WO2000073424A1 - Nouveau vecteur hybride du baculovirus/adenovirus utile pour la sauvegarde, la production et le titrage des vecteurs d'amplicon d'adenovirus haute capacite - Google Patents

Nouveau vecteur hybride du baculovirus/adenovirus utile pour la sauvegarde, la production et le titrage des vecteurs d'amplicon d'adenovirus haute capacite Download PDF

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WO2000073424A1
WO2000073424A1 PCT/US2000/014785 US0014785W WO0073424A1 WO 2000073424 A1 WO2000073424 A1 WO 2000073424A1 US 0014785 W US0014785 W US 0014785W WO 0073424 A1 WO0073424 A1 WO 0073424A1
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adenovirus
vector
virus
baculovirus
genes
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Valeri A. Krougliak
Natalia Cheshenko
Randy C. Eisensmith
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Mount Sinai School Of Medicine
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Definitions

  • This invention pertains to methods for the efficient, large-scale, helper virus-free production of recombinant adenovirus vectors that are devoid of most if not all viral genes using novel baculovirus/adenovirus hybrid helper vectors.
  • adenovirus vectors are a highly efficient means of transferring genes into a wide variety of cell types in vivo. Although the adenovirus has a natural tropism for lung epithelium, adenovirus vectors have quite a broad host cell range.
  • adenovirus vectors In addition to lung and other epithelial-derived tissues (Gilardi et al. 1990) (Stratford-Perricaudet el al. 1990): (Rosenfeld et al. 1991); (Quantin et al. 1992); (Rosenfeld et al. 1992); (Bajocchi et al. 1993); (Yang et al.
  • recombinant adenovirus vectors have been successfully used to transduce normally non-dividing cell types including hepatocytes (Levrero et al. 1991); (Jaffe et al. 1992); (Herz and Gerard 1993); (Ishibashi et al. 1993); (Li et al. 1993) and neuronal and glial cells of the central nervous system (Akli et al. 1993); (Davidson et al.
  • the adenovirus vector system has two limitations that have prevented its application in various forms of somatic gene therapy.
  • the first is the transient expression of genes delivered by this means. Apart from muscle, where low levels of persistent gene expression have been reported (Rosenfeld et al. 1992); (Stratford-Perricaudet et al. 1992); (Vincent et al. 1993), expression of genes delivered by recombinant adenovirus vectors is largely transient in other tissues.
  • hepatocytes were successfully transduced one week after intraportal infusion of a recombinant adenovirus vector expressing ⁇ -galactosidase, only 0.5% to 10% of hepatocytes were still positive for ⁇ - galactosidase activity by 14 to 16 weeks post-infusion (Li et al. 1993). This decline in the proportion of transduced cells was correlated with a decrease in the amount of vector DNA that was present, indicating that the decline in positive cells was not due to inactivation of transcription from the vector.
  • transduced cells become targets for host immune system. These vectors are not always safe, and repeated administration of these vectors is blocked by a strong immune response in the host system. Because cells transduced with El -deleted recombinant adenovirus vectors appear to be specifically targeted due to residual amounts of late viral gene expression (Yang et al. 1994a); (Yang et al. 1994b), several studies have examined whether additional modifications of these vectors to reduce late viral gene expression can significantly increase their persistence in vivo (Engelhardt et al. 1994a;Engelhardt et al. 1994b); (Yang et al. 1994b); (Fang et al. 1995), (Armentano et al. 1995), (Krougliak and Graham 1995); (Wang et al.
  • adenovirus vector modification is the creation of a so-called "gutless”, “gutted” or amplicon vector. This is a vector containing only the cis elements necessary for replication and packaging, but lacking most if not all adenovirus genes. All of the amplicon vectors created thus far (Mitani et al. 1995); (Fisher et al. 1996); (Kochanek et al l 996); (Lieber et al. 1996); (Parks et al. 1996); (Alemany et al. 1997); (Chen et al. 1997);
  • helper viruses or plasmid co-transfection to provide the necessary virus proteins in trans.
  • helper-dependent vectors are produced by simultaneous delivery of both helper and vector genome to a host cell. Both the helper virus and the vector should co-replicate in the same host cells to produce a mixture of a helper virus and a vector. This co-replication allows multiplying the amount of templates for expression of viral genes and producing sufficient amount vector genomes to package in virus particles.
  • helper virus is a first-generation recombinant adenovirus expressing a number of virus genes
  • its presence in the final vector preparation may cause the same adverse effects, which were observed in first-generation adenovirus vectors.
  • complete removal will greatly improve the quality of the vector, making it safer and more persistent.
  • BV baculovirus
  • Ad adenovirus
  • BV/Ad/SV40 hybrid vectors baculovirus hybrid helper virus that is comprised of baculovirus sequences and second viral structural and late viral sequences, where the baculovirus sequences encode the proteins necessary for replication and packaging of gutless viral vectors.
