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WO2001025464A1 - Materiaux et procedes pour la production simplifiee d'aav - Google Patents

Materiaux et procedes pour la production simplifiee d'aav Download PDF

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Publication number
WO2001025464A1
WO2001025464A1 PCT/US1999/022789 US9922789W WO0125464A1 WO 2001025464 A1 WO2001025464 A1 WO 2001025464A1 US 9922789 W US9922789 W US 9922789W WO 0125464 A1 WO0125464 A1 WO 0125464A1
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Prior art keywords
aav
host cell
replication
adenovirus
construct
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Inventor
Varavani Dwarki
Martha Ladner
Jaime Escobedo
Shang-Zhen Zhou
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Novartis Vaccines and Diagnostics Inc
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Chiron Corp
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Priority to AU14414/00A priority Critical patent/AU1441400A/en
Priority to PCT/US1999/022789 priority patent/WO2001025464A1/fr
Priority to JP2001528615A priority patent/JP2003511038A/ja
Priority to EP99974094A priority patent/EP1220938A1/fr
Priority to CA002386722A priority patent/CA2386722A1/fr
Publication of WO2001025464A1 publication Critical patent/WO2001025464A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention is directed to a method for the production of recombinant AAV virions containing a gene of interest. More particularly, the present invention is directed to a method for producing rAAV virions free of wild-type AAV and helper virus. The present invention is useful because it produces a highly pure rAAV virion suitable for evaluation of gene therapy protocols and/or use in gene therapy.
  • Adeno-associated virus is a non-pathogenic, replication-defective parvo virus that has a biphasic life cycle.
  • AAV Adeno-associated virus
  • the AAV genome integrates into the hot cell's genome to establish a latent infection.
  • a helper virus such as adeno virus, herpes simplex virus or vaccinia virus
  • the AAV genome is rescued from latency and is reproduced to establish a lytic infection. See Muzyczka, Curr. Topics Microbiol Immunol. 158 (1992) 97-129; Berns and Linden, BioEssays 17 (1995) 237-245.
  • the helper virus is known to provide the functions needed for AAV replication.
  • AAV stably integrates into human chromosome 19 site specifically. See Kotin, Proc. Natl. Acad. Sci. 87 (1990) 2211-15; Samulski, EMBO J 10 (1991) 3941-50.
  • the AAV genome consists of a 4.7 kb linear, single-stranded, DNA molecule with 145 bp inverted terminal repeats at each end. The remaining, non-repeated sequences encode for the viral proteins, called rep and cap, involved in virus replication and packaging.
  • the AAV ITRs are the only cis elements required for the viral replication, packaging and integration; the rep and cap functions can be provided in trans. See McLaughlin, J. Virol.
  • Recombinant AAV (rAAV) vectors are attractive vehicles for human gene therapy because the vectors do not require AAV coding sequences to be expressed viral coding sequences, the viruses (viral particles) is capable of infecting non-dividing and dividing cells efficiently, it has a broad host range and the virions have high physical stability. See Carter, Curr. Opin. Biotech 3 (1992) 533-39; Bachman, Intervirology 11 (1979) 248-54. The most widely used method of generating rAAV particles is called the invention/transfection method.
  • This method involves transfection of host cells, typically 293 cells, with AAV vector plasmid (i.e., plasmid carrying the gene of interest bounded by the AAV ITRs) and with helper plasmid (i.e., plasmid providing the AAV helper functions rep and cap but lacking the ITRs) and infection with adenovirus or herpes virus.
  • AAV vector plasmid i.e., plasmid carrying the gene of interest bounded by the AAV ITRs
  • helper plasmid i.e., plasmid providing the AAV helper functions rep and cap but lacking the ITRs
  • the resulting AAV vector preparations still may contain low levels of infectious helper virus and proteins that may contribute to the immunogenicity of the composition and present a potential hazard for human administration.
  • the helper virus is a pathogenic virus and poses a health risk to laboratory personnel involved in the manufacturing process.
  • the large amount of helper virus particles and proteins generated during the infection process makes it difficult to achieve high levels of purity.
  • Heat treatment can inactivate infectious adenovirus, but the treatment leads to a 30-40% drop in the tier of functional rAAV virions and it has been difficult to remove all of the adenoviral proteins, even by multiple rounds of CsCl gradient purifications.
  • the "accessory" functions that may be necessary to support AAV replication include adenoproteins E2a and E4, as well as VA I RNA.
  • An alternative method of producing rAAV is disclosed in PCT Patent Publication WO 97/17458, published May 15, 1997.
