Claims for Patent: 5,631,162

Summary for Patent: 5,631,162

Title:	Retroviral vectors for transducing .beta.-globin gene and .beta.-locus control region derivatives
Abstract:	A process and means for the design and the optimization of retroviral vectors transducing human .beta.-globin gene and .beta.-Locus Control Region (.beta.-LCR) derivatives, hereafter referred to as [.beta.-globin/LCR] retroviruses, which successfully meet the following criteria required for gene therapy applications: (1) stability of proviral transmission (low frequency of rearrangements similar to retroviral vectors considered stable in the art) upon infection of cell-lines and murine bone marrow cells, (2) improved viral titer, thereby allowing successful infection of bone marrow cells, and (3) high erythroid expression of the transduced human .beta.-globin gene, are described, along with specific constructs.
Inventor(s):	LeBoulch; Philippe (Cambridge, MA), London; Irving M. (Cambridge, MA), Tuan; Dorothy (Newton, MA)
Assignee:	Massachusetts Institute of Technology (Cambridge, MA)
Application Number:	08/076,090
Patent Claims:	1. A method of making a retroviral vector for transducing .beta.-globin genes and .beta.-LCR sequences comprising the steps of: (a) providing a retroviral vector in combination with a .beta.-globin gene and an effective portion of the HS2 enhancer of the .beta.-LCR, with or without additional .beta.-LCR sites, to achieve transduction and expression of the .beta.-globin gene, and (b) modifying a DNA sequence of the second intron of the .beta.-globin gene, and complementary/reverse splice-signals, polyadenylation signals, or combinations thereof, to remove or inactivate splicing sites or polyadenylation signals of the transduced .beta.-globin gene or .beta.-LCR, to form a retroviral vector characterized by: i) stability of proviral transmission upon infection of cell-lines and murine bone marrow cells, ii) viral titer effective to achieve infection of bone marrow cells, and iii) high erythroid expression of the transduced human .beta.-globin gene. 2. The method of claim 1 wherein the effective viral titer is greater than 10.sup.5 resistant colonies per ml of viral supernatant under standard conditions. 3. The method of claim 1 wherein the high erythroid expression is greater than 50% of a human .beta.-globin to murine .beta..sub.maj -globin mRNA ratio as assessed in dimethyl sulfoxide-induced MEL cells. 4. The method of claim 1 wherein said retroviral vector comprises: (a) a left and right long terminal repeat (LTR), (b) a tRNA primer binding site for initiation of synthesis of viral minus strand strong-stop; (c) a polypurine track primer binding site for initiation of synthesis of viral plus strand strong-stop, and (d) a packaging signal. 5. The method of claim 4 wherein said packaging signal extends into a gag region. 6. The method of claim 4 wherein said right LTR comprises a deletion in a U3 region yielding a self-inactivating vector upon reverse transcription. 7. The method of claim 1 wherein said retroviral vector is a splicing vector, comprising functional splicing signals leading to genomic and sub-genomic transcripts. 8. The method of claim 1 wherein said retroviral vector is not a splicing vector. 9. The method of claim 1 wherein said retroviral vector has a selectable marker. 10. The method of claim 9 wherein said selectable marker is driven by an internal enhancer/promoter. 11. The method of claim 10 wherein said selectable marker is driven by the left LTR. 12. The method of claim 9 wherein said selectable marker is placed in a splicing retroviral vector. 13. The method of claim 9 wherein said selectable marker is selected from the group consisting of a neomycin/G418 resistance gene, a hygromycin resistance gene, a puromycin resistance gene, a phleomycin resistance gene, a dihydrofolate reductase gene, a multidrug-resistance gene, and a gene for an enzyme. 14. The method of claim 9 wherein said selectable marker is a molecule that interacts with a substrate to produce a colored cell. 15. The method of claim 14 wherein said selectable marker is the gene encoding .beta.-galactosidase. 16. The method of claim 9 wherein said selectable marker is a molecule expressed at the cell membrane. 17. The method of claim 1 wherein said modifications are selected from the group consisting of deletions, additions, and substitutions of nucleotides in the DNA sequence of the second intron of the .beta.-globin gene, complementary splice-signals or polyadenylation signals of the transduced .beta.-globin gene or LCR. 18. The method of claim 1 wherein said .beta.-globin gene is modified within the second intron of the .beta.-globin gene, while maintaining correct splicing of this intron as well as normal expression of the .beta.-globin transgene as compared to the non-deleted Intron 2 containing .beta.-globin gene. 19. The method of claim 1 wherein said transduced .beta.-globin gene or .beta.