  • the present invention describes a method of producing gutless viral vectors substatially free from helper virus contamination comprised of the steps of transfecting mammalian cells with a baculovirus hybrid helper virus comprised of baculovirus sequences and second viral structural and late viral sequences and a gutless vector, transducing the mammalian cell, replicating and packaging the gutless vector, and recovering virions.
  • a baculovirus hybrid helper virus comprised of baculovirus sequences and second viral structural and late viral sequences and a gutless vector
  • transducing the mammalian cell replicating and packaging the gutless vector, and recovering virions.
  • the present invention describes a recombinant baculovirus
  • BAC-B4 (VSVG) deposited with the American Type Culture Collection (ATCC, Manassas, Virginia) on May 19, 1999 and receiving ATCC Accession No. PTA-88.
  • Fig. 1 shows the nucleotide sequence in (A) the synthetic loxP site I, (B) synthetic loxP site II and (C) polyadenylation site used in the vectors of the present invention.
  • Fig. 2 (A and B) are schematic representations showing the production of plasmids pL-VK-lox-SPA (2A) and pL/VK-LSL-1 (2B).
  • Fig. 3 is a schematic representation showing the production of plasmid pBAC-112.
  • Fig. 4 is a schematic representation showing the production of plasmid pBAC-B2-2.
  • Fig. 5 is a schematic representations showing the production of plasmid pBAC-B3-2.
  • Fig. 6 is a schematic representation showing the production of plasmid pBAC-B4 (VSVG).
  • Fig. 7 is a schematic representation showing the production of the recombinant baculovirus of the present invention.
  • Fig. 8 is a schematic diagram showing the production of plasmid pAVec4-2.
  • Fig. 9 is is a schematic diagram showing the production of plasmid pAVec9-2.
  • Fig. 10 is a schematic diagram showing the production of plasmid pAVec5.
  • Fig. 11 is a schematic diagram showing the production of plasmid pAVecl3.
  • Fig. 12 is a schematic diagram showing the structures of plasmid pBAC-B4, pBAC- B9, and pBAC-B10.
  • Fig. 13 is a picture of the genetic stability of pBAC-B9 after 4 passages in E. coli.
  • Fig. 14 is a picture of the PCR analysis of vector production in HepG2, Huh7, and A549 cells.
  • the most recent type of adenovirus vector has been proposed as a promising tool for gene therapy. Since these vectors are deleted of most if not all adenovirus genes, they require a helper adenovirus for their propagation. Therefore all gutless vector stocks are contaminated to some degree by the helper virus. This contamination limits the value of currently existing gutless vectors in gene therapy applications.
  • the present invention provides a system that allows production of gutless vectors that are free of helper virus. Unlike the conventional helper-dependent system, the helper-free system does not include a helper adenovirus, which eliminates the necessity for time- and labor-consuming purification of the vector, reduces the potential immunogenicity of the vector, and improves its safety.
  • the term “substantial reduction” or “substantially reduced” refers to complete absence of helper virus in the system or less than about 0.1% helper virus, based on the total content of the helper-free system.
  • antibiotic resistance gene refers to DNA that confers cellular resistance to an antibiotic, liketetracycline.
  • shuffer DNA refers to DNA which does not encode for any protein or has regulatory function.
  • stuffer DNA is intron DNA.
  • the stuffer DNA may be of any length to achieve optimal packaging size, which may range from 27.5to 37.5 kilobases.
  • the term "transducing” refers to the transferring of genes from the vector to the cell.
  • a system for the propagation of gutless vectors is described herein.
  • the adenovirus genes essential for the replication and packaging of the gutless vector are delivered into producer cells by a new recombinant baculovirus-adenovirus hybrid.
  • the ability of baculovirus to carry large inserts (>15 kb) and to transduce a variety of mammalian cells was advantageously used.
  • Recombinant baculoviruses, variants BacAd-B3-2 and BacAd-B3-2 (VSVG) have been produced by the present invention.
  • VSVG baculovirus genome
  • Pseudotyping is the process of altering a virus's envelope proteins so that the virus can bind to receptor molecules other than its natural receptor, allowing it to infect cell types that are different from those that it normally infects.
  • This process can produce a virus that is completely pseudotyped (i.e., its natural envelope protein is completely replaced by a heterologous envelope protein) or only partially pseudotyped (i.e. a second envelope protein is added to the original one).
  • BAC-B4 baculovirus/adenovirus hybrids described herein
  • VSVG baculovirus/adenovirus hybrids described herein
  • the latter is packaged into adenovirus virions while the helper genome remains unpackable.
  • a gutless vector expressing ⁇ -galactosidase was rescued.
  • the vector obtained was completely free of helper virus as confirmed by passaging the resulting vector in 293 cells.
  • the VSVG-pseudotyped helper was slightly more efficient in rescue of the gutless vector, likely due to increased ability to infect the 293-Cre cells.
  • the baculovirus-based helper system allows for efficient rescue and production of helper-free gutless vectors. Since the vectors generated in this system are free of helper this system will be a very useful alternative to current methods for the production on gutless vector.
