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US-20260125656-A1 - BACULOVIRUS EXPRESSION SYSTEM

US20260125656A1US 20260125656 A1US20260125656 A1US 20260125656A1US-20260125656-A1

Abstract

The invention relates to a method for producing a recombinant baculovirus comprising n exogenous genes in an insect cell, by means of homologous recombination of a replication-deficient baculovirus genome and n transfer vectors, each comprising one of the n exogenous genes, n being an integer at least equal to 2.

Inventors

  • Martine Cerutti
  • SYLVIE JULIANT
  • SYLVIE THERY

Assignees

  • CENTRE NATIONAL DE RESEARCHE SCIENTIFIQUE (CNRS)
  • INSTITUT NATIONAL DE LA RESEARCHE AGRONOMIQUE

Dates

Publication Date
20260507
Application Date
20250703
Priority Date
20160805

Claims (19)

  1. 1 . A recombinant baculovirus comprising n nucleotide sequences of formula (I): [exogenous gene]-[spacer nucleotide sequence]-[functional gene essential for viral replication] (I), said spacer nucleic acid sequence consists of 0 to 600 bp, said functional gene essential for viral replication is selected from 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), lef-2 (ORF6); n being an integer at least equal to 2, said recombinant baculovirus does not comprise a nucleic acid sequence allowing it to replicate within a bacterial cell.
  2. 2 . The recombinant baculovirus according to claim 1 , not comprising n genes non-essential for viral replication selected from 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), ORF5.
  3. 3 . The recombinant baculovirus according to claim 1 genome selected from or derived from the BmNPV genome, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SIMNPV, SeMNPV, or TeNPV, preferably AcMNPV.
  4. 4 . The recombinant baculovirus according to claim 1 , wherein n is an integer ranging from 2 to 30, for example ranging from 2 to 10.
  5. 5 . The recombinant baculovirus according to claim 1 , expressing n exogenous polypeptides forming a single protein comprising n distinct subunits, or several proteins.
  6. 6 . The recombinant baculovirus according to claim 5 , characterized in that the expressed protein is an antibody, a protein complex forming Virus-Like-Particles (VLP), a pair of viral proteins, a peptide hormone, a bispecific antibody, a set of three viral proteins, a multienzyme complex, or a protein complex.
  7. 7 . The recombinant baculovirus according to claim 1 , characterized in that the n nucleotide sequences of formula (I) are distributed throughout the genome of the recombinant baculovirus.
  8. 8 . The recombinant baculovirus according to claim 1 , characterized in that the n nucleotide sequences of formula (I) are not duplicated on the genome of the recombinant baculovirus.
  9. 9 . A cell comprising a recombinant baculovirus according to claim 1 .
  10. 10 . The cell according to claim 9 , characterized in that it is an insect cell, preferably selected from Sf9, Sf21, Tn5-b14, and AcMNPV baculovirus-sensitive lepidopteran cell lines.
  11. 11 . A recombinant baculovirus comprising n nucleotide sequences of formula (I): [exogenous gene]-[spacer nucleotide sequence]-[functional gene essential for viral replication] (I), said spacer nucleic acid sequence consists of 0 to 600 bp, said functional gene essential for viral replication is selected from 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), lef-2 (ORF6); n being an integer at least equal to 2, said recombinant baculovirus does not comprise a nucleic acid sequence allowing it to replicate within a bacterial cell.
  12. 12 . The recombinant baculovirus according to claim 11 , not comprising n genes non-essential for viral replication selected from 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), ORF5.
  13. 13 . A set of homologous recombination elements comprising: a) a replication-deficient baculovirus genome in which n genes essential for viral replication are non-functional; b) n transfer vectors each comprising: i) a nucleic acid sequence that restores the function of one of the n non-functional genes essential for viral replication, ii) optionally an exogenous gene, n being an integer at least equal to 2.
  14. 14 . A cell comprising a recombinant baculovirus according to claim 11 .
  15. 15 . Use of a recombinant baculovirus according to claim 11 for the production of n exogenous polypeptides encoded by n exogenous genes.
  16. 16 . A cell comprising a recombinant baculovirus according to claim 12 .
  17. 17 . A cell comprising a set of homologous recombination elements according to claim 13 .
  18. 18 . Use of a recombinant baculovirus according to claim 12 for the production of n exogenous polypeptides encoded by n exogenous genes.
