Claims for Patent: 5,861,299

Summary for Patent: 5,861,299

Title:	Genetic construct of which protein coding DNA comprises introns and is designed for protein production in transgenic animals
Abstract:	Proteinaceous products can be produced by transgenic animals having genetic constructs integrated into their genome. The construct comprises a 5\'-flanking sequence from a mammalian milk protein gene (such as beta-lactoglobulin) and DNA coding for a heterologous protein other than the milk protein (for example a serin protease such as alpha.sub.1 -antitrypsin or a blood factor such as Factor VIII or IX). The protein-coding DNA comprises at least one, but not all, of the introns naturally occurring in a gene coding for the heterologous protein. The 5\'-flanking sequence is sufficient to drive expression of the heterologous protein.
Inventor(s):	Archibald; Alan Langskill (Edinburgh, GB), Clark; Anthony John (Midlothian, GB), Harris; Stephen (Edinburgh, GB), McClenaghan; Margaret (Edinburgh, GB), Simons; Jonathan Paul (Edinburgh, GB), Whitelaw; Christopher Bruce Alexander (Edinburgh, GB)
Assignee:	PPL Therapeutics (Scotland) Limited (Edinburgh, GB6)
Application Number:	08/343,884
Patent Claims:	1. A cell containing a vector, other than a chromosome, said vector comprising a genetic construct, said genetic construct comprising (i) a 5'-flanking sequence from a mammalian milk protein gene and (ii) DNA coding for human alpha.sub.1 -antitrypsin, wherein the protein-coding DNA includes, counting from the 5' end, all but the first complete naturally occurring introns in a gene coding for human alpha.sub.1 -antitrypsin, wherein the first naturally occurring intron of said gene coding for human alpha.sub.1 -antitrypsin is absent in its entirety, wherein said 5'-flanking sequence is operatively linked to said DNA coding for human alpha.sub.1 -antitrypsin, and wherein (a) said 5'-flanking sequence controls and directs expression of the DNA encoding human alpha.sub.1 -antitrypsin, when present in a cell of the mammary gland, for secretion into the milk of a non-human transgenic animal whose germline incorporates said construct, at levels greater than is achieved by expression of a corresponding intron-less construct and (b) said cell does not itself express human alpha.sub.1 -antitrypsin. 2. A cell containing a vector, other than a chromosome, said vector comprising a genetic construct, said genetic construct comprising (i) a 5'-flanking sequence from a mammalian milk protein gene and (ii) DNA coding for human factor IX wherein the protein-coding DNA comprises, counting from the 5' end, the first, but only the first, complete intron naturally occurring in a gene coding for human factor IX wherein said 5'-flanking sequence is operatively linked to said DNA coding for human factor IX, and wherein (a) said 5'-flanking sequence controls and directs expression of the DNA encoding human factor IX, when present in a cell of the mammary gland, for secretion into the milk of a non-human transgenic animal whose germline incorporates said construct and (b) said cell does not itself express human factor IX. 3. A cell as claimed in claim 1 which is a bacterial cell. 4. A cell as claimed in claim 2 which is a bacterial cell. 5. A cell as claimed in claim 3 which is Escherichia coli. 6. A cell as claimed in claim 4 which is Escherichia coli. 7. A cell as claimed in claim 1, wherein said milk protein gene is a beta-lactoglobulin gene. 8. A cell as claimed in claim 2, wherein said milk protein gene is a beta-lactoglobulin gene. 9. A cell, containing a vector, other than a chromosome, said vector comprising a genetic construct, said genetic construct comprising (i) a 5'-flanking sequence from a mammalian milk protein gene and (ii) DNA coding for human alpha.sub.1 -antitrypsin, wherein the protein-coding DNA comprises, counting from the 5' end, all but the first complete naturally occurring introns in a gene coding for human alpha.sub.1 -antitrypsin, wherein the first naturally occurring intron of said gene coding for human alpha.sub.1 -antitrypsin is absent in its entirety, wherein said 5'-flanking sequence is operatively linked to said DNA coding for human alpha.sub.1 -antitrypsin, and wherein (a) said 5'-flanking sequence controls and directs expression of the DNA encoding human alpha.sub.1 -antitrypsin, when present in a cell of the mammary gland, for secretion into the milk of a non-human transgenic animal whose germline incorporates said construct, at levels greater than is achieved by expression of a corresponding intron-less construct and (b) said cell does not itself express human alpha.sub.1 -antitrypsin, wherein said 5'-flanking sequence is an upstream region of the .beta.-lactoglobulin transcription start site consisting of the SphI-BglII 800 base pair fragment of pSS1tgSpDELTA-AvaII, or a fragment that retains the functional properties of the SphI-BglII 800 base pair fragment of pSS1tgSpDELTA-AvaII. 