EP-4735584-A1 - CLICK CHEMISTRY ASSISTED CELL SURFACE ENGINEERING FOR RESTORING LENTIVIRUS ENTRY

Abstract

The present disclosure is directed to methods of labeling the surface of cells with a ligand for improved transduction. The cells may be labeled with a recombinant LDLR or anti-VSV-G antibody, such as by click chemistry. Further provided herein are methods of gene therapy, screening, and disease models using the present surface labeled cells.

Inventors

• MA, Leyuan
• CHAN, Letitia

Assignees

• The Children's Hospital of Philadelphia
• The Trustees of The University of Pennsylvania

Dates

Publication Date

20260506

Application Date

20240628

Claims (16)

1. WHAT IS CLAIMED: 1. A method of chemically labeling a cell comprising attaching to the surface of said cell a recombinant low density lipoprotein receptor (LDLR) protein or anti-vesicular stomatitis virus envelope glycoprotein (VSV-G) antibody or fragment thereof by performing click chemistry.
2. 2. The method of claim 1, wherein the cell has low, undetectable or essentially no expression of LDLR.
3. 3. The method of claim 1 or 2, wherein the anti-VSV-G antibody is I1, M55, 8G5F11 or IE9F9 antibody.
4. 4. The method of any of claims 1-3, wherein performing click chemistry comprises: (a) conjugating Sulfo-NHS DBCO to recombinant LDLR or anti-VSV-G antibody to obtain DBCO-labeled protein; (b) conjugating azide to cell surface proteins of said cell using 6-azidohexanoic acid-NHS ester to obtain an azide-labeled cell; and (c) mixing the DBCO-protein with the azide-labeled cell to obtain a LDLR or anti- VSV-G antibody labeled cell.
5. 5. The method of claim 4, wherein the mixing is performed at 0.1 mg/mL.
6. 6. The method of claim 2, wherein the cell with low or essentially no expression of hLDLR is a human naïve T cell, a resting human T cell, a human B cell, a human epithelial cell, a murine immune cell, a canine immune cell, a monkey immune cell, or a cancer cell line.
7. 7. The method of claim 6, wherein the murine immune cell, monkey immune cell, or canine immune cell is a T cell, NK cell, or hematopoietic cell.
8. 8. The method of claim 6, wherein the murine immune cell, monkey immune cell, or canine immune cell is a T cell.
9. 9. The method of claim 6, wherein the cancer cell line is SUP-B15 or Raji.
10. 10. The method of any of claims 1-7, further comprising transducing the cell with lentivirus. 21 4895-2359-0761, v. 2
11. 11. The method of claim 10, wherein the lentivirus comprises VSV-G as the envelope protein.
12. 12. The method of claim 10, wherein transduction efficiency is at least 80%.
13. 13. A chemically labeled cell having a recombinant LDLR or anti-VSV-G antibody or fragment thereof attached to the surface.
14. 14. The cell of claim 13, wherein the cell is produced by the method of any of claims 1-12.
15. 15. The cell of claim 13, wherein the cell has been transduced by a VSV-G lentivirus.
16. 16. Use of the chemically labelled cell transduced with the VSV-G lentivirus for genomic screening or gene therapy. 22 4895-2359-0761, v. 2

Description

DESCRIPTION CLICK CHEMISTRY ASSISTED CELL SURFACE ENGINEERING FOR RESTORING LENTIVIRUS ENTRY PRIORITY CLAIM This application claims benefit of priority to U.S. Provisional Application Serial No. 63/511,490, filed June 30, 2023, the entire contents of which are hereby incorporated by reference. STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with government support under grant number AI164319 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND 1. Field of the Disclosure The present disclosure relates generally to the fields of molecular biology. More particularly, the disclosure relates to surface labeled cells for improved transduction, such as for gene therapy. 2. Background In current laboratory practice, transduction efficiency of certain cell types using VSV- G pseudotyped lentiviruses is quite low. For example, in murine T cells the transduction efficiency ranges from 5-10%. In canine hematopoietic cells, there is also low transduction efficiency, typically less than 1%. Given that many CRISPR libraries and genetic engineering tools are all lentivirus-based, it is extremely impactful to expand the application of these tools to a wider variety of cell types and animal models for immunology research as well as pre- clinical evaluation of cell-based immunotherapies. Thus, there is an unmet need for improved methods of lentivirus transduction. 1 4895-2359-0761, v. 2 SUMMARY Thus, in accordance with the present disclosure, there is a method of chemically labeling a cell comprising attaching to the surface of said cell a recombinant low density lipoprotein receptor (LDLR) protein or anti-vesicular stomatitis virus envelope glycoprotein (VSV-G) antibody or fragment thereof by performing click chemistry. In some aspects, the cell has low, undetectable or essentially no expression of LDLR, such as by measuring with flow cytometry with an anti-LDLR antibody. In some aspects, the anti-VSV-G antibody is I1 antibody. In certain aspects, performing click chemistry comprises: (a) conjugating Sulfo-NHS DBCO to recombinant LDLR or anti-VSV-G antibody to obtain DBCO-labeled protein; (b) conjugating azide to cell surface proteins of said cell using 6-azidohexanoic acid-NHS ester to obtain an azide-labeled cell; and (c) mixing the DBCO-protein with the azide-labeled cell to obtain a LDLR or anti- VSV-G antibody labeled cell. In some aspects, the mixing is performed at 0.1 mg/mL. In certain aspects, the cell with low or essentially no expression of hLDLR is a human naïve T cell, a resting human T cell, a human B cells, a human epithelial cell, a murine immune cell, a canine immune cell, a monkey immune cell, or a cancer cell line. In certain aspects, the murine immune cell or canine immune cell is a T cell, NK cell, or hematopoietic cell. In some aspects, the murine immune cell, monkey immune cell, or canine immune cell is a T cell. In certain aspects, the cancer cell line is SUP-B15 or Raji. In some aspects, the method further comprises transducing the cell with lentivirus. In certain aspects, the lentivirus comprises VSV-G as the envelope protein. In some aspects, transduction efficiency is at least 80%. A further embodiment provides a chemically labeled cell having a recombinant LDLR or anti-VSV-G antibody or fragment thereof attached to the surface. In some aspects, the cell is produced by the present embodiments and aspects thereof (e.g., a method of chemically labeling a cell comprising attaching to the surface of said cell a recombinant low density lipoprotein receptor (LDLR) protein or anti-vesicular stomatitis virus envelope glycoprotein (VSV-G) antibody or fragment thereof by performing click chemistry). In certain aspects, the cell has been transduced by a VSV-G lentivirus. 2 4895-2359-0761, v. 2 Further embodiments provide use of the present chemically labeled cell having a recombinant LDLR or anti-VSV-G antibody or fragment thereof attached to the surface for gene therapy, lentivirus-based in vitro gene delivery, human naïve T cell engineering, and genetic screens using murine T cells for T cell immunology and cancer immunology research. In additional embodiments, the present lentivirus can be used for pre-clinical studies using the customized lentiviruses or a lentivirus library for genomic screens (e.g., RNAi, CRISPR, ORF library). For example, the desired non-transducible cells can be surface-labeled with fluorescein followed by the addition of VSVG/aFITC dual-envelope pseudotyped lentivirus using routine transduction method (e.g., spin transduction). Further embodiments provide methods for using the present lentivirus for pre-clinical studies using a gene-modified mouse, canine, or monkey T cells. Another embodiment provides use of naïve, resting or activated human T cells pre-labeled with fluorescein followed by addition of VSVG/aFITC dual envelope pseudotyped lentivirus using routine transduction method (e.g., spin trans