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EP-4735583-A1 - CELL SURFACE ENGINEERING OF TAILORED THERAPEUTIC GENE DELIVERY

EP4735583A1EP 4735583 A1EP4735583 A1EP 4735583A1EP-4735583-A1

Abstract

The present disclosure is directed to methods of improving the transduction efficiency of non-transducible cells, such as cells with low hLDLR. The cells may be labeled with 5'FAM and transduced with a scFv-truncated VSV-G pseudotyped lentivirus which comprises an anti-fluorescein scFv. Further provided are methods of gene therapy, cell engineering, genetic screening, and disease models using the fluorescein-labeled cells transduced with the scFv-truncated VSV-G pseudotyped lentivirus.

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 (20)

  1. 1. A composition comprising a cell conjugated to fluorescein, wherein the cell has low or essentially no expression or undetectable expression of human low density lipoprotein receptor (hLDLR).
  2. 2. A composition comprising a cell expressing a VSV-G chimeric protein conjugated to conjugated to fluorescein.
  3. 3. The composition of claim 1, wherein the cell has low, undetectable or essentially no expression of human low density lipoprotein receptor (hLDLR), such as measured by flow cytometry.
  4. 4. The composition of any one of claims 1-3, wherein the fluorescein is 5- carboxyfluorescein (5-FAM), FITC, or 6- carboxyfluorescein (6’FAM).
  5. 5. The composition of any one of claims 1-3, wherein the fluorescein is NHS-5’FAM, Maleimide-5’FAM, or l,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)- Poly-ethylene-glycol(PEG)-Fluorescein.
  6. 6. The composition of any one of claims 1-5, wherein the cell with low or essentially no expression of hLDLR is a human naive T cell, resting human T cells, a human B cell, human epithelial cell, murine immune cell, or canine immune cell, monkey immune cell, or cancer cell line (e.g., SUP-B15 or Raji).
  7. 7. The composition of claim 6, wherein the murine immune cell, monkey immune cell, or canine immune cell is a T cell, NK cell, or a hematopoietic cell.
  8. 8. The composition of any one of claims 1-7, wherein the 5-FAM is conjugated to N- hydroxysuccinimide at the 5’ carbon (NHS-5’-FAM).
  9. 9. The composition of any one of claims 1-7, wherein the 5’FAM is conjugated to an antibody targeting a cell surface protein.
  10. 10. The composition of claim 9, wherein the cell surface protein is CD71 on K562 cells, CD3, CD4, CD8, CD5, or CD7 on T cells, or CD19, CD20, or CD22 on B cells.
  11. 11. The composition of any one of claims 1-10, wherein the cell has been transduced with a pseudotyped lentivirus.
  12. 12. The composition of claim 11, wherein the pseudotyped lentivirus is scFv-truncated vesicular stomatitis virus envelope glycoprotein (VSV-G) pseudotyped lentivirus.
  13. 13. The composition of claim 12, wherein the VSV-G pseudotyped lentivirus comprises a VSV-G hinge, intracellular domain and transmembrane domain fused to an antifluorescein scFv.
  14. 14. The composition of claim 13, wherein the anti-fluorescein scFv is positioned at the N terminal of the lentivirus.
  15. 15. The composition of claim 13 or claim 14, wherein the anti-fluorescein scFv comprises 2A9M derived from fluorescein targeting antibody FITC-E2.
  16. 16. A nucleic acid encoding a VSV-G chimeric protein linked to an anti-fluorescein antibody or fragment thereof.
  17. 17. The nucleic acid of claim 16, wherein the nucleic acid has a sequence at least 90% identical to SEQ ID NO:2.
  18. 18. The nucleic acid of claim 16, wherein the anti-fluorescein antibody is an anti- fluorescein scFv.
  19. 19. The nucleic acid of claim 18, wherein the anti-fluorescein scFv comprises the antibody clone FITC-E2, 4m5.3, or 4-4-20.
  20. 20. The nucleic acid of claim 18, wherein the anti-fluorescein scFv is positioned at the N terminus of the chimeric protein.

