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EP-4739770-A1 - MATERIALS AND METHODS FOR VIRAL ENGINEERING

EP4739770A1EP 4739770 A1EP4739770 A1EP 4739770A1EP-4739770-A1

Abstract

The present invention provides materials and methods for viral engineering, including the production of vectors and viral particles useful in, for example, gene therapy.

Inventors

  • HARAN, Kumudhini Preethi
  • SUKUMAR, Madhusudhanan
  • COSTA, Andreia

Assignees

  • Janssen Biotech, Inc.

Dates

Publication Date
20260513
Application Date
20240703

Claims (20)

  1. 1. A method for generating a multi pseudotyped virus, the method comprising: a. providing a mammalian cell line; b. adding cell medium to the mammalian cell line; c. contacting the mammalian cell line with a composition comprising at least one packaging plasmid encoding at least one glycoprotein; d. incubating the mammalian cell line with the composition; and e. harvesting the multi pseudotyped virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the harvesting of the multi pseudotyped virus yields a higher titer of virus as compared to a method of producing a virus comprising a single pseudotype.
  2. 2. The method of claim 1, wherein the culturing is performed at a temperature of about 37°C and in a humidified incubator with about 5% to about 10% CO2 for about 24 to 36 hours.
  3. 3. The method of claim 1 or 2, wherein the composition comprising at least one packaging plasmid encoding at least one glycoprotein further comprises a transfer vector comprising a gene encoding a transgene.
  4. 4. The method of any one of claims 1-3, wherein the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of an ecotropic virus envelope glycoprotein and an amphotropic envelope glycoprotein.
  5. 5. The method of any one of claims 1-3, wherein the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of RD114, VSV- G, 4070A, gap70, 10A1, and GALV.
  6. 6. The method of any one of claims 1-5, wherein the at least one glycoprotein is two glycoproteins, three glycoproteins, or four glycoproteins.
  7. 7. The method of any one of claims 1-6, wherein the at least one glycoprotein comprises a second glycoprotein selected from the group consisting of consisting of an ecotropic virus envelope glycoprotein and an amphotropic envelope glycoprotein; and is different from the first glycoprotein.
  8. 8. The method of any one of claims 1-6, wherein the at least one glycoprotein comprises a second glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV; and is different from the first glycoprotein.
  9. 9. The method of claim 8, wherein the two glycoproteins are RD114 and VSV-G.
  10. 10. The method of claim 8, wherein the two glycoproteins are GALV and VSV-G.
  11. 11. The method of claim 8, wherein the two glycoproteins are GALV and 10A1.
  12. 12. The method of any one of claims 1-11, wherein the at least one packaging plasmid encoding at least one glycoprotein is at a ratio of about 1/5 to 1/10 compared to total DNA.
  13. 13. A method for generating a multi pseudotyped virus, the method comprising: a. providing a mammalian cell line for transfection and viral production; b. culturing the mammalian cell line; wherein the culturing is performed at a temperature of about 37 °C and in a humidified incubator with about 5% to about 10% CO2; c. treating the mammalian cell line with Trypsin; d. adding cell medium to the mammalian cell line; e. contacting the mammalian cell line with a composition comprising i. a transfer vector comprising a transgene; and ii. at least one packaging plasmid encoding at least one glycoprotein; f. incubating the mammalian cell line with the composition; and g. harvesting the multi pseudotyped virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the harvesting of the multi pseudotyped virus yields a higher titer of virus as compared to a method of producing a virus comprising a single pseudotype.
  14. 14. The method of claim 13, wherein the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of an ecotropic virus envelope glycoprotein and an amphotropic envelope glycoprotein.
  15. 15. The method of claim 13, wherein the first envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV.
  16. 16. The method of any one of claims 13-15, wherein the at least one glycoprotein is two glycoproteins, three glycoproteins, or four glycoproteins.
  17. 17. The method of any one of claims 13-16, wherein the second envelope glycoprotein is selected from the group consisting of an ecotropic virus envelope glycoprotein and an amphotropic envelope glycoprotein; and wherein the second envelope glycoprotein is different from the first envelope glycoprotein.
  18. 18. The method of any one of claims 13-16, wherein the second envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV; and wherein the second envelope glycoprotein is different from the first envelope glycoprotein
  19. 19. The method of any one of claims 13-18, wherein the two glycoproteins are RD 114 and VSV-G.
  20. 20. The method of any one of claims 13-18, wherein the two glycoproteins are GALV and VSV-G.

