. The control group does not package any virus, and only uses unmodified NK cells as a blank control for packaging virus-transduced NK cells in other groups.

Table 6. The efficiency of NK transduction by BaEV lentivirus and BaEV::VSV-G chimeric lentivirus packaged in different cell lines and the in vitro functional test of CAR-NK

From the results in Figure 13 and Table 6, it can be seen that viruses containing both VSV-G and BaEV-G in the envelope are more effective in immune cells than viruses containing only BaEV-G envelope glycoprotein or variants thereof. Cells that are difficult to transduce have higher infection efficiency (reflected in CAR positivity rate). Moreover, the CAR-containing immune effector cells constructed by transducing viruses containing VSV-G and BaEV-G all had good killing power (reflected in the killing rate and expression of IFN-γ), and using BaEV::VSV- Engineered immune effector cells constructed with G chimeric lentivirus have stronger killing power than lentivirus packaged with BaEV-G or its variants.

In addition, the results of this example also prove that the cell line expressing BaEV-G or its variant protein constructed by lentivirus infection of host cells is similar to the cell line expressing BaEV-G or its variant constructed by transposition. It can be used for efficient lentivirus packaging, and the lentivirus can efficiently transduce immune cells and other cells that are difficult to transduce.

Example 6. Construction of 293T-BaEV-Rless cell line using PB transposon system and its application

The codon-optimized BaEV-Rless coding sequence was ligated into the PB transposon plasmid through double enzyme digestion. After enzyme digestion and sequencing verification, the corresponding plasmid pPBK-CAG-BaEVRless-WPRE was extracted. The concentration of the plasmid should not be lower than 1000ng/ul;

Mix 1e7 293T cells with 2.5ug pPBK-CAG-BaEVRless plasmid and 2.5ug transposase plasmid 2P in a total volume of 100ul electroporation medium, and perform electroporation through the Lonza 4D electroporation system. The electroporation program is DG130 and the electroporation medium is OPTI-MEM;

The electroporated 293T cells were expanded and cultured, and positive single cells were sorted into a 96-well plate (i.e. 1 cell/well) by flow cytometry. The antibody used for flow cytometry labeling was a mouse polyclonal antibody against the extracellular region of BaEV. , the fluorescent secondary antibody is rabbit anti-mouse Alexa 647. The expression abundance of BaEV in different clones detected by flow cytometry after expansion of positive single clones is shown in Figure 14. The abundance is divided into three levels: high, medium and low, as shown in the right panel of Figure 14.

Select some clones from the aforementioned three levels of high, medium and low for subsequent virus packaging and titer determination. The lentiviral plasmid of PCAR-19B (described in detail in patent: CN107226867A) and three lentiviral packaging plasmids pMDLg/pRRE, pRSV-Rev, and pMD2.G were transfected into the above-mentioned positive clones by calcium transfer method, and the crude virus extract was used to directly transfect The titer test was performed and the results are shown in Table 7.

Table 7. Titers of BaEV::VSV-G chimeric chronic diseases packaged by different 293T-BaEV-Rless (PB) cell clones in cell culture supernatants

Select clones with higher titers in the above experiments, package the viruses in large quantities, and concentrate them according to the method in Implementation Case 1. After concentration, the titers are tested. As shown in Figure 15, 293T-BaEV(PB)-1 and 293T-BaEV(PB)-2 are lentiviruses packaged using No. 11 and No. 19 293T-BaEVRless (PB) clones in Table 7, respectively. 293T- BaEV(LV) represents the lentivirus packaged in the 293T cell line expressing BaEVRless constructed using lentiviral transduction of the 293T cell line. 293T represents the lentivirus packaged by direct co-transfection of the BaEVRless packaging plasmid. It can be seen that cell lines expressing BaEV-G and its various variants (including the aforementioned BaEV-MoRV-tail envelope glycoprotein and the BaEVRless envelope glycoprotein of this embodiment) were constructed by transposition and used to perform slow-motion experiments. Virus packaging can improve packaging efficiency and titer compared to direct transfection of plasmids containing nucleic acids encoding envelope glycoproteins for packaging.

