IP Library › Granted Patent US 12,188,052
Granted Patent B2
US 12,188,052 · App. 16/758,811 · Granted Jan 7, 2025

Baculovirus expression system

Inventors: Martine Cerutti (Saint Christol les Ales, FR); Sylvie Juliant (Saint Christol les Ales, FR); Coralie Bernon (Boisset-et-Gaujac, FR)
Assignees: CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS); INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE
C12N7/00C12N15/86C12P21/00C12N2710/14043C12N2710/14051
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Quick Facts
Patent No.
US 12,188,052
App. No.
16/758,811
Granted
Jan 7, 2025
Kind
B2
Abstract

Method for preparing, in an insect cell, a recombinant baculovirus comprising one or more transgene(s) each encoding a protein maturation enzyme and n transgenes each encoding a polypeptide of interest, by homologous recombination between a replication deficient baculovirus genome which comprises one or more transgene(s) each encoding a protein maturation enzyme and n transfer vectors each comprising one of the n transgenes each encoding a polypeptide of interest, n being an integer at least equal to 2.

Claims (46)

1. A method for producing a recombinant baculovirus of which the genome comprises: one or more transgene(s) each encoding a protein maturation enzyme, and n transgenes each encoding a polypeptide of interest to be expressed at a different locus, said method comprising the steps of:

a) preparing, in an insect cell, a recombinant baculovirus genome capable of replicating which comprises said one or more transgene(s), each encoding a protein maturation enzyme, and said n transgenes each encoding a polypeptide of interest, by homologous recombination between:

a1) a replication deficient baculovirus genome in which n genes essential for viral replication located in different regions of the viral genome are non-functional and which comprises said one or more transgene(s), each encoding a protein maturation enzyme, and

a2) n transfer vectors each comprising:

i) a nucleotide sequence enabling to restore the function of one of the n non-functional genes essential for viral replication located in different regions of the viral genome,

ii) one of said n transgenes encoding a polypeptide of interest, each polypeptide of interest to be expressed at a different locus,

the set of nucleotide sequences i) of the n transfer vectors being capable of restoring the replication of the replication deficient baculovirus genome,

n being an integer at least equal to 2; and

b) generating a recombinant baculovirus in an insect cell which comprises the recombinant baculovirus genome obtained at step a),

wherein said homologous recombination between the replication deficient baculovirus genome and the n transfer vectors takes place in a single transfection step in the insect cell.

2. The method according to claim 1 , wherein said replication deficient baculovirus genome of step a1) is prepared, in a bacterial cell, by homologous recombination between:

a replication deficient baculovirus genome in which n genes essential for viral replication are non-functional, and

one or more nucleotide sequence(s) each comprising one or more transgene(s) each encoding a protein maturation enzyme.

3. The method according to claim 1 , wherein the genes essential for viral replication are selected from the group consisting of: 1629 (ORF9), Pk1 (ORF10), lef-1 (ORF14), ORF34, lef-11 (ORF37), p47 (ORF40), lef8 (ORF50), DNAJ domain (ORF51), ORF53, vp1054 (ORF54), Lef-9 (ORF62), DNA Pol (ORF65), lef-3 (ORF67), ORF73, ORF75, ORF81, p95 (ORF83), vp39 (ORF89), lef-4 (ORF90), p33 (ORF92), helicase (ORF95), vp80 (ORF104), ORF106-107, odv-ec43 (ORF109), gp64/67 (ORF128), ORF132, ORF133, odv-ec27 (ORF144), ORF146, ie1 (ORF147), and lef-2 (ORF6).

4. The method according to claim 1 , wherein the n non-functional genes essential for viral replication are each adjacent to a gene not essential for viral replication.

5. The method according to claim 4 , wherein the gene not essential for viral replication is selected from the group consisting of: Ph (ORF 8), ORF11, ORF13, egt (ORF15), v-ubiquitin (ORF35), 39K (ORF36), ORF38, p43 (ORF39), lef-12 (ORF41), pcna (ORF49), ORF52, ORF55, Fp (ORF61), ORF63, gp37 (ORF64), ORF68, ORF72, ORF74, ORF82, cg30 (ORF88), ORF91, pif-4 (ORF96), he65 (ORF105), ORF108, ORF110, cathepsin (ORF127), p24 (ORF129), pp34 (ORF131), ORF134, ORF145, odv-e56 (ORF148), and ORF5.

6. The method according to claim 1 , wherein the n transgenes encoding a polypeptide each recombine at the locus of a gene not essential for viral replication adjacent to a non-functional gene essential for viral replication.

7. The method according to claim 1 , wherein the protein maturation enzyme is selected from the group consisting of: a signal peptidase, a furin, a proprotein convertase, a glycosyltransferase, a glycosidase, a chaperone protein, an isomerase disulphide, an acyltransferase, a methyltransferase, a hydroxylase, a transglutaminase, a farnesyltransferase, a geranylgeranyl-transferase, a N-myristoyltransferase, a palmityltransferase, a phosphatase, a transpeptidase, a carboxylase, and a ubiquitin ligase.

8. The method according to claim 2 , wherein the one or more transgene(s), each encoding a protein maturation enzyme, each recombines at the locus of a gene not essential for viral replication.

