Claims for Patent: 8,603,780

Summary for Patent: 8,603,780

Title:	Methods and compositions for enhanced expression and secretion of proteins
Abstract:	Optimized signal peptide coding sequences for enhanced expression and secretion of protein from a cell and related compositions and methods are described. The optimized signal peptide coding sequence encodes an mRNA that contains at least one hairpin structure immediately downstream of the initiation codon. Methods for obtaining the optimized signal peptide coding sequences and methods for enhanced expression and secretion of proteins using the optimized signal peptide coding sequences are also described.
Inventor(s):	Wang; Zhuying (Monmouth Junction, NJ), Liu; Xiaowu (Nanjing, CN)
Assignee:	Nanjingjinsirui Science & Technology Biology Corp. (Nanjing, CN)
Application Number:	13/550,120
Patent Claims:	1. An isolated nucleic acid molecule for enhanced recombinant expression and secretion of a polypeptide from a cell, the nucleic acid molecule comprising a first polynucleotide sequence encoding a signal peptide operably linked to a second polynucleotide sequence encoding the polypeptide, wherein the first polynucleotide sequence and the second polynucleotide sequence are not endogenously operably linked in the cell, the first polynucleotide encodes a messenger RNA (mRNA) that comprises at least one hairpin structure immediately downstream of the initiation codon, and when expressed in the cell, the signal peptide directs the secretion of the polypeptide from the cell, resulting in enhanced recombinant expression and secretion of the polypeptide from the cell. 2. The isolated nucleic acid molecule of claim 1, wherein the first polynucleotide sequence encodes a signal peptide having an amino acid sequence identical to that of the native signal peptide for the polypeptide in the cell. 3. The isolated nucleic acid molecule of claim 1, wherein the first polynucleotide sequence encodes a signal peptide having an amino acid sequence different from that of the native signal peptide for the polypeptide in the cell. 4. The isolated nucleic acid molecule of claim 1, wherein the at least one hairpin structure has a free energy (.DELTA.G) of about -30 Kcal/mol to about -6.8 Kcal/mol. 5. The isolated nucleic acid molecule of claim 1, wherein the at least one hairpin structure has a stem of about 6 to 25 base pairs. 6. The isolated nucleic acid molecule of claim 1, wherein the mRNA comprises two hairpin structures immediately downstream of the initiation codon. 7. The isolated nucleic acid molecule of claim 6, wherein the mRNA comprises about 1 to 10 nucleotides that participate in the formation of either of the two hairpin structures. 8. The isolated nucleic acid molecule of claim 6, wherein the distance between the two hairpin structures is about 1 to 10 nucleotides. 9. The isolated nucleic acid molecule of claim 1, wherein the first polynucleotide sequence has at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 1. 10. A vector comprising the nucleic acid molecule of claim 1. 11. A recombinant cell comprising the nucleic acid molecule of claim 1. 12. A method for obtaining enhanced recombinant expression and secretion of a polypeptide from the cell, the method comprising: obtaining a nucleic acid molecule according to claim 1; introducing the nucleic acid molecule into the cell to obtain a recombinant cell; and growing the recombinant cell under conditions to allow enhanced recombinant expression and secretion of the polypeptide from the cell. 13. The method of claim 12, further comprising isolating the polypeptide from the extracellular environment of the cell. 14. The method of claim 12, wherein the polypeptide is selected from the group consisting of Herceptin (trastuzumab), Erbitux, Avastin (bevacizumab), Humira (adalimumab), Rituxan (rituximab), Lantus (insulin glargine), Factor IX, Factor VIII, .gamma.-Interferon, Interleukin 2, Human growth hormone (HGH), Tissue plamsinogen activator (TPA), Epo (Erythropoietin), Aranesp, Orencia, Stelare, and Vectibix. 15. An isolated nucleic acid molecule comprising a polynucleotide sequence encoding a signal peptide, the polynucleotide sequence having at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 1. 16. The isolated nucleic acid molecule of claim 15 comprising SEQ ID NO: 1. 17. The isolated nucleic acid molecule of claim 16 further comprising a second polynucleotide sequence encoding a polypeptide operably linked to SEQ ID NO:1. 18. A method of obtaining an optimized polynucleotide that encodes an optimized signal peptide for enhanced recombinant expression and secretion of a polypeptide, the method comprising: obtaining a polynucleotide encoding a signal peptide; and substituting one or more nucleotides in the polynucleotide to obtain the optimized polynucleotide encoding the optimized signal peptide, and the optimized polynucleotide encoding a messenger RNA comprising at least one hairpin structure immediately downstream of the initiation codon. 19. The method of claim 18, wherein the substituting step comprises a synonymous substitution. 20. The method of claim 19, wherein the substituting step comprises a non-synonymous substitution.

Details for Patent 8,603,780

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Sanofi-aventis U.s. Llc	LANTUS	insulin glargine	Injection	021081	04/20/2000		2031-07-15
Sanofi-aventis U.s. Llc	LANTUS	insulin glargine	Injection	021081	04/25/2007		2031-07-15
Genentech, Inc.	RITUXAN	rituximab	Injection	103705	11/26/1997		2031-07-15
Idec Pharmaceuticals Corp.	RITUXAN	rituximab	Injection	103737	02/19/2002		2031-07-15
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	09/25/1998		2031-07-15
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	02/10/2017		2031-07-15