IP Library › Granted Patent US 9,493,803

Granted Patent B2

US 9,493,803 · App. 14/134,177 · Granted Nov 15, 2016

Artificial introns

Inventor: Barbara Enenkel (Warthausen, DE)

Assignee: Boehringer Ingelheim International GmbH

C12P21/00 C07K16/00 C12N15/67 C12N15/85 C07K2317/52 C07K2319/00 C07K2319/30 C12N2830/42 C12N2830/85

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Quick Facts

Patent No.: US 9,493,803
App. No.: 14/134,177
Granted: Nov 15, 2016
Kind: B2

External Links

USPTO Patent Center↗ Full Patent PDF↗

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.

Claims (299)

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:

CAG:C

CAG:T

AAG:C

AAG:T

TAG:C

TAG:T

GlnLeu

GlnPhe

LysLeu

LysPhe

PheSer

CAGCTN

CAGTTY

AAGCTN

AAGTTY

TTTAGC

TTTAGT

GlnPro

GlnLeu

LysPro

LysLeu

SerSer

CAGCCN

CAGTTR

AAGCCN

AAGTTR

TCTAGC

TCTAGT

GlnHis

GlnSer

LysHis

LysSer

TyrSer

CAGCAY

CAGTCN

AAGCAY

AAGTCN

TATAGC

TATAGT

GlnGln

GlnTyr

LysGln

LysTyr

CysSer

CAGCAR

CAGTAY

AAGCAR

AAGTAY

TGTAGC

TGTAGT

GlnArg

GlnCys

LysArg

LysCys

LeuSer

CAGCGN

CAGTGY

AAGCGN

AAGTGY

CTTAGC

CTTAGT

SerAla

GlnTrp

GlnAla

LysTrp

ProSer

TCAGCN

CAGTGG

CAAGCN

AAGTGG

CCTAGC

CCTAGT

ProAla

SerVal

LysAla

GlnVal

HisSer

CCAGCN

TCAGTN

AAAGCN

CAAGTN

CATAGC

CATAGT

ThrAla

ProVal

GluAla

LysVal

ArgSer

ACAGCN

CCAGTN

GAAGCN

AAAGTN

CGTAGC

CGTAGT

AlaAla

ThrVal

LeuSer

GluVal

IleSer

GCAGCN

ACAGTN

YTAAGC

GAAGTN

ATTAGC

ATTAGT

PheSer

AlaVal

SerSer

LeuSer

ThrSer

TTCAGC

GCAGTN

TCAAGC

YTAAGT

ACTAGC

ACTAGT

SerSer

PheSer

ProSer

SerSer

AsnSer

TCCAGC

TTCAGT

CCAAGC

TCAAGT

AATAGC

AATAGT

TyrSer

SerSer

GlnSer

ProSer

SerSer

TACAGC

TCCAGT

CAAAGC

CCAAGT

AGTAGC

AGTAGT

CysSer

TyrSer

ArgSer

GlnSer

ValSer

TGCAGC

TACAGT

MGAAGC

CAAAGT

GTTAGC

GTTAGT

LeuSer

CysSer

IleSer

ArgSer

AlaSer

CTCAGC

TGCAGT

ATAAGC

MGAAGT

GCTAGC

GCTAGT

ProSer

LeuSer

ThrSer

IleSer

AspSer

CCCAGC

CTCAGT

ACAAGC

ATAAGT

GATAGC

GATAGT

HisSer

ProSer

LysSer

ThrSer

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.

Assignments (1)

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 8, 2014

From: ENENKEL, BARBARA

To: BOEHRINGER INGELHEIM INTERNATIONAL GMBH

Reel/Frame 032623/0647 →

Priority Claims (1)

EP 12199814 · Dec 31, 2012 · regional

Continuity (1)

Related Publication 20140242637A1 · Aug 28, 2014