  • BAC-B9 and BAC-B10 have been produced. Both have structures similar to BAC-B4(VSVG) with the addition of adenovirus E1A and E1B regions (BacB9) or 5.7 kb stuffer sequence into the adenovirus E3 region (BacBlO).
  • BAC-B9 contains the Ad5 El region, the El A promoter and the entire packaging signal were removed and replaced by the RSV promoter. Additionally, the El region is placed outside of the excising region and in an orientation that is opposite to normal.
  • BAC-B9 and BAC-B10 contained inserts of approximately 43 kb and 45 kb, respectively.
  • Serial passaging followed by Southern blotting analysis of the restriction pattern of the viral DNA in each passage showed that no genetic changes affecting the inserted sequences were detected after four subsequent large- scale preparations of BAC-B9 (Fig. 13). Similar data were obtained for BAC-B10 (data not shown).
  • BAC-B9 to complement replication of gutless adenovirus vectors in the non-complementing cell lines Huh7, HepG2 and A549 was evaluted.
  • PCR analysis of DNA revealed the presence of gutless vector DNA in crude lysates of the co-infected cells (Fig. 14). Vector DNA in the cells was not detected when infected with crude lysates only
  • helper-free system can be adapted for use in non-complementing cell lines.
  • the present invention provides for a method of producing adenovirus vectors, adeno- associated virus based vectors, herpes virus-based vectors, retrovirus and lentivirus vectors, and SV40-based vectors, all of which could be free of contaminating helper virus.
  • the adenovirus genes expressed from the vector of the present invention are derived from the plasmid pBHGlO (Microbix Biosystems, Inc., Toronto, ON), which is presently used to rescue El -deleted adenovirus vectors in conjunction with the El- transcomplementing 293 cell line.
  • pBHGlO Morobix Biosystems, Inc., Toronto, ON
  • a baculovirus vector was rescued in which pBHGlO was flanked by two loxP sites (Hoess and Abremski. 1984).
  • Cre recombinase the pBHGlO sequences are excised from the baculovirus genome, circularized, and replicated to high copy number.
  • Efficient trans-complementation of the adenovirus amplicon vector then occurs, with packaging and release of the recombinant adenovirus amplicon vector from the cell.
  • the pBHGlO-derived adenovirus sequences are prevented from being packaged into infectious virus particles through deletion of the packaging signal.
  • packaging of the adenovirus helper sequences are further prevented through the incorporation of additional sequences, so that the size of the adenovirus genome exceeds the packaging capacity of the adenovirus particle (>108% of the wild-type genome; Bett et al., 1993).
  • the minimal requirements for an adenovirus amplicon vector that is produced consists of a plasmid containing all of the cis elements necessary for replication and packaging (the adenoviral ITRs and the packaging signal), an expression cassette containing the therapeutic gene, inert "stuffer DNA" sequences to achieve a vector of appropriate size for optimal packaging into adenovirus particles, and as few genes, viral or otherwise, as are necessary for the induction of amplification and expression of the helper genome.
  • Optimal packaging size ranges from 27.5 to 37.5 kilobases.
  • the only gene that is absolutely essential in the vector in its initial embodiment is the pTP gene.
  • Recombinant adeno-associated virus (rAAV) vectors based on AAV type 2 can safely transduce various cell types and result in persistent gene expression in vivo.
  • the genome of the wild- type AAV contains two inverted terminal repeats (ITRs) with a packaging signal and encodes for two family of proteins, called rep and cap.
  • ITRs inverted terminal repeats
  • rep and cap For efficient propagation of AAV, some adenovirus products, namely the El, E4, DBP and VA R A, are also required in trans.
  • the rAAV devoid of all viral genes can be propagated if all adenovirus helper functions as well as AAV rep and cap are expressed in trans.
  • the helper function are provided by co-transfection of plasmids encoding all helper genes or by using appropriate vectors expressing rep and cap.
  • An alternative embodiment is the production of recombinant baculovirus-based helper virus for AAV production.
  • This virus can be generated to contain an AAV fragment encoding rep and cap genes, which can be obtained from plasmid pAV2 (ATCC No. 37216). Additionally, the adenovirus E4 region, the DBP-expressing cassette and the adenovirus fragment containing the VA1-RNA gene are incorporated in the recombinant baculovirus genome.
  • the recombinant baculovirus is generated using Bac-to-Bac technology (Gibco Life Technologies) or any other system for generating the recombinant baculoviruses.
  • This virus can be used to produce the rAAV by one of the following ways: a) El -complementing cell line (293 , PerC6 or other) is transfected with the plasmid DNA containing an rAAV vector genome followed by the infection by the B AC. AAV/ Ad hybrid virus, b) E 1 -complementing cell line (293 , PerC6 or other) is co-infected with the helper virus-free rAAV vector (generated as described above or by any other method) and by the
  • B AC. AAV/ Ad hybrid virus, or c) El -complementing cell line (293 , PerC6 or other) is first modified to contain an integrated copy of the rAAV vector genome, and such a modified cell line then will be infected by the B AC/ AAV/ Ad hybrid virus.