  • the accessory functions capable of supporting rAAV virion production are provided in the from of one or more vectors containing the adenovirus VA sequence, the adenovirus E4 ORF6 coding region and/or the adenovirus E2a 72 kD coding region.
  • the present invention is directed to a method for producing replication- defective AAV virions that avoids the production of live adenovirus, the necessity of monitoring for multiple adenoviral accessory functions, and the need for cumbersome purification protocols. More particularly, the present invention is directed to a method for producing replication-defective recombinant AAV virions substantially free of wild- type AAV and helper adenovirus, comprising: a.
  • an AAV vector that is free of AAV coding sequences and that comprises a heterologous gene operatively positioned between two AAV ITRs, (ii) a replication-defective AAV helper construct having at least one gene encoding an AAV capsid protein, and (iii) an adenoplasmid accessory construct having a full adenoviral genome that either lacks a packaging signal or that contains sufficient additional nucleotides to be rendered unpackagable, to produce a transformed host cell; b. culturing the transformed host cell to produce replication- defective recombinant AAV virions having the heterologous gene; and c. lysing the cultured host cell to obtain replication-defective recombinant AAV virions substantially free of wild-type AAV and adenovirus particles.
  • the host cells that are suitable for use in the method of the present invention are mammalian host cells, preferably human host cells.
  • the invention is directed to a method of producing purified recombinant AAV virions, comprising: a. introducing into a suitable host cell (i) an AAV vector that is free of AAV coding sequences and that comprises a heterologous gene operatively positioned between two AAV ITRs, (ii) a replication-defective AAV helper construct having at least one gene encoding an AAV capsid protein, and (iii) an adenoplasmid accessory construct having a full adenoviral genome that either lacks a packaging signal or that contains sufficient additional nucleotides to be rendered unpackagable, to produce a transformed host cell; b.
  • step (c) culturing the transformed host cell to produce replication- defective recombinant AAV virions having the heterologous gene; c. lysing the cultured host cell to obtain replication-defective recombinant AAV virions substantially free of wild-type AAV and adenovirus particles; d. applying the lysate of step (c) to a column comprising sulfonated cellulose; and e. recovering purified replication-defective recombinant AAV virions substantially free of host cell proteins and host cell debris.
  • the lysate from step (c) is subjected to cesium chloride equilibrium gradient centrifugation, and the purified replication-defective rAAV virions containing the heterologous gene are recovered.
  • the AAV vector, the replication- defective AAV helper construct and the adenoplasmid accessory construct are combined either simultaneously or sequentially.
  • the advantage of the triple transfection protocol utilized in the methods of the present invention is the significant purity of rAAV preparations after CsCl gradient purification.
  • the Western plot analysis shows that the triple transfection method, while producing equivalent amounts of rAAV particles as the standard transfection/infection protocol, results in much lower adenovirus protein production in both the initial lysates and in the final purified product.
  • the adenoplasmid accessory construct used in the methods lacks a packaging signal, there are no adenovirus particles in the purified material.
  • This protocol also eliminates the health and safety concerns raised by the use of live adenovirus and allows production of rAAV particles in a safe manner. Importantly, the method simplifies the downstream purification process, thereby enabling relatively efficient and economical large-scale manufacturing.
  • the present invention is directed to a method for producing replication-defective recombinant AAV virions substantially free of wild- type AAV and helper adenovirus, comprising: a. introducing into a suitable host cell (i) an AAV vector that is free of AAV coding sequences and that comprises a heterologous gene operatively positioned between two AAV ITRs, (ii) a replication defective AAV helper construct having at least one gene encoding an AAV capsid protein, and (iii) an adenoplasmid accessory construct having a full adenoviral genome that either lacks a packaging signal or that contains sufficient additional nucleotides to be rendered unpackagable, to produce a transformed host cell; b.
  • the above described method further comprise the steps of: d. applying the lysate of step (c) to a column comprising sulfonated cellulose; and e. recovering purified replication-defective recombinant AAV virions substantially free of host cell proteins and host cell debris.
  • the host cells that are suitable for use in the method of the present invention are mammalian host cells, preferably human host cells.
  • the AAV vector, replication-defective AAV helper construct and adenoplasmid accessory construct of step (a) of the method of the present invention are prepared using conventional methods of virology, molecular biology, microbiology and recombinant DNA techniques. Such techniques are well known and explained fully in the literature, including, for example, in Sambrook, Molecular Cloning: A Laboratory Manual (Current Ed.); DNA Cloning: A Practical Approach (D. Glover, ed.); Oligonucleotide Synthesis (Current Ed., N. Gait, ed.); Nucleic Acid Hybridization (Current Ed., B. Hames and S.
