Claims for Patent: 9,249,201

Summary for Patent: 9,249,201

Title:	Heterologous intron within a signal peptide
Abstract:	The invention concerns the field of recombinant gene engineering. It concerns novel introns and compositions comprising such introns as well as a method to improve expression of polypeptides from nucleic acids such as cloned genes with heterologous introns, especially genes encoding antibodies and antibody derived fragments, and the production of various polypeptides in eukaryotic host cells using said novel intron sequences as heterologous introns.
Inventor(s):	Enenkel; Barbara (Warthausen, DE)
Assignee:	Boehringer Ingelheim International GmbH (Ingelheim am Rhein, DE)
Application Number:	14/134,169
Patent Claims:	1. A transcription unit or expression vector comprising at least one signal peptide gene sequence operatively linked to a gene of interest, whereby said signal peptide gene sequence comprises at least one heterologous intron located within a signal peptide exon, whereby the 5' and 3' ends of said exon are defined as occuring in a corresponding native signal peptide gene sequence. 2. The transcription unit or expression vector of claim 1, wherein the coding region of the gene of interest comprises at least one immunoglobulin domain. 3. The transcription unit or expression vector of claim 1, whereby said gene of interest encodes an immunoglobulin. 4. The transcription unit or expression vector of claim 1, wherein said heterologous intron is a nucleic acid sequence at least 90% identical to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. 5. The transcription unit or expression vector of claim 1, wherein the signal peptide sequence is derived from an immunoglobulin heavy or light chain gene. 6. The transcription unit or expression vector of claim 1, whereby the signal peptide gene sequence encodes an amino acid sequence selected from the group consisting of: SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. 7. The transcription unit or expression vector of claim 1, wherein the heterologous intron is located at a position resulting in a functional splice donor site and a functional splice acceptor site. 8. The transcription unit or expression vector of claim 1, wherein the heterologous intron is positioned within the nucleotide sequence successions CAG:C, CAG:T, AAG:C, AAG:T, TAG:T or TAG:C, preferably CAG:C, CAG:T, AAG:C or AAG:T, whereby the colons denote the site of intron insertion. 9. The transcription unit or expression vector of claim 8, wherein the nucleotide sequence successions encode the amino acid pairs selected from the group consisting of: TABLE-US-00003 CAG:C CAG:T AAG:C AAG:T TAG:C TAG:T GlnLeu GlnPhe LysLeu LysPhe PheSer PheSer CAGCTN CAGTTY AAGCTN AAGTTY TTTAGC TTTAGT GlnPro GlnLeu LysPro LysLeu SerSer SerSer CAGCCN CAGTTR AAGCCN AAGTTR TCTAGC TCTAGT GlnHis GlnSer LysHis LysSer TyrSer TyrSer CAGCAY CAGTCN AAGCAY AAGTCN TATAGC TATAGT GlnGln GlnTyr LysGln LysTyr CysSer CysSer CAGCAR CAGTAY AAGCAR AAGTAY TGTAGC TGTAGT GlnArg GlnCys LysArg LysCys LeuSer LeuSer CAGCGN CAGTGY AAGCGN AAGTGY CTTAGC CTTAGT SerAla GlnTrp GlnAla LysTrp ProSer ProSer TCAGCN CAGTGG CAAGCN AAGTGG CCTAGC CCTAGT ProAla SerVal LysAla GlnVal HisSer HisSer CCAGCN TCAGTN AAAGCN CAAGTN CATAGC CATAGT ThrAla ProVal GluAla LysVal ArgSer ArgSer ACAGCN CCAGTN GAAGCN AAAGTN CGTAGC CGTAGT AlaAla ThrVal LeuSer GluVal IleSer IleSer GCAGCN ACAGTN YTAAGC GAAGTN ATTAGC ATTAGT PheSer AlaVal SerSer LeuSer ThrSer ThrSer TTCAGC GCAGTN TCAAGC YTAAGT ACTAGC ACTAGT SerSer PheSer ProSer SerSer AsnSer AsnSer TCCAGC TTCAGT CCAAGC TCAAGT AATAGC AATAGT TyrSer SerSer GlnSer ProSer SerSer SerSer TACAGC TCCAGT CAAAGC CCAAGT AGTAGC AGTAGT CysSer TyrSer ArgSer GlnSer ValSer ValSer TGCAGC TACAGT MGAAGC CAAAGT GTTAGC GTTAGT LeuSer CysSer IleSer ArgSer AlaSer AlaSer CTCAGC TGCAGT ATAAGC MGAAGT GCTAGC GCTAGT ProSer LeuSer ThrSer IleSer AspSer AspSer CCCAGC CTCAGT ACAAGC ATAAGT GATAGC GATAGT HisSer ProSer LysSer ThrSer GlySer GlySer CACAGC CCCAGT AAAAGC ACAAGT GGTAGC