Clone No. 26 was expanded and cultured for large-system lentivirus packaging. PBNK was transduced at MOI=3 after activation for 2 days, and the CAR-NK positive rate was detected 3 days after transduction. The results are shown in Figure 16. It can be seen that the BaEV envelope lentivirus packaged using the protocol of the present application has a high transduction efficiency for cells that are difficult to transduce, such as NK cells.

Example 7. 293T-BaEV-Rless cell line is used to package lentiviruses with different target genes.

The genes encoding CD123-CAR, mbIL15, and mbIL12 were ligated into the lentiviral transfer plasmid pBKL2 through double enzyme digestion. After verification by double enzyme digestion and sequencing, pBKL2-CD123-CAR, pBKL2-CD123-CAR, and mbIL12 were obtained respectively. Three plasmids, pBKL2-mbIL15 and pBKL2-mbIL12; perform virus packaging, concentration and titer determination according to the lentivirus packaging method in Example 6. The virus titer is shown in Figure 17. It can be seen that the protocol of this application can be used for lentiviral packaging of various target genes.

The above lentivirus was used to transduce PBNK activated for 2 days at MOI=3 to obtain mbIL12-NK, mbIL15-NK and CD123-CAR-NK cells respectively, and the positive rate was detected by flow cytometry three days after transduction. mbIL12-NK and mbIL15-NK were labeled with IL12-PE (anti-IL12 monoclonal antibody with PE label) and CD215-PE (anti-CD215 monoclonal antibody with PE label) respectively, and CD123-CAR-NK was labeled with rCD123-his+anti His -Detection was performed after APC labeling, and the results are shown in Figure 18. It can be seen that the lentivirus carrying various target genes constructed using this protocol can efficiently construct engineered immune effector cells.

Example 8. Preparation of CAR-γδ T cells using BaEV chimeric lentivirus with different structures

γδT cells were activated in vitro for 8 days. BaEV-Rless::VSV-G chimeric lentivirus was used to transduce γδT cells at MOI=1, and VSV-G lentivirus was used as a control group to transduce γδT cells at MOI=5. On the 5th day after lentiviral transduction, the CAR protein was labeled with PE-coupled L protein, and CAR-γδT cells were detected by flow cytometry. The results are shown in Figure 19 (BaEV-Rless:VSV-G chimeric lentivirus and VSV- Comparison of G lentivirus transduction efficiency of γδ T cells): Chimeric lentivirus has a higher transduction rate. That is, compared with the packaging method of enveloped lentivirus in the prior art (only one envelope glycoprotein, VSV-G, is used to package enveloped viruses), VSV-G and BaEV-G or variants thereof are used to co-package chimeric envelopes. Viruses can increase the transduction efficiency of the enveloped virus.

Example 9.293T-BaEV-Rless packaging retrovirus and the efficiency of BaEV-Rless retrovirus in transducing mouse B cells

The 293T-BaEV-Rless cell line was transfected with 10 μg each of retroviral packaging element-related plasmids pCL-Eco and MSGV1-GFP. Refer to Example 5 for virus packaging and concentration methods. Use CHO cells to determine the retrovirus titer and detection method. And the calculation method refers to Example 1. The results show that the virus titer is 1.4e8Tu/ml; B cells isolated from mouse spleen are cultured in vitro for 3 days and the above retrovirus is added according to MOI=1, 3, 6; 24h after the addition of retrovirus , the GFP positive rate of B cells was detected by flow cytometry; the results are shown in Figure 20, which shows that the virus packaged using the protocol of the present application can efficiently transduce B cells. and can efficiently transduce primary cells.

Example 10 Comparison of transduction efficiency of BaEV-Rless and BaEV-Rless::VSV-G chimeric lentivirus

BaEV-Rless and BaEV-Rless::VSV-G chimeric lentivirus packaging, concentration and titer detection were carried out according to the method of Example 6. The titer results are shown in Table 8. The results show that the titer of the chimeric lentivirus The titer is comparable to that of BaEV-Rless. It shows that the method of packaging enveloped viruses through cell lines expressing BaEV-G is applicable to various BaEV-G variants (such as BaEV-Rless, BaEV-MoRV-tail, BaEV/TR, etc.), or chimeric enveloped viruses ( For example, the packaging of BaEV-G and its various variants and chimeric enveloped viruses of VSV-G), and there is no restriction on the type of target gene being packaged. This method is universal.

