Retroviral vector particles and methods for their generation and use
The present invention relates to methods of host cell transduction utilizing ecotropic retroviral vector particles. The retroviral vector particle may comprise an envelope of Friend murine leukaemia virus, in particular the envelope encoded by molecular clone PVC-211 and the host cell may be engineered to recombinantly express the Reel receptor. The retroviral vector particles and methods of the invention can be used to introduce expressible polynucleotide sequences of interest into host cells with high efficiency. This results in protein production methods with higher yield (mg/L) and a reduction in manufacturing costs that could be used in a range of applications including for example, the production of therapeutic proteins, vaccines and antibodies.
1. A method for transducing a host cell, said method comprising transducing the host cell with a retroviral vector particle comprising an envelope of Friend murine leukaemia virus, wherein the envelope of Friend murine leukaemia virus is encoded by molecular clone PVC-211 (SEQ ID NO: 1) and wherein the host cell recombinantly expresses a Rec1 receptor.
2. The method of claim 1 , wherein susceptibility of the host cell to transduction is increased.
3. The method according to claim 1 , wherein the retroviral vector particle further comprises an expressible heterologous nucleotide sequence encoding a protein of interest.
4. The method according to claim 3 , further comprising the step of culturing the host cell under conditions to produce the protein of interest.
5. The method according to claim 4 , wherein said culturing step produces the protein of interest at a concentration of at least 1 mg/L.
6. The method according to claim 1 , wherein the host cell is a hamster cell.
7. The method according to claim 1 , wherein the host cell is a Chinese hamster ovary (CHO) cell.
8. The method according to claim 3 , wherein the protein of interest is selected from the group consisting of: interferons, erythropoietin, Factor VIII, clotting factors, antibodies, insulin, chemokines, cytokines, growth factors, angiogenesis modulatory factors, apoptosis modulatory factors and vaccines.
9. The method according to claim 3 , wherein the retroviral vector particle comprises at least two heterologous nucleotide sequences coding for at least two different proteins of interest.
10. The method according to claim 9 , wherein the at least two proteins of interest are an immunoglobulin heavy chain and an immunoglobulin light chain.
11. The method according to claim 1 , wherein the retroviral vector particle is produced by a retroviral packaging system comprising:
a) an envelope construct comprising a promoter operably linked to an envelope coding sequence of a Friend murine leukaemia virus, wherein the envelope of Friend murine leukaemia virus is encoded by molecular clone PVC-211 (SEQ ID NO: 1);
b) a packaging construct comprising a promoter operably linked to a nucleotide sequence encoding a retroviral gag and pol; and
c) a retroviral transfer vector.
12. The method of claim 11 , wherein the retroviral packaging system is expressed in a host cell under conditions effective to produce a retroviral vector particle.
13. A host cell line transduced with a retroviral vector particle comprising an envelope of Friend murine leukaemia virus encoded by the molecular clone PVC-211 (SEQ ID NO: 1), wherein the host cell line recombinantly expresses a Rec1 receptor.
14. The host cell line according to claim 13 , wherein the host cell line produces a protein of interest encoded by the retroviral vector.
15. The host cell line according to claim 14 , wherein the protein of interest is produced by the cell line at a concentration of at least 1 mg/L.
16. A system for transducing a host cell, comprising:
a) a retroviral vector particle comprising an envelope of Friend murine leukaemia virus encoded by the molecular clone PVC-211 (SEQ ID NO: 1); and
b) a target host cell which recombinantly expresses a Rec1 receptor.
17. The host cell line of claim 13 , wherein the cell is a hamster cell.
18. The host cell line of claim 17 , wherein the hamster cell is a Chinese hamster ovary (CHO) cell.
19. The method of claim 1 , wherein the Rec1 receptor has the sequence of SEQ ID NO: 32.
20. The system of claim 16 , wherein the Rec1 receptor has the sequence of SEQ ID NO: 32.