  • the adenovirus El region will also be incorporated into the baculovirus, so that the propagation of the rAAV can be performed in any AAV -permissive cell line such as HeLa, Vero or other (available from ATCC).
  • a third alternative is that the recombinant baculovirus-based helper virus for AAV production is generated to contain at least one of the following: an AAV fragment encoding rep or rep and cap genes, the adenovirus El and E4 regions, the DBP-expressing cassette and the adenovirus fragment containing the VA1-RNA gene.
  • the elements listed above and not incorporated into the BAC/AAV hybrid virus must be provided by a cell line, in which the propagation occurs.
  • AAV rep and cap genes in the recombinant Bac/ AAV/Ad hybrid virus can be flanked by two sites for a specific recombinase (such as loxF sites for Cre-recombinase or FRT sites for Flp recombinase) and contain a eukaryotic replication elements (such as Epstein-
  • Barr virus EBN A- 1 gene and oriP sequence or any other viral or non- viral eukaryotic replication origins). This allows excision of the AAV genes from the baculovirus backbone in the presence of corresponding recombinase (Cre recombinase if loxP sites were used), that is provided either the producing cell line or expressed by the Bac/ AAV hybrid virus, and replication of the excised sequences resulting in increase of the copy number of AAV genes in producing cells and in increase of vector yields.
  • recombinase Cre recombinase if loxP sites were used
  • lentivirus vectors such as a vector based on feline immunodeficiency virus (FIV) (available through ATCC with Accession No. VR-233) using the baculovirus-based helper
  • FIV vectors a vector based on feline immunodeficiency virus (FIV) (available through ATCC with Accession No. VR-233)
  • the genetic elements required for production of the FIV vectors are incorporated into a recombinant baculovirus using the Bac-to-Bac technology (Gibco Life Technologies) or any other system for generating the recombinant baculoviruses.
  • the recombinant baculovirus helper virus contains the following genetic elements: a) FIV structural cassette expressing gag and pol genes and driven by strong mammalian promoter (for example, EF-l ⁇ promoter (Invitrogen)), b) the envelop gene (VSV G or any other capable of incorporating into FIV particles), and c) a vector genome, consisting of 5' LTR modified maximize transcription in producing cells, 3 ' LTR and the transgene driven by the FIV LTR promoter or by an internal promoter, such as PGK.
  • strong mammalian promoter for example, EF-l ⁇ promoter (Invitrogen)
  • VSV G envelop gene
  • a vector genome consisting of 5' LTR modified maximize transcription in producing cells, 3 ' LTR and the transgene driven by the FIV LTR promoter or by an internal promoter, such as PGK.
  • the recombinant baculovirus containing all three of the mentioned elements is used to transduce mammalian cells (for example, 293 cells or HepG2 cells or many other). This transduction converts the transduced cells into lentivirus-producing cells, and the resulting lentivirus (FIV) vector will be accumulated in cultural medium.
  • mammalian cells for example, 293 cells or HepG2 cells or many other.
  • the remaining ones can be delivered to the producing cells by other means (other baculoviruses, DNA transfection, etc.) or be provided by the producing cell.
  • VK-33 cells are a derivative cell line obtained through the stable transformation of 293 cells (Microbix Biosystems, Inc., Toronto, ON or ATCC CRL-1573) with the plasmid pTet-on (Clontech Laboratories Inc., Palo Alto CA), which contains both the tetracycline transactivator protein gene and the neomycin resistance gene, and the plasmid pVK58, which was constructed by insertion of an E4-encoding PCR fragment derived from the wild-type Ad 5 genome (ATCC# VR-5) between the Bglll and Nhel sites of the plasmid pBI-EGFP (Clontech Laboratories Inc., Palo Alto CA).
  • the 293 -Cre cell line is described in Lieber et al., 1996. This cell line could be created through stable transformation of 293 cells (Microbix Biosystems, Inc., Toronto, ON or ATCC# CRL-1573) with any one of the plasmids sold by Clontech Laboratories Inc. that contain a Cre expression cassette. The cells are then super-infected with the BV/Ad hybrid helper virus at a multiplicity of infection (moi) of 100-2000. Cre expression from the vector leads to the release of the Ad helper genome from the hybrid helper. When transcomplemented by the El gene products provided by the 293 cells, the helper genome replicates to high copy number and expresses large quantities of the Ad proteins required to package the adenovirus amplicon vector.
  • moi multiplicity of infection
  • any cell type that is transducible by recombinant baculovirus vectors could serve as a suitable cell for the production of recombinant adenovirus amplicon vectors using this system.
  • Non-limiting examples of such cells include A549 (ATCC# CCL-185) or HepG2 (ATCC# HB-8065).
  • This novel system has many advantages over existing systems for the production of high-capacity recombinant adenovirus vectors.