Table 8: Comparison of titers of BaEV-Rless and BaEV-Rless::VSV-G chimeric lentivirus

The above lentivirus was used to transduce PBNK cells activated for 2 days at MOI=3. Three days after transduction, the positive rate of NK cells was detected by flow cytometry. For the detection method, please refer to Example 7. The results are shown in Figure 21, as shown below. It can be seen from the figure that in the enveloped virus packaging scheme provided by this application, the use of enveloped viruses packaged with VSV-G and BaEV-G or their variants can further improve the transduction of difficult-to-transduce cells such as immune cells. Guidance efficiency.

The sequences used in the above examples of the present application are shown in the sequence listing below. It should be understood that the following sequences are only exemplary sequences for the embodiments of the present application, and are not intended to limit any of the embodiments of the present application. The nucleic acid sequence in the following sequence listing may represent a DNA sequence or an RNA sequence. When it represents an RNA sequence, the "T" represents uridine.

Attached: sequence information

Claims

A chimeric viral envelope glycoprotein or polypeptide comprising the extracellular region of the baboon endogenous retrovirus (BaEV) envelope glycoprotein, the transmembrane region of the BaEV envelope glycoprotein, and the murine endogenous retrovirus ( MoRV) envelope glycoprotein tail domain.
The chimeric viral envelope glycoprotein or polypeptide according to claim 1, wherein the extracellular region sequence of the BaEV envelope glycoprotein includes the amino acid sequence shown in SEQ ID NO: 1 or a functional derivative thereof, or includes An amino acid sequence that is approximately 70% or more identical to the amino acid sequence shown in SEQ ID NO: 1.
The chimeric viral envelope glycoprotein or polypeptide according to claim 1 or 2, wherein the transmembrane region sequence of the BaEV envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 19 Or a functional derivative thereof, or an amino acid sequence containing more than about 70% identity with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 19.
The chimeric viral envelope glycoprotein or polypeptide according to any one of claims 1-3, wherein the protease cleavage site of the tail domain of the MoRV envelope glycoprotein is replaced by an HIV protease cleavage site.
The chimeric viral envelope glycoprotein or polypeptide according to claim 4, wherein the HIV protease cleavage site includes the amino acid sequence shown in SEQ ID NO: 9 or a functional derivative thereof, or includes the same amino acid sequence as SEQ ID NO: 9 The amino acid sequences shown are amino acid sequences having about 70% or more identity.
The chimeric viral envelope glycoprotein or polypeptide according to any one of claims 1-5, wherein the tail domain of the MoRV envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 3 or 20 Or a functional derivative thereof, or an amino acid sequence containing more than about 70% identity with the amino acid sequence shown in SEQ ID NO: 3 or 20.
The chimeric viral envelope glycoprotein or polypeptide according to claim 1, which includes the amino acid sequence shown in SEQ ID NO: 4 or 21 or its functional derivative, or includes the amino acid sequence shown in SEQ ID NO: 4 or 21 The amino acid sequence has about 70% or more identity to the amino acid sequence.
Nucleic acid molecule encoding a chimeric viral envelope glycoprotein or polypeptide according to any one of claims 1-7.
The nucleic acid molecule according to claim 8, which comprises a polynucleotide sequence as shown in SEQ ID NO: 8 or 27 and a polynucleotide having about 70% or more identity with the nucleotide sequence as shown in SEQ ID NO: 8 or 27 nucleotide sequence.
A vector comprising a nucleic acid molecule according to claim 8 or 9, preferably the vector is a plasmid, a virus toxic particles, or artificial nanoparticles.
Cells comprising the chimeric viral envelope glycoprotein or polypeptide according to any one of claims 1-7, and/or the nucleic acid molecule according to claims 8 or 9, or the vector according to claim 10 .
The cell of claim 11, wherein the cell is a 293T cell or a derivative thereof.
A composition for virus packaging, comprising:

Vesicular stomatitis virus (VSV) envelope glycoprotein or nucleic acid encoding said VSV envelope glycoprotein; and

The chimeric viral envelope glycoprotein or polypeptide or nucleic acid encoding the same according to any one of claims 1-7.
The chimeric viral envelope glycoprotein or polypeptide according to any one of claims 1 to 7, the nucleic acid according to claim 8 or 9, the vector according to claim 10, and the vector according to claim 11 or 12 Use of the cells or the composition according to claim 13 for virus packaging.
Use according to claim 14, wherein said virus is a retrovirus, preferably a lentivirus.
A chimeric viral envelope glycoprotein or polypeptide comprising the extracellular region of the BaEV envelope glycoprotein, the transmembrane region of the BaEV envelope glycoprotein, and the tail domain of the BaEV envelope glycoprotein or murine leukemia virus (MLV ) The tail domain of an envelope glycoprotein, wherein the protease cleavage site in the tail domain is replaced by an HIV protease cleavage site.
Nucleic acid molecule encoding a chimeric viral envelope glycoprotein or polypeptide according to claim 16.
A method of packaging fake viruses, including:

Introduce into target cells the chimeric viral envelope glycoprotein or polypeptide according to any one of claims 1-7 and 16 or the nucleic acid molecule according to any one of claims 8, 9 and 17, for the purpose Genetically encoded nucleic acids and viral packaging elements; or

Construct a cell line that stably expresses the chimeric viral envelope glycoprotein or polypeptide according to any one of claims 1 to 7, and introduce the nucleic acid encoding the target gene and the viral packaging element into the cell line.
The method of claim 18, wherein the pseudovirus is a retrovirus, preferably a virus derived from HIV.
The method according to claim 18 or 19, wherein the step of constructing a cell line stably expressing the chimeric viral envelope glycoprotein or polypeptide includes: using a transposition method to convert the nucleic acid encoding the chimeric viral envelope glycoprotein or polypeptide into When inserted into the cell genome of the cell line, it is preferred that the transposon system used for transposition is selected from the group consisting of: PB transposon system, SB transposon system or ΦC31 integrase system.
The method according to claim 18 or 19, wherein the step of constructing a cell line stably expressing the chimeric virus envelope glycoprotein or polypeptide includes: using a transduction method to encode the chimeric virus envelope glycoprotein. The nucleic acid of the white or polypeptide is inserted into the cell genome of the cell line. Preferably, the sensitizing agent DEAE or polybrene is used during the transduction process.
The method of any one of claims 18-21, wherein the target cell or cell line further comprises or expresses VSV envelope glycoprotein.
The method according to any one of claims 18-22, wherein the target cell or cell line is a 293T cell or a derivative thereof.
A pseudoviral particle for transduction of cells, which contains the chimeric viral envelope glycoprotein or polypeptide according to any one of claims 1-7 and 16, and the chimeric viral envelope glycoprotein or polypeptide according to any one of claims 8, 9 and 17. A nucleic acid molecule as claimed in claim 1, or a composition according to claim 13.
The pseudoviral particle according to claim 24, which is a retrovirus, preferably a virus derived from HIV.
The pseudoviral particle according to claim 24 or 25, comprising a nucleic acid coding sequence encoding a chimeric antigen receptor.
The pseudovirion according to claim 26, wherein the chimeric antigen receptor is a chimeric antigen receptor that specifically binds CD19 or CD123.
Use of the pseudovirus according to any one of claims 24 to 27 for introducing foreign genes into cells.
The use according to claim 28, wherein the foreign gene is a chimeric antigen receptor gene.
Use according to claim 28 or 29, wherein said cells are immune cells, preferably T cells, NK cells or dendritic cells.
Cells transduced using pseudovirions according to any one of claims 24-27.
The cell according to claim 31, which is an NK cell, an αβ T cell, a γδ T cell, a DC cell or a hematopoietic stem cell.
Use of the cells according to claim 31 or 32 in the preparation of cell therapy drugs.