9. The method according to claim 8 , wherein the gene not essential for viral replication is selected from the group consisting of: ptp (ORF1), ctx (ORF3), ORF4, ORF7, odv-e26 (ORF16), ORF17, ORF18, ORF19, ARIF-1 ORF20-21, pif2 (ORF22), protein F (ORF23), iap1 (ORF27), lef6 (ORF28), ORF29, ORF30, sod (ORF31), fgf (ORF32), gta (ORF42), ORF43, ORF44, ORF45, odv-e66 (ORF46), ORF47, ORF56, ORF57, chaB-like (ORF58/59), chaB-like (ORF60), mtase (ORF69), hcf-1 (ORF70), iap2 (ORF71), ORF86, ORF87, ORF111, ORF114, pif3 (ORF115), ORF116, ORF117, pif1 (ORF119), ORF120, ORF121, ORF122, pk2 (ORF123), ORF124, lef7 (ORF125), chitinase (ORF126), gp16 (ORF130), p35 (ORF135), p26 (ORF136), p10 (ORF137), p74 (ORF138), ORF149, ORF150, ie2 (ORF151), pe38 (ORF153) and ORF154.

10. The method according to claim 1 , wherein the insect cell is selected from the group consisting of: Sf9, Sf21, Tn5-b14, lepidoptera cell lines sensitive to the baculovirus AcMNPV.

11. The method according to claim 2 , wherein the bacterial cell is E. coli selected from DH10B and EL350.

12. The method according to claim 1 , wherein the replication deficient baculovirus genome is obtained from a baculovirus genome selected from or derived from the genome of one of the following baculovirus: BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SIMNPV, SeMNPV and TeNPV.

13. The method according to claim 1 , wherein n is an integer ranging from 2 to 31.

14. A recombinant baculovirus or recombinant baculovirus genome, susceptible to be obtained by the production method according to claim 1 , comprising:

a) One or more transgene(s) each encoding a protein maturation enzyme, and

b) n nucleotide sequences of formula (I):

[transgene encoding a polypeptide of interest]−[spacer nucleotide sequence]−[gene essential for functional viral replication]  (I),

said spacer nucleic acid sequence being constituted of 0 to 600 base pairs,

said gene essential for functional viral replication being selected from the group consisting of: 1629 (ORF9), Pk1 (ORF10), lef-1 (ORF14), ORF34, lef-11 (ORF37), p47 (ORF40), lef8 (ORF50), DNAJ domain (ORF51), ORF53, vp1054 (ORF54), Lef-9 (ORF62), DNA Pol (ORF65), lef-3 (ORF67), ORF73, ORF75, ORF81, p95 (ORF83), vp39 (ORF89), lef-4 (ORF90), p33 (ORF92), helicase (ORF95), Vp80 (ORF104), ORF106-107, odv-ec43 (ORF109), gp64/67 (ORF128), ORF132, ORF133, odv-ec27 (ORF144), ORF146, ie1 (ORF147), and lef-2 (ORF6); and

n being an integer at least equal to 2.

15. The recombinant baculovirus or recombinant baculovirus genome according to claim 14 , wherein said recombinant baculovirus or recombinant baculovirus genome do not comprise n genes not essential for viral replication chosen from the group consisting of: Ph (ORF 8), ORF11, ORF13, egt (ORF15), v-ubiquitin (ORF35), 39K (ORF36), ORF38, p43 (ORF39), lef-12 (ORF41), pcna (ORF49), ORF52, ORF55, Fp (ORF61), ORF63, gp37 (ORF64), ORF68, ORF72, ORF74, ORF82, cg30 (ORF88), ORF91, pif-4 (ORF96), he65 (ORF105), ORF108, ORF110, cathepsin (ORF127), p24 (ORF129), pp34 (ORF131), ORF134, ORF145, odv-e56 (ORF148), and ORF5.

16. A set of homologous recombination elements comprising:

a) a replication deficient baculovirus genome in which n genes essential for viral replication located in different regions of the viral genome are non-functional and which comprises one or more transgene(s) each encoding a protein maturation enzyme;

b) n transfer vectors each comprising:

i) a nucleic acid sequence enabling to restore the function of one of the n non-functional genes essential for viral replication,

ii) a transgene encoding a polypeptide of interest, each polypeptide of interest expressed at a different locus,

n being an integer at least equal to 2.

17. A cell comprising a recombinant baculovirus or a recombinant baculovirus genome according to claim 14 .

18. A process for the production of n polypeptides of interest comprising producing the polypeptides from a recombinant baculovirus or a recombinant baculovirus genome according to claim 14 .

19. The method according to claim 10 , wherein the insect cell is Sf9.

20. The method according to claim 12 , wherein the replication deficient baculovirus genome is obtained from a baculovirus genome selected from or derived from AcMNPV.

21. The method according to claim 13 , wherein n is equal to or greater than 3.

22. A cell comprising a set of homologous recombination elements according to claim 16 .

23. A process for the production of n polypeptides of interest comprising producing the polypeptides in a cell according to claim 17 .

24. The method according to claim 8 , wherein the one or more transgene(s), each encoding a protein maturation enzyme each recombine at the locus of a gene not essential for viral replication non-adjacent to a non-functional gene essential for viral replication.

Assignments (2)
CHANGE OF NAME Recorded Jul 12, 2023
From: INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE
To: INSTITUT NATIONAL DE RECHERCHE POUR L'AGRICULTURE, L'ALIMENTATION ET L'ENVIRONNEMENT
Reel/Frame 064257/0266 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 18, 2022
From: CERUTTI, MARTINE; JULIANT, SYLVIE; BERNON, CORALIE
To: CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS); INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE
Reel/Frame 061458/0076 →
Priority Claims (1)
FR 1760068 · Oct 25, 2017 · national
Continuity (1)
Related Publication 20200263144A1 · Aug 20, 2020
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