  • the first is that the vectors produced in this system are substantially free of contaminating helper virus.
  • helper virus could provoke immunological or other responses in the host that could jeopardize both the efficacy and safety of the adenovirus vector, this is a significant benefit of the system of the present invention.
  • the second advantage is that the recombinant BV/Ad hybrid helper virus can be utilized to produce adenovirus amplicon vectors in almost any cultured human cell type. By eliminating the requirement for El- transcomplementing 293 cells, contamination of the adenovirus amplicon vector by replication- competent adenovirus (RCA) can be avoided. In addition, by eliminating the need for a specific cell type, both monolayer and suspension cell culturing can be used for the production of the adenovirus amplicon vector, providing greater flexibility and the potential for further cost reductions relative to existing systems.
  • a third advantage of this system is the fact that the recombinant BV/Ad hybrid helper virus can be propagated to high titers easily, cheaply and with a minimum of human labor in cultured insect cells, thereby greatly reducing production costs.
  • Non-limiting examples of suitable insect cells include Sf9 (ATCC# CRL-1711) or S£21 (Invitrogen, Inc.).
  • the production system also can be easily expanded, with a minimum of human labor, to quickly, easily and cheaply produce vectors on a scale required for human clinical trials. This is certainly not the case with system most commonly employed, which requires highly time-consuming serial passaging of the vector in the presence of empirically- determined amounts of helper virus (Parks et al., 1996; Morsy et al., 1998; Schiedner et al., 1998).
  • Another advantage of this production system is that it can be adapted to enable titration of adenovirus amplicon vectors. Current vector production systems can only estimate indirectly the amount of virus DNA that is in a given preparation, but cannot accurately gauge the proportion of this vector DNA that is present in fully-infectious virus particles.
  • the vectors of the present invention can be used in numerous applications such as to treat inoperable cancers, reduce cholesterol, and reverse atherosclerosis.
  • the genes necessary for the treatment of most of these conditions are known, and preliminary data are available showing that transient correction of these phenotypes can be achieved through the delivery of these genes by transient, E 1 -deleted adenovirus vectors. It is believed that long-term corrections of these disease phenotypes will be achieved when these same genes are delivered by adenovirus amplicon vectors, which have now been shown to stably persist for periods exceeding one year in immunocompetent, non-human primates.
  • baculovirus BAC-B4 can be modified by incorporation of an expression cassette for El outside of the loxP-flanked helper adenovirus genome so that production of recombinant adenovirus vectors is no longer restricted to VK-33 cells, but can occur in any other Ad-permissive cell line, such as A549 (ATCC# CCL- 185) or HepG2 (ATCC# HB-8065), transformed to express the tetracycline transactivator protein (Clontech Laboratories Inc., Palo Alto, CA).
  • Ad-permissive cell line such as A549 (ATCC# CCL- 185) or HepG2 (ATCC# HB-8065) transformed to express the tetracycline transactivator protein (Clontech Laboratories Inc., Palo Alto, CA).
  • Ad-permissive cell line such as A549 (ATCC# CCL- 185) or HepG2 (ATCC# HB-8065) transformed to express the
  • the modified baculovirus described above can be further engineered to add an expression cassette for the tetracycline transactivating protein
  • the modified baculovirus described above can be further engineered to add an expression cassette for the tetracycline transactivating protein outside the loxP-flanked helper adenovirus genome so that production of recombinant adenovirus vectors is no longer restricted to cell lines transformed by this gene, but can occur in any cell line.
  • the advantage to this embodiment is similar to that described above.
  • the Cre-recombinase expressing cassette can be controlled by a constitutive mammalian promoter that does not work in insect cells (such as the HCMV immediately early promoter) (Invitrogen Corporation, Carlsbad CA) which can be incorporated into the backbone of the baculovirus described above. Th alteration will remove all restrictions from the cell line, so that any cell line that is sensitive to baculovirus infection and permissive to adenovirus infection such as A549 (ATCC# CCL-185) or HepG2 (ATCC# HB- 8065) can be used for the production of recombinant gutless adenovirus vectors that are free of contaminating helper virus.
  • a constitutive mammalian promoter that does not work in insect cells (such as the HCMV immediately early promoter) (Invitrogen Corporation, Carlsbad CA) which can be incorporated into the backbone of the baculovirus described above. Th alteration will remove all restrictions from the cell line, so that any cell line that
  • This recombinant baculovirus can also be modified by replacing Ad5 sequences with homologous sequences from adenoviruses of different serotypes, such as hAd40 (ATCC# VR-
  • Baculovirus BAC-B4 can be modified by the introduction of the SV40 origin of replication together with the SV40 T-Ag gene controlled by a mammalian promoter into the excisable adenovirus sequence, so that after the Cre-mediated excision, the adenovirus helper genome will be able to replicate using either the S V40 or adenovirus origins of replication.
