Alphaviral vectors and cell lines for producing recombinant proteins

• US 10,011,847 B2
• Filed: 05/20/2014
• Issued: 07/03/2018
• Est. Priority Date: 05/20/2013
• Status: Active Grant

• Current Assignee: 3P Biopharmaceuticals, S.L.
• Original Assignee: 3P Biopharmaceuticals, S.L.
• Inventors: Aranda, Alejandro, Bezunartea Bezunartea, Jaione, Smerdou Picazo, Cristian, Molero Sanchez, Damaso, Mistiene, Edita, Soldevila Fabrega, Andreu, Serrano Perez-Nievas, Paula
• Primary Examiner(s): Kelly, Robert M
• Application Number:
  US14/892,829
• Publication Number:
  US 20160208286A1
• Time in Patent Office: 1,505 Days
• Field of Search: None
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2770/36141 Use of virus, viral particle or viral elements as a vector
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2840/203 having an IRES
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

The present invention relates to polynucleotides and alphaviral vectors for the expression of genes of interest in mammalian cells. Additionally, the invention relates to cells which comprise said polynucleotides and alphaviral vectors and are capable of stably expressing one or more genes of interest. The invention also relates to methods for obtaining said cells, to methods for expressing a gene of interest in said cells, and to methods for replacing the gene of interest stably expressed by said cells with another gene of interest.

Claims

1. A polynucleotide comprising

(i) a transcription regulatory sequence,

(ii) a DNA sequence complementary to an alphavirus replicon, wherein said alpharivus replicon comprises, ordered from the 5′

to 3′

end;

a 5′

sequence capable of directing replication of the alphavirus,

a nucleotide sequence encoding a polyprotein comprising the sequences of non-structural proteins nsp1, nsp2, nsp3 and nsp4 of an alphavirus,

an alphavirus subgenomic promoter, and

an alphavirus 3′

untranslated sequence,

 

wherein the DNA sequence complementary to an alphavirus replicon is operatively bound to said transcription regulatory sequence, and wherein said alphavirus replicon complementary sequence comprises a first recognition sequence for a site-specific recombinase located between the sequence complementary to the alphavirus replicon subgenomic promoter and the sequence complementary to the alphavirus replicon 3′

untranslated sequence,

(iii) a transcription termination sequence postitioned 3′

with respect to the 3′

end of the sequence complementary to said alphavirus replicon, and

(iv) a second recognition sequence for a site-specific recombinase positioned 3′

with respect to the transcription termination sequence.

2. The polynucleotide according to claim 1, wherein the recognition sequences for a site-specific recombinase are heterospecific sequences.

3. The polynucleotide according to claim 1, wherein the recognition sequences for a site-specific recombinase are LoxP sequences.

4. The polynucleotide according to claim 3, wherein the first and second LoxP sequences are selected from the group consisting of the pairs

(a) SEQ ID NO:

9 and SEQ ID NO;

10,

(b) SEQ ID NO;

11 and SEQ ID NO;

12,

(c) SEQ ID NO;

9 and SEQ ID NO;

12,

(d) SEQ ID NO;

11 and SEQ ID NO;

10,

(e) SEQ ID NO;

10 and SEQ ID NO;

9,

(f) SEQ ID NO;

12 and SEQ ID NO;

11,

(g) SEQ ID NO;

10 and SEQ ID NO;

11, and

(h) SEQ ID NO;

12 and SEQ ID NO;

9.

5. The polynucleotide according to claim 4, wherein the transcription regulatory sequence comprises a eukaryotic promoter or a minimal variant thereof.

6. The polynucleotide according to claim 5, wherein the promoter is the minimal albumin gene promoter or a functionally equivalent variant thereof.

7. The polynucleotide according to claim 1, wherein the transcription regulatory sequence has regulable activity.

8. The polynucleotide according to claim 7, wherein the transcription regulatory sequence having regulable activity additionally comprises an inducing agent responsive element.

9. The polynucleotide according to claim 8, wherein the inducing agent responsive element is a ligand responsive element.

10. The polynucleotide according to claim 9, wherein the ligand responsive element is a tetracycline or tetracycline analogue responsive element.

11. The polynucleotide according to claim 5, wherein the transcription regulatory sequence comprises the nucleotide sequence SEQ ID NO:

5.

12. The polynucleotide according to claim 11, wherein the transcription regulatory sequence comprises the nucleotide sequence SEQ ID NO:

8, and wherein the transcription regulatory sequence does not comprise the sequence SEQ ID NO;

4.

13. The polynucleotide according to claim 1, wherein the DNA sequence complementary to an alphavirus replicon further comprises a heterologous sequence positioned 3′

with respect to the sequence complementary to the replicon subgenomic promoter and with respect to the first recognition sequence for a site-specific recombinase.

14. The polynucleotide according to claim 13, wherein said heterologous sequence is selected from the group consisting of

(i) a stuffer sequence,

(ii) a sequence comprising a multiple cloning site,

(iii) a sequence of a gene of interest operatively bound to the sequence complementary to the alphavirus replicon subgenomic promoter, and

(iv) any combination thereof.

15. The polynucleotide according to claim 14, wherein said heterologous sequence is a sequence of a gene of interest operatively bound to the sequence complementary to the alphavirus replicon subgenomic promoter, and wherein the gene of interest encodes a protein of interest or a precursor thereof.

16. The polynucleotide according to claim 14, wherein the polynucleotide comprises at least one sequence of a second gene of interest, wherein said sequence of a second gene of interest is positioned 3′

with respect to the first recognition sequence for a site-specific recombinase in the polynucleotide, and wherein said sequence of a second gene of interest is operatively bound to a sequence complementary to an additional subgenomic promoter or is operatively bound with the sequence of the first gene of interest by means of an IRES or a sequence encoding a post-translational proteolytic cleavage site.