  • Gene transfer, gene therapy or gene delivery refer to methods, techniques or systems for reliably inserting into a host cell a heterologous or a foreign DNA or a DNA not normally expressed.
  • the resultant insertion can be by integration of transferred genetic material into the host cell genomic DNA, by extrachromosomal replication and expression of transferred replicons or in a non-integrated manner.
  • Vector means any genetic element that is capable of replication when associated with the proper control elements and that can transfer DNA or RNA sequences between cells. Examples include plasmids, phages, transposons, cosmids, chromosomes, viruses, and virions and include cloning and expression vehicles and viral vectors.
  • the replication-defective AAV virions produced by the method of the present invention comprise a gene (DNA) encoding a therapeutic protein operably positioned between a pair of adeno-associated virus inverted terminal repeats ("AAV ITRs").
  • AAV ITRs are art-recognized regions found at each end of the AAV genome that function together in cis as recognition signals for DNA replication and for packaging the AAV vector into an AAV coat.
  • the nucleotide sequences of the AAV ITR regions for the various AAV serotypes i.e., AAV-1 to AAV-7 are known in the art and vary in size with the serotpe. Typically, the AAV ITRs range in size from about 125-145 bp.
  • the AAV ITRs of Applicants' recombinant replication-defective retrovirion need not be identical to the nucleotide sequence of the native, i.e., wild-type, sequence, but may be altered by insertion, deletion or substitution of nucleotides.
  • the two AAV ITRs may be derived from any of the AAV serotypes, for example AAV-1, AAV -2, AAV-3, AAV-4, AAV- 5 and AAV-7, and need not be identical to or derived from the same serotype, so long as they permit integration of the heterologous sequence of interest into the recipient cell genome when AAV rep gene products are present in the cell.
  • the AAV rep coding region is the art-recognized region of the AAV genome that encodes the proteins required for replication of the viral genome and for insertion of the viral genome into a host genome during latent infection.
  • the rep coding region includes at least the four genes encoding the two long forms of rep (rep 78 and rep 68) and the two short forms of rep (rep 52 and rep 40).
  • the rep coding region may be derived from any AAV serotype or from a functional homologue such as the human herpes virus 6 rep gene.
  • the region need not include all of the native sequence, but may be altered by insertion, deletion or substitution of nucleotides, so long as the sequence that is present provides for sufficient integration when expressed in a suitable recipient cell.
  • the AAV vector and virions utilized in the present invention lack one or more of the rep proteins so as to render it replication- defective. More preferably, the
  • the AAV vector of the present invention lacks all four of the rep proteins.
  • the AAV cap coding region is the art-recognized region of the AAV genome that encodes the capsid or coat proteins, VP1, VP2 and VP3, that package the viral genome. For more details, see, for example, Muzyczka, Current Topics in Microbiol. 158 (1992) 97-129 and Kotin, Human Gene Therapy 5 (1994) 793-801.
  • the cap coding region may be derived from any AAV serotype or from a functional homologue.
  • the cap coding region may be altered by insertion, deletion or substitution of nucleotides, so long as the sequence present provide for sufficient packaging when expressed in a suitable recipient cell.
  • cap coding region is preferably not included in the AAV vectors and the replication-defective AAV virions employed in the present invention, it needs to be included in a helper vector that is expressed in a packaging cell that recognizes and packages the ITRs and the gene(s) positioned therebetween.
  • AAV vector means a vector derived from an adeno-associated virus serotype that includes at least those sequences required in cis for replication and packaging, for example, a pair of functional ITRs flanking a heterologous (i.e., non-AAV) nucleotide sequence.
  • any AAV vector of any serotype can be employed in the method of this invention.
  • vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, PCT Patent Publication WO 93/09239 or simply a pair of AAV-7 ITRs having one or more genes operatively positioned therebetween.
  • the AAV ITRs employed in the vectors and virions of the present invention may be the native (wild-type) AAV ITRs or they may be modified. If the ITRs are modified, they are preferably modified at their D-sequences.
  • the native D- sequences of the AAV ITRs are sequences of twenty consecutive nucleotides in each AAV ITR (i.e., there is one sequence at each end) which are not involved in HP formation.
  • the D-sequences of the ITRs are modified by the substitution of nucleotides, such that 5-18 native nucleotides, preferably 10-18 native nucleotides, most preferably 10 native nucleotides, are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native, i.e., exogenous, nucleotides.
  • One preferred sequence of five native nucleotides that are retained is 5' CTCCA 3'.
  • the exogenous or non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence at the same position.