  • helper-free gutless adenovirus vector could further increase the number of copies of the helper genome per cell and improve the yields of the helper-free gutless adenovirus vector.
  • the 2842 bp BamHI-Sphl fragment of the plasmid pBRSV (ATCC# 45019), containing the SV40 origin of replication and the T-antigen gene could be subcloned into a unique Clal site of pBAC-B4 (VSVG), generating a new plasmid that would express the SV40 T-antigen, and thus, upon Cre-mediated excision from the baculovirus backbone, would be able to replicate episomally to high copy number using the SV40 replication origin.
  • Baculovirus BAC-B4 can be modified to insert a fragment of DNA containing the AAV rep and cap genes (Samulski et al., 1987), which are required for the production of adeno- associated virus (AAV)-based vectors.
  • This modified virus will now contain all of the AAV and Ad genes necessary for the production of helper virus-free AAV-based vectors.
  • baculovirus BAC-B4 (VSVG) was deposited with the American Type Culture Collection (ATCC, Manassas, Virginia) on May 19, 1999 and received ATCC Accession No. PTA-88.
  • nucleic acid manipulations used in practicing the present invention employ methods that are well known in the art, as disclosed in, e.g., Molecular Cloning, A Laboratory
  • Preferred disclosed vectors, restriction enzymes, ligases, nucleases, and the like are commercially available from numerous sources, such as Sigma Chemical Co. (St. Louis, MO),
  • Example 1 The present invention is described below in specific examples which are intended to further describe the invention without limiting the scope thereof.
  • Example 1
  • Method for the construction of a novel baculovirus vector that, by efficiently delivering a replicating, packaging-deficient helper genome into cultured mammalian cells, can be used for the rescue, propagation and titration of a wide range of vectors derived from recombinant viruses, including but not limited to adenoviruses, adeno-associated viruses and lentiviruses.
  • baculovirus-based approach to the rescue, production and titration of virus- derived vectors described in this application could be applied to any number of viruses and cell lines
  • the following example documents the construction of a baculovirus/adenovirus hybrid vector and 293 cell lines.
  • This hybrid can be used to produce high-titers of recombinant adenovirus amplicon vectors (i.e., high-capacity vectors that are free of most, if not all, viral genes) that are substantially free of contaminating helper virus.
  • the first step in the construction process was the design and synthesis of two loxP sites and a polyadenylation signal ( Figure 1).
  • the synthetic ologonucleotides containing two loxP sites and a polyadenylation signal were sequentially introduced into the cloning vector pLitmus29 ( Figures 2A and 2B; New England BioLabs).
  • the resulting plasmid, pVK-LSL-1 ( Figure 2B) was modified by insertion of a reporter gene encoding the Zeo-GFP fusion protein, obtained from plasmid pTracer CMV (Invitrogen Corporation, Carlsbad CA), yielding pBAC- 1.2 ( Figure 3).
  • the Spel-Xbal fragment of this latter plasmid was inserted into the pFastBacl vector (GIBCO Life Technologies, Rockville MD), which is a component of Bac-to-Bac system designed for the quick generation of recombinant baculoviruses ( Figure 4).
  • the resulting plasmid, pBAC-B2-2 was modified further through the insertion of a 32-kb Xbal-Cal fragment derived from pBHGlO (Microbix Biosystems, Inc., Toronto, ON) to produce pBAC-B3-2 ( Figure 5).
  • pBHGl 0 contains all of the genes necessary for the replication and packaging of the adenovirus genome.
  • VSV vesicular stomatitis virus
  • pBAC-B4 recombinant plasmid pBAC-B4
  • Figure 7 recombinant baculovirus [BAC-B4 (VSVG); Figure 7] . This was done according to the standard procedures supplied with the commercially - available Bac-to-Bac system (GIBCO Life Technologies, Rockville MD). Briefly, pBAC-B4
  • VSVG E coli DH1 OBac cells
  • E coli DH1 OBac cells E coli DH1 OBac cells
  • bacmid E coli DH1 OBac cells
  • the bacmid also contains a Tn7 integration site.
  • DHlOBac carries a helper plasmid encoding transposon trans-functions.
  • the transposition that occurs in the DHlOBac cells results in incorporation of the entire insert flanked with Tn7L and Tn7R from pBAC-B4 (VSVG) into the bacmid.
  • This resulting recombinant bacmid is then isolated from the E. coli and used to transfect the insect cell line Sf9 (ATCC# CRL-1711).
  • pBacB4 was digested with Pad, which cuts at a unique site located within the ⁇ 3 deletion that is present in the fragment of the Ad genome contained in the Bac/ Ad hybrid vector.
  • a 5.8 kb Avrll fragment obtained from the human PAH gene was isolated and subcloned into the Pad site.
  • the PAH gene fragment can be of no specific source or sequence composition.
  • the Avrll and Pad ends in these fragments were joined together using a synthetic Avrll-Pad adapter consisting of following two oligonucleotides: 5'- CTAGGCTCACTGACGCCATAAT- 3 ' and 5 ' - TATGGCGTCAGTGAGC-3 ' .