-LCR contain partial deletions, substitutions, mutations or modifications of at least one of the complementary/reverse 5' splice-sites or 3' splice-sites or branchpoint signals or polyadenylation signals. 20. The method of claim 1 wherein a transcriptionally active HS2 fragment is incorporated, in a single or duplicated form or in association with other .beta.-LCR or heterologous enhancer sequences, in any position or orientation. 21. The method of claim 1 wherein a transcriptionally active HS3 fragment is incorporated, in a single or duplicated form or in association with other .beta.-LCR derivatives or heterologous enhancer sequences, in any position or orientation. 22. The method of claim 1 wherein a transcriptionally active HS4 fragment is incorporated, in a single or duplicated form or in association with other .beta.-LCR or heterologous enhancer sequences, in any position or orientation. 23. A retroviral vector for transducing .beta.-globin genes and .beta.-LCR sequences comprising: (a) a left and a right long terminal repeat (LTR), (b) a tRNA primer binding site for initiation of synthesis of vital minus strand strong-stop, (c) a polypurine track primer binding site for initiation of synthesis of viral plus strand strong-stop, (d) a packaging signal, (e) a .beta.-globin gene, and (f) an effective portion of the HS2 enhancer of the .beta.-LCR to achieve transduction and expression of the .beta.-globin gene, wherein the DNA sequence of the second intron of the .beta.-globin gene is modified, and complementary/reverse splice-signals or polyadenylation signals of the transduced .beta.-globin gene or .beta.-LCR are modified, or both are modified, to inactivate or remove splicing sites or polyadenylation signals within the vector so that the retroviral vector exhibits stability of proviral transmission upon infection of cell-lines and murine bone marrow cells, viral titer effective to achieve infection of bone marrow cells, and high erythroid expression of the transduced human .beta.-globin gene. 24. The retroviral vector of claim 23 wherein the effective viral titer is greater than 10.sup.5 resistant colonies per ml of viral supernatant under standard conditions and the high erythroid expression is greater than 50% of a human .beta.-globin to murine .beta..sub.maj -globin mRNA ratio as assessed in dimethyl sulfoxide-induced MEL cells. 25. The retroviral vector of claim 23 wherein said packaging signal extends into a gag region. 26. The retroviral vector of claim 23 wherein said retroviral vector is a splicing vector comprising functional splicing signals leading to genomic and sub-genomic transcripts. 27. The retroviral vector of claim 23 wherein said retroviral vector further comprises a selectable marker. 28. The retroviral vector of claim 23 wherein said modifications are selected from the group consisting of deletions, additions, and substitutions of nucleotides in the DNA sequence of the second intron of the .beta.-globin gene, complementary splice-signals or polyadenylation signals of the transduced .beta.-globin gene or LCR. 29. The retroviral vector of claim 23 wherein said selectable marker is driven by an internal enhancer/promoter. 30. The retroviral vector of claim 23 wherein said selectable marker is driven by the left LTR. 31. The retroviral vector of claim 23 wherein said selectable marker is placed in a splicing retroviral vector. 32. The retroviral vector of claim 23 wherein said selectable marker is selected from the group consisting of a neomycin/G418 resistance gene, a hygromycin resistance gene, a puromycin resistance gene, a phleomycin resistance gene, a dihydrofolate reductase gene, a multidrug-resistance gene, and a gene for an enzyme. 33. The retroviral vector of claim 23 wherein said selectable marker is a molecule that interacts with a substrate to produce a colored cell. 34. The retroviral vector of claim 33 wherein said selectable marker is the gene encoding .beta.-galactosidase. 35. The retroviral vector of claim 23 wherein said selectable marker is a molecule expressed at the cell membrane. 36. The retroviral vector of claim 23 wherein said right LTR comprises a deletion in a U3 region yielding a self-inactivating vector upon reverse transcription. 37. The retroviral vector of claim 23 wherein said .beta.-globin gene is modified within the second intron of the .beta.-globin gene, while monitoring correct splicing of this intron as well as normal expression of the .beta.-globin transgene as compared to the non-deleted Intron 2 containing .beta.-globin gene. 38. The retroviral vector of claim 23 wherein said transduced .beta.-globin gene or .beta.-LCR sequences contain partial deletions, substitutions, mutations or modifications of at least one of the complementary/reverse 5' splice-sites or 3' splice-sites or branchpoint signals or polyadenylation signals.

Details for Patent 5,631,162

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2039-02-26