Description

Materials and Methods for Viral Engineering CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No. 63/511,977, filed July 5, 2023, which is incorporated by reference herein in its entirety. BACKGROUND [0002] A retrovirus is an RNA viral molecule encapsulated in a viral protein envelope which infects a host cell, reverse transcribes its RNA molecule into DNA, and integrates its genome stably into the host cell's genome. Systems for packaging retroviral vectors into viral particles to form virions have been developed to facilitate the transfer of exogenous genes into target cells for the purpose of gene therapy. A retroviral vector is DNA or RNA that has been modified to serve as a vector for recombinant DNA. Retroviral vectors can transfer genes into a wide variety of cell types from many different species. SUMMARY [0003] The present invention relates, in part, to methods of engineering viruses to comprise two or more envelope glycoproteins. [0004] In some embodiments, the invention relates to methods for generating multi pseudotyped viruses, the method comprising providing a mammalian cell line, adding cell medium to the mammalian cell line, contacting the mammalian cell line with a composition comprising at least one packaging plasmid encoding at least one glycoprotein, incubating the mammalian cell line with the composition, and harvesting the multi pseudotyped virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the harvesting of the multi pseudotyped virus yields a higher titer of virus as compared to a method of producing a virus comprising a single pseudotype. [0005] In one embodiment, the culturing is performed at a temperature of about 37°C and in a humidified incubator with about 5% to about 10% CO2 for about 24 to 36 hours. In one embodiment, the composition comprising at least one packaging plasmid encoding at least one glycoprotein further comprises a transfer vector comprising a gene encoding a transgene. In one embodiment, the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of an ecotropic virus envelope glycoprotein and an amphotropic envelope glycoprotein. In one embodiment, the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV. In one embodiment, the at least one glycoprotein is two glycoproteins, three glycoproteins, or four glycoproteins. In one embodiment, the at least one glycoprotein comprises a second glycoprotein selected from the group consisting of consisting of an ecotropic virus envelope glycoprotein and an amphotropic envelope glycoprotein; and is different from the first glycoprotein. In one embodiment, the at least one glycoprotein comprises a second glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV; and is different from the first glycoprotein. In one embodiment, the two glycoproteins are RD114 and VSV-G. In one embodiment, the two glycoproteins are GALV and VSV-G. In one embodiment, the two glycoproteins are GALV and 10A1. In one embodiment, the at least one packaging plasmid encoding at least one glycoprotein is at a ratio of about 1/5 to 1/10 compared to total DNA. [0006] In some embodiments, the invention relates to a method for generating a multi pseudotyped virus, the method comprising, providing a mammalian cell line for transfection and viral production, culturing the mammalian cell line; wherein the culturing is performed at a temperature of about 37 °C and in a humidified incubator with about 5% to about 10% CO2, treating the mammalian cell line with Trypsin, adding cell medium to the mammalian cell line, contacting the mammalian cell line with a composition comprising i) a transfer vector comprising a transgene and ii) at least one packaging plasmid encoding at least one glycoprotein, incubating the mammalian cell line with the composition, and harvesting the multi pseudotyped virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the harvesting of the multi pseudotyped virus yields a higher titer of virus as compared to a method of producing a virus comprising a single pseudotype. [0007] In some embodiments, the method further comprises filtering the multi pseudotyped virus, wherein the filtering is performed with low protein binding filters. In one embodiment, the filtering is performed with filters comprising cellulose acetate or polysulfonate. In some embodiments, the method further comprises concentrating the virus. In some embodiments, the method produces about 20, 30, 40, 50, 60, 70, 80 or 90 times more titres of the multi pseudotyped virus than a method of producing a virus comprising a single envelope glycoprotein, wherein the single envelope glycoprotein comprises RD114, VSV-G, GALV, or 10A1. [0008] In some embodiments, the virus