  19. 19 . Use of a cell according to claim 14 for the production of n exogenous polypeptides encoded by n exogenous genes.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a Divisional of U.S. application Ser. No. 16/321,634, filed on Jan. 29, 2019 which is a U.S. national phase application filed under 35 U.S.C. § 371 from International Patent Application No. PCT/FR2017/052191, filed Aug. 4, 2017, which claims priority to French Application 165711, filed Aug. 5, 2016, each of which is incorporated by reference herein in their entireties. TECHNICAL FIELD The invention concerns a process for the preparation of a recombinant baculovirus comprising at least two exogenous genes, homologous recombination sets used in this preparation process, the recombinant baculoviruses obtained by this process, and the use of the recombinant baculoviruses for the production of polypeptides. REFERENCE TO A SEQUENCE LISTING XML This application contains a Sequence Listing which has been submitted electronically in XML format. The Sequence Listing filed in electronic form is hereby incorporated by reference. Said XML file, created on Aug. 9, 2025, is named BNT242002USPC01.xml and is 4,937 bytes in size. TECHNOLOGICAL BACKGROUND Baculoviruses are a family of rod-shaped viruses, specific to arthropods, composed of four genera (Alphabaculovirus, Betabaculovirus, Deltabaculovirus, Gammabaculovirus) comprising 49 species. Baculoviruses are unable to replicate in mammalian or other vertebrate cells. The baculovirus genome consists of a circular, double-stranded DNA molecule of between 80 and 180 kbp in size. The baculovirus genome is associated with highly basic proteins of 6.5 kDa within a helically symmetrical nucleocapsid which contains a 39 kDa capsid protein. The size of the genome determines the length of the nucleocapsid. The nucleocapsid is further packaged within a lipoprotein envelope to form the virus particle or virion. These structures may be occluded within a crystalline matrix or polyhedron consisting largely of a single protein (polyhedrin) of about 30 kDa. Polyhedra are large structures ranging in size from 1 to 15 μm in diameter with an outer polysaccharide envelope which confers additional protection. Baculoviruses with a genetically modified genome are used in biotechnology for the production of recombinant proteins (i.e. recombinant baculoviruses). After entering an insect cell, these recombinant baculoviruses will use the insect cell's machinery to produce the recombinant protein. Recombinant baculoviruses are obtained by inserting one or more genes from other species (e.g. humans, other vertebrates, plants, bacteria and viruses) into the genome of a parental baculovirus. These genes are placed under the control of a viral or cellular promoter (e.g. the polyhedrin gene promoter) to generate a recombinant baculovirus. The promoter allows transcription of the foreign gene into messenger RNA which, in turn, is translated into protein in the recombinant baculovirus-infected insect cell. The advantage of using this system is that the level of recombinant protein production in recombinant baculovirus-infected insect cells can be very high. The recombinant protein can then be purified from the infected cells if the protein is intracellular or from the culture medium if the protein is secreted. The baculovirus expression system is widely used in industry and in research laboratories. In addition to the high productivity of the baculovirus expression system, this system is also highly valued because it allows the production of biologically active recombinant proteins. Indeed, insect cells generally allow suitable post-translational modifications to be obtained. Despite all these advantages, the baculovirus expression system is difficult to implement on an industrial scale for the production of several distinct recombinant polypeptide sequences, for example proteins comprising several distinct subunits, such as antibodies or pairs of viral proteins. Indeed, the processes used for the production of recombinant baculoviruses are complex and require a large number of steps that do not allow homogeneous and stable recombinant baculovirus genomes to be obtained. The homogeneity and stability of recombinant baculovirus genomes are necessary criteria in order to envisage industrial development. There is therefore a need to develop processes that are easy to implement, that allow the production of recombinant proteins comprising several distinct subunits, such as antibodies, in baculovirus expression systems, and that can be developed on an industrial scale in particular. On the basis of the above, the applicant has developed a process for preparing homogeneous and stable recombinant baculoviruses which is particularly efficient and easy to implement, and which makes it possible to envisage industrial development, notably for the production of several distinct recombinant polypeptide sequences, particularly proteins comprising several distinct subunits. SUMMARY OF THE INVENTION In a first aspect, the invention concerns a process for preparin