  • the product of this ligation was the plasmid pBacB 10.
  • the plasmid pBacB 10 then was used to generate a baculovirus/adenovirus hybrid using the Bac- to-Bac technology (GIBCO Life Technologies) as previously described.
  • the El region (positions 499-3510) was subcloned in the EcoR V site of the cloning vector pZero-2 (Invitrogen) using PCR cloning with the following two primers: 5'- CTCTTGAGTGCCAGCGAGTAGAGTT-3' and 5'-CTCAATCTGTATCTTCATCGCTAG-3' to generate pZeroEl .
  • the structural integrity of the cloned fragment was confirmed by sequencing.
  • the expression vector pREP7 (Invitrogen) was modified by replacing the Xbal- Acc65I fragment containing the RSV promoter with a fragment containing the PGK promoter that was derived from the commercially available plasmid pMSCVneo (Clontech).
  • the resulting plasmid was dubbed pREP-PGK.
  • An Xbal/Spel fragment containing El was excised from pZero-E 1 and inserted into a unique Nhel site in pREP-PGK to generate the plasmid pPGK-E 1.
  • the whole PGK-driven expression cassette was then excised from pPGK-El by Sail digestion.
  • a Sall-Clal adapter consisting of the two oligonucleotides 5'-TCGAATGACGGATCCAT-3' and 5'-CGATGGATCCGTCAT-3' was attached to the ends of the fragment.
  • the modified fragment then was inserted into a unique Clal site of pBAC-B i 0 to produce pBAC-B9.
  • the recombinant baculovirus BAC-B9 was generated from pBAC-B9 using the same Bac-to-Bac technology (GIBCO Life Technologies) as previously described.
  • the adenovirus cis elements necessary for virus replication and packaging are located at the termini of the adenovirus genome.
  • the regions are referred to as inverted terminal repeats or ITRs.
  • pAVec4-2 was then digested with Sail and an expression cassette consisting of the bacterial lacZ gene driven by the human elongation factor l ⁇ (EFla) promoter, excised from plasmid pAVec2, was inserted to form the plasmid pAVec5 ( Figure 10).
  • PAvec5 was then digested with Pmel and Ascl, and an expression cassette in which the Cre recombinase gene (Life Technologies, Inc., Rockville MD) was driven by the tetracycline-inducible TRE promoter
  • this vector forms a linear molecule in which the two adenovirus ITRs flank a lacZ expression cassette, a tet inducible Cre expression cassette, and a 19 kb "stuffer" DNA fragment obtained from an intronic region of the human phenylalanine hydroxylase gene.
  • Cre expression from this vector is driven by the tet-inducible promoter system, which has an absolute requirement for the tet transactivator protein, Cre expression from this vector can occur only in cells that express this protein, such as VK-33.
  • Mammalian VK-33 cells (293 cells transformed with expression cassettes for the "tet- on” transactivator and tet-inducible GFP) were grown at 37 ° C in 60mm dishes to approximately 70-80% confluence.
  • the growth media (90% MEM + 10% Fetal Bovine Serum + 1% Penicillin/Streptomycin/Fungizone; all obtained from GIBCO) was removed and the cells were washed with phosphate-buffered saline (PBS) or fresh serum-free MEM (GIBCO).
  • PBS phosphate-buffered saline
  • GIBCO fresh serum-free MEM
  • Adsorption of the virus was performed by incubating the cells with the virus suspension for 2 hours at 37 °C in the presence of 5% CO 2 . The cells were rocked gently every 15 minutes during the infection period. After two hours of incubation, the baculovirus inoculum was removed and fresh MEM growth media was added. The infected cells then were immediately transfected with the adenovirus vector DNA, which was prepared as follows. Purified DNA of the pAVecl3 plasmid was linearized by digestion with the restriction enzyme Swal and purified using the QIAquick Gel Extraction Kit (Qiagen Inc). This digestion released the adenovirus ITRs, making them functional for initiating adenovirus replication. The purified plasmid DNA was transfected into 293 cells using either a standard calcium phosphate procedure or the Effectene Transfection Reagent (Qiagen Inc.). At 24 hours after transfection, the transfection media was removed and replaced by fresh media.
  • the cells were harvested and a crude lysate of the transfected cells was prepared.
  • One "blue-forming unit” is the amount of a ⁇ -galactosidase-expressing adenovirus vector necessary to produce one 293 cell that is positive for ⁇ -galactosidase expression after transduction of the cells by the vector followed by X-gal staining.
  • Example 5 Titration of the adenovirus vector.
  • the crude lysates from the propagation procedure described above were clarified by centrifugation at 700 rpm for ten minutes at 4°C and filtered through 0.8 micron filter units. 0.1 ml of the crude extract was used for infection of 293 cells.
