Virus-based vector compositions useful for transducing eukaryotic cells
The present invention provides viral vector compositions of high titer and purity, as well as methods for production of said compositions. The methods of the invention incorporate multiple features, such as production of viral vector particles in serum free media and multiple harvesting steps following transduction of the producer cell which provides for enhanced production of said viral vectors. The viral vector compositions of the invention, by virtue of their high titer and purity, minimize the deleterious phenotypic changes that typically occur following transduction of target cells, such as loss of a sub-populations of transduced cells, and effects on proliferation, differentiation, reprogramming or functionality of transduced cells.
1. A method for producing a purified RNA based viral vector composition, said method comprising:
(i) culturing a viral vector transfected producer cell in serum-free medium, said producer cell modified to complement deletions in the viral genome upon which the viral vector is based, under suitable conditions to permit the production of viral vector particles;
(ii) collecting the supernatant containing said viral vector particles at multiple times up to 72 hours post-transfection, wherein the first collection occurs between 24 and 36 hours post-transfection;
(iii) determining the ratio of total physical particles/transducing units (PP/TU) for each of the supernatants collected in (ii), wherein the PP/TU ratio of each of the collected supernatants is measured to be from 900:1 to 200:1;
(iv) pooling said collected supernatants; and
(v) subjecting said pooled supernatants to tangential ultrafiltration and/or diafiltration,
wherein the ratio of total physical particles/transducing units (PP/TU) in the RNA based viral vector composition is no greater than the PP/TU of any of the collected supernatants of (ii).
2. The method of claim 1 , further comprising a step of ion-exchange chromatography.
3. The method of claim 1 , wherein the ultrafiltration is operated on polysulfone hollow-fiber cartridges.
4. The method of claim 1 , wherein the retentate obtained following tangential ultrafiltration and/or diafiltration is treated with a nuclease.
5. The method of claim 4 , wherein the nuclease is benzonase or a DNase.
6. The method of claim 2 , resulting in a preparation of purified RNA based viral vector particles wherein the removal of DNA contaminants is at least 98% of such contaminants present in the initial serum-free culture medium, and the removal of cellular proteins contaminants is at least 99% of the cellular proteins contained in the initial serum-free culture medium.
7. The method of claim 1 , resulting in a preparation of purified RNA based viral vector particles, wherein the removal of DNA contaminants is less than 80% up to 90% of the content of such contaminants present in the initial serum-free culture medium, and the removal of cellular proteins contaminants up to 98% of the content of such contaminants present in the initial serum-free culture medium.
8. A purified RNA based viral vector composition produced using the method of claim 1 wherein the RNA based viral vector composition is one wherein the ratio of total physical particles/transducing units (PP/TU) is between 200:1 up to 600:1.
9. A purified RNA based viral vector composition produced using the method of claim 2 wherein the RNA based viral vector composition is one wherein the ratio of total physical particles/transducing units (PP/TU) is between 100:1 up to 400:1.
10. The method of claim 1 , wherein the multiple collections are conducted at 8-16 hour intervals.
11. The purified RNA based viral vector composition of claim 8 having a specific activity of greater than 1.88×10 7 TU/mg protein and greater than 6.88×10 6 TU/μg DNA.
12. The purified RNA based viral vector composition of claim 9 having a specific activity of greater than 7.96×10 8 TU/mg protein and greater than 1.61×10 7 TU/μg DNA.