10. A cell, containing a vector, other than a chromosome, said vector comprising a genetic construct, said genetic construct comprising (i) a 5'-flanking sequence from a mammalian milk protein gene and (ii) DNA coding for human factor IX wherein the protein-coding DNA comprises, counting from the 5' end, the first, but only the first, complete intron naturally occurring in a gene coding for human factor IX wherein said 5'-flanking sequence is operatively linked to said DNA coding for human factor IX, and wherein (a) said 5'-flanking sequence controls and directs expression of the DNA encoding human factor IX, when present in a cell of the mammary gland, for secretion into the milk of a non-human transgenic animal whose germline incorporates said construct and (b) said cell does not itself express human factor IX, wherein said 5'-flanking sequence is an upstream region of the .beta.-lactoglobulin transcription start site consisting of the SphI-BglII 800 base pair fragment of pSS1tgSpDELTA-AvaII, or a fragment that retains the functional properties of the SphI-BglII 800 base pair fragment of pSS1tgSpDELTA-AvaII. 11. A cell, containing a vector, other than a chromosome, said vector comprising a genetic construct, said genetic construct comprising (i) a 5'-flanking sequence from a mammalian milk protein gene and (ii) DNA coding for human alpha.sub.1 -antitrypsin, wherein the protein-coding DNA comprises, counting from the 5' end, all but the first complete naturally occurring introns in a gene coding for human alpha.sub.1 -antitrypsin, wherein the first naturally occurring intron of said gene coding for human alpha.sub.1 -antitrypsin is absent in its entirety, wherein said 5'-flanking sequence is operatively linked to said DNA coding for human alpha.sub.1 -antitrypsin, and wherein (a) said 5'-flanking sequence controls and directs expression of the DNA encoding human alpha.sub.1 -antitrypsin, when present in a cell of the mammary gland, for secretion into the milk of a non-human transgenic animal whose germline incorporates said construct, at levels greater than is achieved by expression of a corresponding intron-less construct and (b) said cell does not itself express human alpha.sub.1 -antitrypsin, wherein said 5'-flanking sequence is an upstream region of the .beta.-lactoglobulin transcription start site consisting of the 4.2 kilobase pair SalI-SphI DNA fragment isolated from the 5' flanking sequences of the .beta.-lactoglobulin gene or a fragment that retains the functional properties of the 4.2 kilobase pair SalI-SphI DNA fragment. 12. A cell, containing a vector, other than a chromosome, said vector comprising a genetic construct, said genetic construct comprising (i) a 5'-flanking sequence from a mammalian milk protein gene and (ii) DNA coding for human factor IX wherein the protein-coding DNA comprises, counting from the 5' end, the first, but only the first, complete intron naturally occurring in a gene coding for human factor IX wherein said 5'-flanking sequence is operatively linked to said DNA coding for human factor IX, and wherein (a) said 5'-flanking sequence controls and directs expression of the DNA encoding human factor IX, when present in a cell of the mammary gland, for secretion into the milk of a non-human transgenic animal whose germline incorporates said construct and (b) said cell does not itself express human factor IX, wherein said 5'-flanking sequence is an upstream region of the .beta.-lactoglobulin transcription start site consisting of the 4.2 kilobase pair SalI-SphI DNA fragment isolated from the 5' flanking sequences of the .beta.-lactoglobulin gene or a fragment that retains the functional properties of the 4.2 kilobase pair SalI-SphI DNA fragment. 13. A cell as claimed in claim 9, wherein the human alpha.sub.1 -antitrypsin is expressed at a level at least eight fold greater than is achieved by expression of a corresponding intron-less construct. 14. A cell as claimed in claim 9, wherein the human alpha.sub.1 -antitrypsin is expressed at a level at least one hundred fold greater than is achieved by expression of a corresponding intron-less construct. 15. A cell as claimed in claim 11, wherein the human alpha.sub.1 -antitrypsin is expressed at a level at least eight fold greater than is achieved by expression of a corresponding intron-less construct. 16. A cell as claimed in claim 12, wherein the human alpha.sub.1 -antitrypsin is expressed at a level at least one hundred fold greater than is achieved by expression of a corresponding intron-less construct.

Details for Patent 5,861,299

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		2008-11-11
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2008-11-11
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2008-11-11