GGTAGT ArgSer HisSer ValSer LysSer LeuAla LeuVal CGCAGC CACAGT GTAAGC AAAAGT YTAGCN YTAGTN IleSer ArgSer AlaSer ValSer IleAla IleVal ATCAGC CGCAGT GCAAGC GTAAGT ATAGCN ATAGTN ThrSer IleSer GluSer AlaSer ValAla ValVal ACCAGC ATCAGT GAAAGC GCAAGT GTAGCN GTAGTN AsnSer ThrSer GlySer GluSer AACAGC ACCAGT GGAAGC GAAAGT SerSer AsnSer GlySer AGCAGC AACAGT GGAAGT ValSer SerSer GTCAGC AGCAGT AlaSer ValSer GCCAGC GTCAGT AspSer AlaSer GACAGC GCCAGT GlySer AspSer GGCAGC GACAGT GlySer GGCAGT. colon: site of intron insertion, N = any base,Y = C or T, R = A or G, M = C or A 10. The transcription unit or expression vector according to claim 1, wherein said transcription unit or expression vector further comprises at least one promoter and at least one terminator, optionally said transcription unit or expression vector further comprises at least one selection marker and/or at least one enhancer. 11. A host cell comprising the transcription unit or expression vector according to claim 1. 12. The host cell of claim 11, wherein said cell is a eukaryotic cell. 13. A method of producing a protein of interest encoded by a polynucleotide sequence comprising at least one signal peptide gene sequence operatively linked to said polynucleotide sequence comprising the following steps: a) introducing at least one heterologous intron sequence into said signal peptide gene sequence by placing the intron into a nucleotide sequence of a signal peptide encoding exon, whereby the 5' and 3' ends of the exon are defined as occuring in a corresponding native signal peptide gene sequence, and b) introducing the heterologous intron-containing signal peptide gene sequence of step a) into a transcription unit or a expression vector encoding a protein of interest, and c) transfecting a cell with said transcription unit or said vector of step b), and d) cultivating said cell of step c) under conditions which allow expression of said protein of interest. 14. A method of producing a protein of interest encoded by a polynucleotide sequence comprising at least one domain of an immunoglobulin gene comprising: a) transfecting a cell with the transcription unit or expression vector of claim 1, b) cultivating said cell of step a) under conditions which allow expression of said protein of interest. 15. The method according to claim 13, whereby the heterologous intron sequence is at least 90% identical to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. 16. The method of claim 13, additionally comprising the following step of isolating and purifying said protein of interest. 17. A method of generating a recombinant host cell/production cell comprising: a) transfecting a cell with the transcription unit or expression vector of claim 1, and b) selecting a recombinant host cell/production cell. 18. A kit comprising a vector including at least parts of a transcription unit comprising a first nucleotide sequence comprising at least one heterologous intron located within a signal peptide encoding exon, whereby the 5' and 3' ends of said exon are defined as occuring in a corresponding native signal peptide gene sequence, and whereby the nucleotide sequence of this transcription unit comprises suitable recognition sites for restriction enzymes allowing the integration or operative linkage of a second nucleotide sequence coding for a gene of interest, and instructions and optionally a vector map, and optionally a host cell, and optionally a cultivation medium for the cultivation of a host cell, and/or optionally a selection medium for selecting and cultivating a transfected host cell. 19. The transcription unit or expression vector of claim 3, whereby said gene of interest encodes an IgG1, IgG2, or IgG4, or an Fc fusion protein, or a single chain format or another antibody-derived molecule.

Details for Patent 9,249,201

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		2032-12-31
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2032-12-31
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2032-12-31