Claims for Patent: 5,641,670

Summary for Patent: 5,641,670

Title:	Protein production and protein delivery
Abstract:	The invention relates to constructs comprising: a) a targeting sequence; b) a regulatory sequence; c) an exon; and d) an unpaired splice-donor site. The invention further relates to a method of producing protein in vitro or in vivo comprising the homologous recombination of a construct as described above within a cell. The homologously recombinant cell is then maintained under conditions which will permit transcription and translation, resulting in protein expression. The present invention further relates to homologously recombinant cells, including primary, secondary, or immortalized vertebrate cells, methods of making the cells, methods of homologous recombination to produce fusion genes, methods of altering gene expression in the cells, and methods of making a protein in a cell employing the constructs of the invention.
Inventor(s):	Treco; Douglas A. (Arlington, MA), Heartlein; Michael W. (Boxborough, MA), Selden; Richard F. (Wellesley, MA)
Assignee:	Transkaryotic Therapies, Inc. (Cambridge, MA)
Application Number:	08/243,391
Patent Claims:	1. A homologously recombinant cell having incorporated therein a new transcription unit, wherein the new transcription unit comprises an exogenous regulatory sequence, an exogenous exon and a splice-donor site, operatively linked to the second exon of an endogenous gene, wherein the homologously recombinant cell comprises said exogenous exon in addition to exons present in said endogenous gene. 2. The homologously recombinant cell of claim 1 wherein the exogenous exon comprises a CAP site. 3. The homologously recombinant cell of claim 1 wherein the exogenous exon further comprises the nucleotide sequence ATG. 4. The homologously recombinant cell of claim 3 wherein the exogenous exon further comprises encoding DNA in-frame with the targeted endogenous gene. 5. The homologously recombinant cell of claim 4 wherein the encoding DNA is different from the encoding DNA of the first exon of the endogenous gene. 6. The homologously recombinant cell of claim 4 wherein the exogenous regulatory sequence, exogenous exon and splice-donor site are upstream of the coding region of the endogenous gene. 7. The homologously recombinant cell of claim 6 wherein the exogenous regulatory sequence, exogenous exon and splice-donor site are upstream of the endogenous regulatory sequence of the endogenous gene. 8. The homologously recombinant cell of claim 1 wherein the endogenous regulatory sequence is deleted. 9. The homologously recombinant cell of claim 4 wherein the endogenous gene encodes a hormone, a cytokine, an antigen, an antibody, an enzyme, a clotting factor, a transport protein, a receptor, a regulatory protein, a structural protein or a transcription factor. 10. The homologously recombinant cell of claim 4 wherein the endogenous gene encodes a protein selected from the group consisting of erythropoietin, calcitonin, growth hormone, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, .beta.-interferon, .gamma.-interferon, nerve growth factors, FSH.beta., TGF-.beta., tumor necrosis factor, glucagon, bone growth factor-2, bone growth factor-7, TSH-.beta., interleukin 1, interleukin 2, interleukin 3, interleukin 6, interleukin 11, interleukin 12, CSF-granulocyte, CSF-macrophage, CSF-granulocyte/macrophage, immunoglobulins, catalytic antibodies, protein kinase C, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, urokinase, antithrombin III, DNAse, .alpha.-galactosidase, tyrosine hydroxylase, blood clotting factor V, blood clotting factor VII, blood clotting factor VIII, blood clotting factor IX, blood clotting factor X, blood clotting factor XIII, apolipoprotein E, apolipoprotein A-I, globins, low density lipoprotein receptor, IL-2 receptor, IL-2 antagonists, alpha-1 antitrypsin, immune response modifiers, and soluble CD4. 11. The homologously recombinant cell of claim 1 wherein the cell is a eukaryote. 12. The homologously recombinant cell of claim 11 wherein the cell is of fungal, plant or animal origin. 13. The homologously recombinant cell of claim 12 wherein the cell is of vertebrate origin. 14. The homologously recombinant cell of claim 13 wherein the cell is a primary or secondary mammalian cell. 15. The homologously recombinant cell of claim 13 wherein the cell is a primary or secondary human cell. 16. The homologously recombinant cell of claim 13 wherein the cell is an immortalized mammalian cell. 17. The homologously recombinant cell of claim 13 wherein the cell is an immortalized human cell. 18. The homologously recombinant cell of claim 13 wherein the cell is selected from the group consisting of: HT1080 cells, HeLa cells, derivatives of HeLa cells, MCF-7 breast cancer cells, K-562 leukemia cells, KB carcinoma cells, 2780AD ovarian carcinoma cells, Raji cells, Jurkat cells, Namalwa cells, HL-60 cells, Daudi cells, RPMI 8226 cells, U-937 cells, Bowes Melanoma cells, WI-38VA13 subline 2R4 cells, and MOLT-4 cells. 19. The homologously recombinant cell of claim 18 wherein the endogenous gene encodes erythropoietin. 20. The homologously recombinant cell of claim 19 which expresses erythropoietin. 21. The homologously recombinant cell of claim 4 wherein the encoding DNA is the same as the encoding DNA of the first exon of erythropoietin. 22. The homologously recombinant cell of claim 4 wherein the encoding DNA is different from the encoding DNA of the first exon of erythropoietin. 23. The homologously recombinant cell of claim 22 wherein the encoding DNA is the same as the encoding DNA of the first exon of human growth hormone. 24. The homologously recombinant cell of claim 1 which expresses a fusion protein comprising amino acids encoded by the exogenous exon and amino acids encoded by the endogenous gene. 25. The homologously recombinant cell of claim 5 wherein the regulatory sequence is a promoter, an enhancer, a scaffold-attachment region or a transcription factor binding site. 26. The homologously recombinant cell of claim 25 wherein the exogenous regulatory sequence is a promoter. 27. The homologously recombinant cell of claim 25 wherein the exogenous regulatory sequence is a regulatory sequence of the mouse metallothionein-I gene, a regulatory sequence of an SV-40 gene, a regulatory sequence of a cytomegalovirus gene, a regulatory sequence of a collagen gene, a regulatory sequence of an actin gene, a regulatory sequence of an immunoglobulin gene, a regulatory sequence of the HMG-CoA reductase gene or a regulatory sequence of the EF-1.alpha. gene. 28. The DNA plasmid pREPO18. 29. A homologously recombinant cell comprising the dhfr gene, the neo gene, the CMV promoter, hGH exon 1 and an unpaired splice-donor site targeted to a position upstream of the endogenous erythropoietin regulatory region. 30. The homologously recombinant cell of claim 29 produced by the integration of DNA from pREPO18.

Details for Patent 5,641,670

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Microbix Biosystems Inc.	KINLYTIC	urokinase	For Injection	021846	01/16/1978		2014-06-24
Nps Pharmaceuticals, Inc.	NATPARA	parathyroid hormone	For Injection	125511	01/23/2015		2014-06-24