  • E. coli ⁇ -galactosidase cells infected by serial dilutions of the vector could be identified by staining with 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal) dissolved to a concentration of
  • phosphate-buffered saline Life Technologies, Inc., Rockville MD
  • the cells were fixed by a five minute exposure to 0.5%) glutaraldehyde diluted in PBS at 20 hours after infection, rinsed several time with PBS, and then stained by exposure to the X-gal solution for five hours at 37°C.
  • the number of positively-stained cells multiplied by the dilution factor corresponded to the infections titer of the recombinant adenovirus vector produced by this method.
  • helper-dependent adenovirus vector system removal of helper virus by Cre- mediated excision of the viral packaging signal. Proc.Natl.Acad.Sci. U.S.A. 93, 13565- 13570.
  • helper-dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging. J. Virol. 71, 3293- 3298.
  • Adenovirus type 5 precursor terminal protein-expressing 293 and HeLa cell lines J. Virol. 69, 4079-4085.

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Abstract

La présente invention porte sur un virus assistant hybride du baculovirus recombinant qui comprend des séquences du baculovirus, des secondes séquences structurales virales et des dernières séquences virales. L'invention porte également sur un procédé de production de vecteurs n'exprimant plus de gènes viraux et qui ne risquent pratiquement pas d'être contaminés par le virus assistant. Cette invention porte en outre sur un baculovirus recombinant BAC-B4 (VSVG).
PCT/US2000/014785 1999-05-28 2000-05-26 Nouveau vecteur hybride du baculovirus/adenovirus utile pour la sauvegarde, la production et le titrage des vecteurs d'amplicon d'adenovirus haute capacite WO2000073424A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002064170A1 (fr) * 2001-02-09 2002-08-22 Children's Medical Center Corporation Methode pour traiter le cancer et augmenter le taux d'hematocrite
WO2003029448A1 (fr) * 2001-09-29 2003-04-10 Yun, Chae-Ok Adenovirus recombinant a effet therapeutique ameliore et composition pharmaceutique comprenant ledit adenovirus recombinant
WO2005112541A3 (fr) * 2004-05-20 2006-03-23 Proyecto Biomedicina Cima Sl Vecteur hybride adenovirus-alphavirus destine a l'administration de maniere efficace et a l'expression de genes therapeutiques dans des cellules tumorales
CN112852746A (zh) * 2021-02-04 2021-05-28 中吉智药(南京)生物技术有限公司 基于Cre重组酶诱导的大规模慢病毒基因药物制备系统及方法
CN114787345A (zh) * 2019-10-23 2022-07-22 吉恩迈生物科技有限公司 基于辅助质粒的无肠腺病毒生产系统

Citations (2)

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WO1998022607A1 (fr) * 1996-11-22 1998-05-28 Rhone-Poulenc Rorer S.A. Procede de production de virus recombinants
WO1998055640A1 (fr) * 1997-06-04 1998-12-10 Oxford Biomedica (Uk) Limited Particules de vecteur de retrovirus produites dans un systeme d'expression de baculovirus

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998022607A1 (fr) * 1996-11-22 1998-05-28 Rhone-Poulenc Rorer S.A. Procede de production de virus recombinants
WO1998055640A1 (fr) * 1997-06-04 1998-12-10 Oxford Biomedica (Uk) Limited Particules de vecteur de retrovirus produites dans un systeme d'expression de baculovirus

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002064170A1 (fr) * 2001-02-09 2002-08-22 Children's Medical Center Corporation Methode pour traiter le cancer et augmenter le taux d'hematocrite
WO2003029448A1 (fr) * 2001-09-29 2003-04-10 Yun, Chae-Ok Adenovirus recombinant a effet therapeutique ameliore et composition pharmaceutique comprenant ledit adenovirus recombinant
US9175309B2 (en) 2001-09-29 2015-11-03 Industry-University Cooperation Foundation Hanyang University Recombinant adenovirus with enhanced therapeutic effect and pharmaceutical composition comprising said recombinant adenovirus
WO2005112541A3 (fr) * 2004-05-20 2006-03-23 Proyecto Biomedicina Cima Sl Vecteur hybride adenovirus-alphavirus destine a l'administration de maniere efficace et a l'expression de genes therapeutiques dans des cellules tumorales
ES2292271A1 (es) * 2004-05-20 2008-03-01 Proyecto De Biomedicina Cima, S.L. Un vector hibrido adenovirus-alfavirus para la administracion eficaz y expresion de genes terapeuticos en celulas tumorales.
US7850957B2 (en) 2004-05-20 2010-12-14 Proyecto De Biomecdicina Cima, S.L. Adenovirus/alphavirus hybrid vector for the effective administration and expression of therapeutic genes in tumour cells
CN114787345A (zh) * 2019-10-23 2022-07-22 吉恩迈生物科技有限公司 基于辅助质粒的无肠腺病毒生产系统
CN112852746A (zh) * 2021-02-04 2021-05-28 中吉智药(南京)生物技术有限公司 基于Cre重组酶诱导的大规模慢病毒基因药物制备系统及方法

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