Promoter with an enriched Cytosine-Guanine dinucleotide region, vectors, cellular lines, method for producing recombinant protein

Patent metadata

Field	Value
Publication number	US-11618904-B2
Application number	US-201716314192-A
Country	US
Kind code	B2
Filing date	Jun 9, 2017
Priority date	Jun 29, 2016
Publication date	Apr 4, 2023
Grant date	Apr 4, 2023

Abstract

The present invention relates to the field of genetic engineering, preferably the expression of recombinant proteins (RP). In particular, the invention relates to a promoter and variants thereof having an equal function and more than 90% sequence identity. The promoter comprises a fragment of 1147 base pairs (bp) of a first promoter, promoter of the β-actin gene of the Cricetulus griseus genome, enriched in cytosine-guanine dinucleotides (RegCG). The first promoter can be upstream of a second promoter, cytomegalovirus (CMV) promoter. The invention also relates to vectors, transfected cellular lines and a method for producing RP in mammal cells that have been transfected with vectors containing said promoter or variants thereof.

First claim

The invention claimed is:

1. A promoter for the expression of recombinant proteins (RP), comprising:
a first promoter comprising the sequence of SEQ ID NO: 5 or a variant having the same function and more than 90% sequence identity;
a second promoter comprising a cytomegalovirus (CMV) promoter comprising the sequence of SEQ ID NO: 3 or a variant having more than 90% sequence identity, located downstream of the first promoter, Wherein said second promoter has an enhancer comprising the sequence of SEQ ID NO: 29 and a core comprising the sequence of SEQ ID NO: 30; and
a sequence comprising five (5) tandem glucocorticoid-responsive elements (GRE), said sequence comprising the sequence of SEQ ID NO: 8, located between said first and second promoters.

2. The promoter of claim 1 , further comprising an additional sequence comprising five (5) tandem glucocorticoid-responsive elements (GRE), said sequence comprising the sequence of SEQ ID NO: 8, located between the enhancer and the core of the second promoter.

3. A vector comprising a promoter for the expression of recombinant proteins (RP), comprising:
a first promoter comprising the sequence of SEQ ID NO: 5 or a variant having the same function and more than 90% sequence identity;
a second promoter comprising a cytomegalovirus (CMV) promoter comprising the sequence of SEQ ID NO: 3 or a variant having more than 90% sequence identity, wherein said second promoter has an enhancer comprising the sequence of SEQ ID NO: 29 and a core comprising the sequence of SEQ ID NO: 30, located downstream of the first promoter; and
a sequence comprising five (5) tandem glucocorticoid-responsive elements (GRE), said sequence comprising the sequence of SEQ ID NO: 8 located between said first and second promoters.

4. The vector of claim 3 , wherein the vector comprises a sequence encoding a protein selected from the group consisting of an antibody and a chain of antibody.

5. The vector of claim 3 , wherein the vector encodes an antibody heavy chain (Ab2) comprising the sequence of SEQ ID NO: 22 and an antibody light chain (Ab2) comprising the sequence of SEQ ID NO: 23.

6. The vector of claim 3 , wherein the vector encodes an antibody heavy chain (Ab1) comprising the sequence of SEQ ID NO: 15 and an antibody light chain (Ab1) comprising the sequence of SEQ ID NO: 16.

7. The vector of claim 6 , further comprising an IRES sequence comprising the sequence of SEQ ID NO: 17, located between the antibody heavy chain (Ab1) sequence and the antibody light chain (Ab1) sequence.

8. The vector of claim 3 , wherein the vector comprises a sequence encoding dihydrofolate reductase (DHFR) comprising the sequence of SEQ ID NO: 26.

9. The vector of claim 8 , wherein the vector responds in a dose-dependent manner when dexamethasone is present.

10. The vector of claim 3 , wherein the promoter further comprises an additional sequence comprising five (5) tandem glucocorticoid-responsive elements (GRE), said sequence comprising the sequence of SEQ ID NO: 8, located between the enhancer and the core of the second promoter.

11. The vector of claim 5 , further comprising an IRES sequence comprising the sequence of SEQ ID NO: 17, located between the antibody heavy chain (Ab2) sequence and the antibody light hair (Ab2) sequence.

12. A host cell, comprising the vector of claim 8 .

13. The host cell of claim 12 , wherein the host cell is a mammalian cell.

14. The host cell of claim 13 , wherein the host cell is a Chinese hamster ovary cell (CHO).

15. A host cell, comprising the vector of claim 7 .

16. A host cell, comprising the vector of claim 8 .

17. A method for producing recombinant proteins (RP) comprising culturing in suspension a host cell comprising the vector of claim 3 , wherein said vector further includes a sequence encoding a recombinant protein of interest.

18. A method for producing recombinant proteins (RP), comprising culturing in suspension the host cell of claim 15 .

19. A method for producing recombinant proteins (RP), comprising culturing in suspension the host cell of claim 16 .

Assignees

• Univ Chile
• Bph S A
• Ingenieria Orbicorp Ltda
• Ingeneria Orbicorp Ltda

Inventors

• Aguillon Gutierrez Juan Carlos
• Molina Sampayo Maria Carmen
• Zuñiga Olate Roberto Aquiles
• Guitierrez Gonzalez Matias Fernando
• Collazo Muñoz Norberto Andres
• Teneb Lobos Jaime Camilo

Classifications

• C07K16/241

  Tumor Necrosis Factors · CPC title

• C12N15/85 Primary

  for animal cells · CPC title

• C12N2710/16143

  viral genome or elements thereof as genetic vector · CPC title

• C07K14/4716

  Muscle proteins, e.g. myosin, actin · CPC title

• C12N2830/60

  from viruses · CPC title