Description

DESCRIPTION CELL SURFACE ENGINEERING OF TAILORED THERAPEUTIC GENE DELIVERY PRIORTIY CLAIM This application claims benefit of priority to U.S. Provisional Application Serial No. 63/511,468, filed June 30, 2023, the entire contents of which are hereby incorporated by reference. SEQUENCE LISTING DISCLOSURE This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on June 14, 2024, is named CHOPP0069WO.xml and is 21,586 bytes in size. BACKGROUND 1. Field of the Disclosure The present disclosure relates generally to the fields of molecular biology. More particularly, the disclosure relates to improved methods of transduction, such as for therapeutic gene delivery. 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, there is an unmet need 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. SUMMARY Thus, in accordance with the present disclosure, there is a composition: comprising a cell conjugated to fluorescein, wherein the cell has low or essentially no expression or undetectable expression of human low density lipoprotein receptor (hLDLR), such as wherein the cell has low, undetectable or essentially no expression of human low density lipoprotein receptor (hLDLR), such as measured by flow cytometry; or comprising a cell expressing a VSV-G chimeric protein conjugated to conjugated to fluorescein. The fluorescein may be5-carboxyfluorescein (5-FAM), FITC, or 6- carboxyfluorescein (6’FAM). The fluorescein may be NHS-5’FAM, Maleimide-5’FAM, or 1,2-Distearoyl-sn- glycero-3-phosphoethanolamine (DSPE)-Poly-ethylene-glycol(PEG)-Fluorescein. The cell with low, undetectable or essentially no expression of hLDLR may a human naive T cell, resting human T cells, a human B cell, human epithelial cell, murine immune cell, or canine immune cell, monkey immune cell, or cancer cell line (e.g., SUP-B15 or Raji). The murine immune cell, monkey immune cell, or canine immune cell may be a T cell, NK cell, or a hematopoietic cell. The 5-FAM may conjugated to N-hydroxysuccinimide at the 5’ carbon (NHS-5’-FAM) or may be is conjugated to an antibody targeting a cell surface protein, such as wherein the cell surface protein is CD71 on K562 cells, CD3, CD4, CD8, CD5, or CD7 on T cells, or CD 19, CD20, or CD22 on B cells. The cell may be have been transduced with a pseudotyped lentivirus, such as a scFv-truncated vesicular stomatitis virus envelope glycoprotein (VSV-G) pseudotyped lentivirus, e.g., wherein the VSV-G pseudotyped lentivirus comprises a VSV-G hinge, intracellular domain and transmembrane domain fused to an antifluorescein scFv. The anti-fluorescein scFv may be positioned at the N terminal of the lentivirus. The anti-fluorescein scFv may comprise 2A9M derived from fluorescein targeting antibody FITC-E2. A further embodiment provides a nucleic acid encoding a VSV-G chimeric protein linked to an anti-fluorescein antibody or fragment thereof. In some aspects, the anti-fluorescein antibody is an anti-fluorescein scFv. In some aspects, the anti-fluorescein scFv comprises the antibody clone FITC-E2, 4m5.3, or 4-4-20. In certain aspects, the anti-fluorescein scFv is positioned at the N terminus of the chimeric protein. In some aspects, the anti-fluorescein scFv is a 2A9M scFv derived from fluorescein targeting antibody FITC-E2. In certain aspects, the anti-fluorescein is fused to a VSV-G hinge, transmembrane and intracellular domain. In some aspects, the fusion protein is under control of a constitutive promoter. In certain aspects, the constitutive promoter is CMV, CAG, or EFla. Another embodiment provides a vector comprising the nucleic acid of the present embodiments or aspects thereof (e.g., a cell conjugated to 5 -carboxyfluorescein (5-FAM), wherein the cell has low or essentially no expression of human low density lipoprotein receptor (hLDLR)). In some aspects, the vector is a HIV-1 -derived lentiviral vector. Also provided herein is a host cell comprising the nucleic acid of the present embodiments or aspects thereof (e.g., a nucleic acid encoding a VSV-G chimeric protein linked to an anti-fluorescein antibody or fragment thereof). A further embodiment provides a polypeptide encoded by the nucleic acid of any one of the present embodiments or aspects thereof (e.g., a nucleic acid encoding a VSV-G chimeric protein linked to an anti-fluorescein antibody or fragment thereof). Another embodiment provides a pseudotyped lentivir