Claims for Patent: 9,493,803

Summary for Patent: 9,493,803

Title:	Artificial introns
Abstract:	The invention concerns the field of recombinant gene engineering. It concerns novel artificial introns and compositions comprising such introns as well as a method to improve expression of polypeptides from nucleic acids such as cloned genes, especially genes encoding antibodies and antibody derived fragments, and the production of various polypeptides in eukaryotic host cells using said novel artificial intron sequences.
Inventor(s):	Enenkel; Barbara (Warthausen, DE)
Assignee:	Boehringer Ingelheim International GmbH (Ingelheim am Rhein, DE)
Application Number:	14/134,177
Patent Claims:	1. A transcription unit or expression vector comprising a gene of interest and comprising at least one artificial intron, whereby the artificial intron is i. replacing a native intron sequence within said gene of interest, or ii. positioned at the site of a native intron position within said gene of interest, wherein the gene of interest is an immunoglobulin gene. 2. The transcription unit or expression vector of claim 1, wherein the artificial intron sequence comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:3, and wherein presence of the artificial intron sequence enhances expression of the gene of interest as compared to expression of gene of interest without the artificial intron sequence. 3. The transcription unit or expression vector of claim 1, wherein the artificial intron sequence is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:3. 4. The transcription unit or expression vector of claim 1, wherein said artificial intron is operably linked to a heterologous coding sequence of a gene. 5. The transcription unit or expression vector of claim 1, wherein the gene of interest is an immunoglobulin gene encoding an IgG1, IgG2, or IgG4. 6. The transcription unit or expression vector of claim 1, wherein the gene of interest encodes at least one domain of an immunoglobulin gene. 7. The transcription unit or expression vector of claim 6, wherein said domain is a variable domain. 8. The transcription unit or expression vector of claim 6, wherein said domain is a constant domain of an immunoglobulin gene or a hinge region of an immunoglobulin gene. 9. The transcription unit or expression vector of claim 5, wherein the artificial intron is located within the codon encoding the first amino acid at position 108 in the constant domain CL of immunoglobulin kappa and lambda light chains between the first and second nucleotide of the codon, within the codon encoding the first amino acid at position 118 in the constant domain CH1 of immunoglobulin heavy chains between the first and second nucleotide of the codon, within the codon encoding the first amino acid at position 216 in the hinge region of immunoglobulin heavy chains between the first and second nucleotide of the codon, within the codon encoding the first amino acid at position 231 in the constant domain CH2 of immunoglobulin heavy chains between the first and second nucleotide of the codon and/or within the codon encoding the first amino acid at position 341 in the constant domain CH3 of immunoglobulin heavy chains between the first and second nucleotide of the codon. 10. The transcription unit or expression vector of claim 1, wherein the gene of interest is an Fc-fusion protein. 11. The transcription unit or expression vector of claim 10, wherein the artificial intron is located within the codon encoding the first amino acid at position 216 in the hinge region of an immunoglobulin heavy chain between the first and second nucleotide of the codon if part of the Fc-fusion protein, within the codon encoding the first amino acid at position 231 in the constant domain CH2 of an immunoglobulin heavy chain between the first and second nucleotide of the codon and/or within the codon encoding the first amino acid at position 341 in the constant domain CH3 of an immunoglobulin heavy chain between the first and second nucleotide of the codon. 12. The transcription unit or expression vector of claim 1, wherein the artificial intron is positioned in a signal peptide sequence operably linked to a heterologous coding sequence of a gene. 13. The transcription unit or expression vector according to claim 12, wherein the signal peptide is a signal peptide of an immunoglobulin gene. 14. The transcription unit or expression vector according to claim 13, wherein the artificial intron is located within the codon encoding the amino acid at position -4 counting backwards from the 3' end of the amino acid sequence of the signal peptide of the signal peptide sequence of immunoglobulin heavy and/or light chains between the first and second nucleotide of the codon. 15. The transcription unit or expression vector of claim 1, wherein the artificial 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, and one of the following amino acid pairs is encoded at the intron insertion site: 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 ATCAGC 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 16. A host cell comprising the expression vector of claim 1. 17. The host cell of claim 16, wherein said cell is a eukaryotic cell. 18. A kit comprising a) a vector including a transcription unit comprising a first nucleotide sequence comprising at least one artificial intron located within an immunoglobulin gene or at the position of a native immunoglobulin intron, and wherein the nucleotide sequence of the transcription unit comprises recognition sites for restriction enzymes allowing integration of a second nucleotide sequence coding for another a gene of interest, b) instructions, c) optionally a vector map, and d) optionally a host cell. 19. A kit comprising a) a vector including a transcription unit comprising a first polynucleotide sequence encoding a signal peptide sequence comprising at least one artificial intron boated within said signal peptide sequence at the position of a native intron of the signal peptide gene sequence, and wherein the nucleotide sequence of the transcription unit comprises recognition sites for restriction enzymes allowing integration of a second nucleotide sequence coding for a gene of interest, b) instructions, c) optionally a vector map, and d) optionally a host cell. 20. The kit of claim 18 additionally comprising a) a cultivation medium for the cultivation of a host cell, and/or b) a selection medium for selecting and cultivating a transfected host cell.

Details for Patent 9,493,803

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