Claims
- 1. A synthetic mammalian expression plasmid comprising:
(a) a synthetic or eukaryotic promoter; (b) a codon-optimized-eukaryotic therapeutic gene sequence; (c) a poly adenelation signal; (d) a selectable marker gene promoter; (e) a ribosomal binding site; (f) selectable marker gene sequence; and (g) an origin of replication; wherein the synthetic or eukaryotic promoter, the codon-optimized-eukaryotic therapeutic gene sequence, and the poly adenylation signal comprise therapeutic elements of the synthetic mammalian expression plasmid; the therapeutic elements are operatively linked and located in a first operatively-linked arrangement; the selectable marker gene promoter, the ribosomal binding site, the selectable marker gene sequence, and the origin of replication comprise replication elements of the synthetic mammalian expression plasmid; the replication elements are operatively linked and located in a second operatively-linked arrangement; the first-operatively-linked arrangement and the second-operatively-linked arrangement comprise a circular structure of the synthetic mammalian expression plasmid; and the synthetic mammalian expression plasmid is utilized for plasmid mediated gene supplementation.
- 2. The synthetic mammalian expression plasmid of claim 1, wherein the origin of replication comprises a prokaryotic origin of replication sequence.
- 3. The synthetic mammalian expression plasmid of claim 1, wherein the origin of replication comprises an autonomously replication sequence (“ARS”).
- 4. The synthetic mammalian expression plasmid of claim 1, wherein the origin of replication comprises SeqID#12.
- 5. The synthetic mammalian expression plasmid of claim 1, wherein the ribosomal binding site (“RBS”) comprises a prokaryotic RBS.
- 6. The synthetic mammalian expression plasmid of claim 1, wherein the ribosomal binding site (“RBS”) comprises SeqID#13.
- 7. The synthetic mammalian expression plasmid of claim 1, wherein the poly adenylation signal (“PolyA”) comprises a eukaryotic poly A signal.
- 8. The synthetic mammalian expression plasmid of claim 1, wherein the poly adenylation signal (“PolyA”) comprises SeqID# 10.
- 9. The synthetic mammalian expression plasmid of claim 1, wherein the 5′ UTR comprises a portion of a eukaryotic 5′ UTR.
- 10. The synthetic mammalian expression plasmid of claim 1, wherein the 5′ UTR comprises SeqID# 11.
- 11. The synthetic mammalian expression plasmid of claim 1, further comprising a prokaryotic promoter.
- 12. The synthetic mammalian expression plasmid of claim 11, wherein the prokaryotic promoter comprises PNEO and a transposon fragment (“Tn5”).
- 13. The synthetic mammalian expression plasmid of claim 11, wherein the prokaryotic promoter comprises SeqID#14.
- 14. The synthetic mammalian expression plasmid of claim 1, wherein the selectable marker gene comprises SeqID#3.
- 15. The synthetic mammalian expression plasmid of claim 1, wherein the codon optimized mammalian therapeutic gene sequence comprises at least one species specific codon modification.
- 16. The synthetic mammalian expression plasmid of claim 1, wherein the codon optimized mammalian therapeutic gene sequence comprises a signal peptide.
- 17. The synthetic mammalian expression plasmid of claim 16, wherein the signal peptide is species specific.
- 18. The synthetic mammalian expression plasmid of claim 1, wherein the codon optimized mammalian therapeutic gene sequence comprises a sequence that encodes a modified species specific growth hormone releasing hormone (“GHRH”).
- 19. The synthetic mammalian expression plasmid of claim 1, further comprising a 5′ untranslated region (“UTR”) operatively linked to the first operatively-linked arrangement.
- 20. The synthetic mammalian expression plasmid of claim 19, wherein the 5′ untranslated region (“UTR”) comprises a portion of a human growth hormone 5′UTR.
- 21. The synthetic mammalian expression plasmid of claim 1, wherein the codon optimized mammalian therapeutic gene sequence comprises SeqID#4.
- 22. The synthetic mammalian expression plasmid of claim 1, wherein the codon optimized mammalian therapeutic gene sequence comprises SeqID#5.
- 23. The synthetic mammalian expression plasmid of claim 1, wherein the codon optimized mammalian therapeutic gene sequence comprises SeqID#6.
- 24. The synthetic mammalian expression plasmid of claim 1, wherein the codon optimized mammalian therapeutic gene sequence comprises SeqID#7.
- 25. The synthetic mammalian expression plasmid of claim 1, wherein the codon optimized mammalian therapeutic gene sequence comprises SeqID#8.
- 26. The synthetic mammalian expression plasmid of claim 1, wherein the codon optimized mammalian therapeutic gene sequence comprises SeqID#9.
- 27. A synthetic mammalian expression plasmid comprising SEQID#17.
- 28. A synthetic mammalian expression plasmid comprising SEQID#18.
- 29. A synthetic mammalian expression plasmid comprising SEQID#19.
- 30. A synthetic mammalian expression plasmid comprising SEQID#20.
- 31. A synthetic mammalian expression plasmid comprising SEQID#21.
- 32. A method for plasmid mediated gene supplementation comprising:
delivering into a subject a codon optimized synthetic mammalian expression plasmid; wherein the codon optimized synthetic mammalian expression plasmid encodes a growth hormone releasing hormone (“GHRH”) or functional biological equivalent in the subject.
- 33. The method of claim 32, wherein delivering into the cells of the subject the codon optimized synthetic mammalian expression plasmid is via electroporation.
- 34. The method of claim 32, wherein the cells of the subject are somatic cells, stem cells, or germ cells.
- 35. The method of claim 32, wherein the codon optimized synthetic mammalian expression plasmid consisting of SeqID#17, SeqID#18, SeqID#19, SeqID#20, and SeqID#21.
- 36. The method of claim 32, wherein the encoded GHRH is a biologically active polypeptide; and the encoded functional biological equivalent of GHRH is a polypeptide that has been engineered to contain a distinct amino acid sequence while simultaneously having similar or improved biologically activity when compared to the GHRH polypeptide.
- 37. The method of claim 32, wherein the encoded GHRH or functional biological equivalent thereof facilitates growth hormone (“GH”) secretion in the subject.
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 60/396,247, entitled “Codon Optimized Synthetic Plasmids,” filed on Jul. 16, 2002, the entire content of which is hereby incorporated by reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60396247 |
Jul 2002 |
US |