17. The polynucleotide according to claim 16, wherein the first gene of interest and the second gene of interest encode a first and a second antibody chain, respectively.

18. The polynucleotide according to claim 1, wherein the alphavirus replicon is a Semliki Forest Virus replicon.

19. The polynucleotide according to claim 18, wherein the Semliki Forest Virus replicon comprises at least one mutation conferring said replicon a non-cytopathic phenotype.

20. The polynucleotide according to claim 19, wherein said mutation is located in the region encoding the nsp2 subunit of the viral replicase and wherein said mutation is selected from the group consisting of mutations P718T, R649H and a combination of both.

21. The polynucleotide according to claim 1, additionally comprising a selection gene, wherein said selection gene is located in the region comprised between the first and second recombinase recognition sites, and wherein

(i) if the transcription regulatory sequence which is operatively bound to the alphavirus replicon complementary sequence is a regulable sequence, then the selection gene is located outside said replicon and bound to a constitutive promoter, or

(ii) if the transcription regulatory sequence which is operatively bound to the alphavirus replicon complementary sequence comprises a constitutive promoter, then said selection gene is operatively associated with the gene of interest by means of an IRES or a sequence encoding a post-translational proteolytic cleavage site, or said selection gene is under the control of a second constitutive promoter.

22. The polynucleotide according to claim 21, wherein the constitutive promoter to which the selection gene is bound comprises the nucleotide sequence which is shown in SEQ ID NO:

14.

23. The polynucleotide according to claim 21, wherein the selection gene is a gene conferring antibiotic resistance.

24. The polynucleotide according to claim 23, wherein the gene conferring antibiotic resistance is selected from the group consisting of the neomycin resistance gene and the puromycin resistance gene.

25. The polynucleotide according to claim 1, additionally comprising a polyadenylation signal or a polyadenylation and transcription termination signal positioned 3′

with respect to the second recombinase recognition site.

26. The polynucleotide according to claim 25, wherein the polyadenylation and transcription termination signal comprises the sequence SEQ ID NO:

70.

27. A eukaryotic cell comprising a polynucleotide according to claim 1, wherein the polynucleotide is integrated in the genome thereof.

28. The cell according to claim 27, wherein the transcription regulatory sequence which is operatively bound to the alphavirus replicon complementary sequence is a regulable sequence, and wherein said cell additionally comprises an DNA sequence encoding a transcriptional activator operatively bound to a constitutive promoter, wherein said transcriptional activator is capable of regulating transcription from said transcription regulatory sequence by means of the binding thereof to the ligand responsive site, and wherein the DNA sequence encoding a transcriptional activator is integrated in the genome.

29. An in vitro method for generating a cell line capable of expressing a gene of interest which comprises

(i) contacting a cell with a polynucleotide according to claim 1, wherein said polynucleotide additionally comprises said gene of interest operatively bound to the sequence complementary to the replicon subgenomic promoter, or with an expression vector comprising said polynucleotide, and

(ii) selecting cells that have incorporated said polynucleotide or an expression vector comprising said polynucleotide.

30. A method for expressing a gene of interest which comprises putting a eukaryotic cell in suitable conditions for activating transcription of the sequence complementary to the alphaviral replicon, wherein said cell comprises a polynucleotide according to claim 1 or an expression vector comprising said polynucleotide, wherein said polynucleotide additionally comprises the sequence of the gene of interest operatively bound to the subgenomic promoter.

32. An in vitro method for generating a cell line capable of expressing a gene of interest, comprising the steps of:

(i) introducing in a cell according to claim 27 a vector, wherein;

(a) said cell comprises a heterologous sequence positioned 3′

with respect to the sequence complementary to the replicon subgenomic promoter, wherein said heterologous sequence is a sequence of a gene of interest operatively bound to the sequence complementary to the subgenomic promoter,

(b) the first recognition sequence of the polynucleotide comprising the alphavirus replicon complementary sequence is compatible with the first heterospecific recognition sequence of the vector,

(c) the second recognition sequence of the polynucleotide comprising the alphavirus replicon complementary sequence is compatible with the second heterospecific recognition sequence of the vector,

(d) the cell expresses a specific recombinase of said first and second recognition sequences, and

(e) the sequence necessary for replication of the alphavirus present in the vector coincides with the sequence necessary for replication of the alphavirus which is part of the alphaviral replicon,

(ii) maintaining the cell in suitable conditions to allow substitution by means of specific recombination of the gene of interest which is part of the polynucleotide comprising the alphavirus replicon complementary sequence with the gene of interest which is part of the vector, and

(iii) selecting the cells in which substitution of the first gene of interest with the second gene of interest has occurred,

wherein the vector comprises a DNA sequence comprising, ordered in the 5′

to 3′

direction,

(A) a first recognition sequence for a site-specific recombinase,

(B) a sequence of a gene of interest,

(C) a 3′

sequence necessary for replication of an alphavirus,

(D) a transcription termination sequence,

(E) a sequence of a selection gene operatively bound to a promoter, and

(F) a second recognition sequence for a site-specific recombinase.

31. A vector comprising a DNA sequence comprising, ordered in the 5′

to 3′

direction,

(i) a first recognition sequence for a site-specific recombinase,

(ii) a sequence of a gene of interest,

(iii) a 3′

sequence necessary for replication of an alphavirus,

(iv) a transcription termination sequence,

(v) a sequence of a selection gene operatively bound to a promoter, and

(vi) a second recognition sequence for a site-specific recombinase.