Methods and compositions related to high-titer pseudotyped retroviruses
View Patent ↗The present invention concerns the methods and compositions related to pseudotyped viral vectors. Embodiments of the invention include pseudotyping expression cassettes that include nucleic acid elements for enhancing the titer of pseudotyped viral particles. Embodiments of the invention include novel methods and compositions related to making high titer pseudotyped retroviral vector compositions. A heterologous envelope glycoprotein is typically incorporated into the virus during the budding or virus production process. Certain embodiments of the invention include pseudotyped retroviral vectors comprising a heterologous envelope glycoprotein derived from a Jaagsiekte sheep retrovirus (JSRV env). Pseudotyped viruses or viral particles may have a modified host range that is influenced by the properties of the heterologous envelope glycoprotein. Thus, embodiments of the invention include improved methods and compositions related to pseudotyped viruses suitable for ex vivo and in vivo methods including gene transfer and other therapeutic and experimental methods.
1. A polynucleotide preparation comprising:
(a) a pseudotyping expression cassette comprising:
(i) a first polynucleotide segment comprising a splice acceptor site and encoding all or part of a heterologous env protein;
(ii) a second polynucleotide segment positioned 5′ to the first polynucleotide segment comprising all or part of a retroviral 5′ LTR and a splice donor site; and
(iii) a third polynucleotide segment positioned 3′ to the first polynucleotide segment comprising all or part of a retroviral 3′ LTR;
wherein the pseudotyping expression cassette lacks a GAG and POL encoding nucleic acid sequences; and
(b) a viral expression vector configured for packaging in a pseudotyped viral particle.
2. The preparation of claim 1 , wherein the first polynucleotide segment encodes all or part of a Jaaksiekte sheep retrovirus env protein.
3. The preparation of claim 1 , wherein the second polynucleotide segment encodes all or part of a Jaaksiekte sheep retrovirus 5′ LTR.
4. The preparation of claim 3 , wherein the 5′ LTR comprises a R and U5 region.
5. The preparation of claim 3 , wherein the 5′ LTR comprises a major splice donor site.
6. The preparation of claim 1 , wherein the third polynucleotide segment encodes all or part of a Jaaksiekte sheep retrovirus 3′ LTR.
7. The preparation of claim 6 , wherein the 3′ LTR comprises a U3, R, and U5 region.
8. The preparation of claim 1 , wherein the pseudotyping expression cassette further comprises a promoter.
9. The preparation of claim 8 , wherein the promoter is a CMV IE promoter.
10. The preparation of claim 8 , wherein the pseudotyping expression cassette is incorporated into the genome of a cell.
11. The preparation of claim 1 , wherein the pseudotyping expression cassette is maintained episomally in a cell.
12. The preparation of claim 1 , wherein the viral expression vector is retroviral expression vector.
13. The preparation of claim 12 , wherein the retroviral expression vector is a lentiviral expression vector.
14. The preparation of claim 13 , wherein the lentiviral expression vector is an immunodeficiency virus expression vector.
15. The preparation of claim 14 , wherein the immunodeficiency virus expression vector is a feline immunodeficiency virus expression vector.
16. A viral composition produced by methods comprising:
a) contacting a cell with a pseudotyping expression cassette and a viral vector of claim 1 ; and
b) obtaining a pseudotyped virus produced by the cell.