  • appropriate replacement nucleotides for native D-sequence nucleotide C are A, T and G
  • appropriate replacement nucleotides for native D-sequence nucleotide A are T, G and C.
  • the construction of four such AAV vectors is disclosed in United States Serial No. 08/921,467, filed September 2, 1997.
  • Other employable exemplary vectors are pWP-19 and pWN-1, both of which are disclosed in Nahreini, Gene 124 (1993) 257-62.
  • Another example of such an AAV vector is psub201 as disclosed in Samulski, /. Virol. 61 (1987) 3096.
  • Other suitable AAV vectors are the Double-D ITR vector.
  • an AAV vector employable in the methods of this invention is SSV9AFABTKneo, which contains the ⁇ -fetoprotein (AFP) enhancer and albumin promoter and directs expression of the herpes simplex thymidine kinase (TK) gene predominantly in the liver. Its structure and method for making are disclosed in Su, Human Gene Therapy 7 (1996) 463-70).
  • the replication-defective AAV vectors are packaged into empty AAV capsids to produce the replication-defective AAV virions helper viruses employed in the methods of the present invention.
  • To package the replication-defective AAV vectors which are typically one or more genes positioned between a pair of ITRs, one employs a helper construct or helper virus that has AAV-derived coding sequences that function in trans to enable AAV replication, and that include the AAV rep and cap sequences.
  • the helper virus has AAV coding sequences but lacks the AAV ITRs and thus are not packaged in the capsids that are produced. This helper virus then provides for transient expression of the AAV rep and cap genes missing from the AAV vector.
  • AAV helper constructs For greater details, including exemplary AAV helper constructs, see, for example, Samulski, J. Virol. 63 (1989) 3822-28; McCarty, J. Virol 65 (1991) 2936-45 and U.S. Patent No. 5,139,941.
  • One such AAV helper construct comprises pKS rep/cap, which contains the genes encoding the AAV-2 rep and cap polypeptide sequences.
  • Additional examples of helper viruses, constructs and functions that can be employed include the plasmids pAAV/Ad and pIM29+45 (see Samulski, J. Virol. 63 (1989) 3822-28 and McCarthy, J. Virol 65 (1991) 2936-45) and those disclosed in U.S. Patent No. 5,622,856.
  • Accessory functions and accessory function vectors are non-AAV derived functions and vectors containing sequences encoding such functions upon which AAV is dependent for its replication.
  • Such accessory functions can be derived or obtained from any of the known helper viruses, such as adenovirus, herpesvirus (except herpes simplex virus type-1) and vaccinia virus and include moieties and/or sequences involved in activation of gene transcription, DNA replication, synthesis of cap expression products and capsid assembly. See, for example, Carter, "Adeno- Associated Virus Helper Functions" in CRC handbook of Parvoviruses, Vol. I (1990) (P. Tijssen, ed.); Muzyczka, Current Topics in Microbiol.
  • the adenoplasmid accessory constructs employed in the method of the present invention comprise adenovirus plasmid DNA rendered unable to be packaged into adenovirus particles, for example, the adenoplasmid accessory constructs lack the adenovirus packaging signals required for production of infectious adenovirus particles but contain the adenovirus genes required for rAAV virion production. Alternatively, the adenoplasmid accessory construct are rendered to large to be packaged by the additional heterologous sequences, plasmids or other constructs. Use of such adenoplasmid accessory constructs results in the generation of rAAV virions having similar infectious activity and packaging efficiency as compared to prior art methods.
  • One such construct comprises an adenovirus type 5 plasmid which contains all of the DNA sequence of the serotype 5 adenovirus but lacks the serotype 5 packaging signal which lies between base pairs 194 through 398. See Hearing and
  • An alternative construct comprises an adenovirus type 2 plasmid which contains all of the DNA sequence of the serotype 2 adenovirus but lacks the serotype 2 packaging signal, which lies in a similar location, analogously, constructs comprising any other adenovirus serotype may be used, as long as the packaging signal is removed.
  • exemplary serotypes which can be employed include serotypes Adl, Ad6, Ad8, Ad9, AdlO, Adll, Adl2, Adl3, Adl5, Adl7, Adl9, Ad20, Ad22, Ad23, Ad24, Ad25, Ad26, Ad27, Ad28, Ad29, Ad30, Ad32, Ad33, Ad367, Ad37, Ad38, Ad39, Ad40, Ad41 and Ad42.
  • Another such construct comprises an adenovirus 5 plasmid which contains heterologous sequences making it too large to be packaged.
  • adenovirus 5 plasmid which contains heterologous sequences making it too large to be packaged.
  • plasmid pBR322 or one of its derivatives at base pair (bp) 1339 (3.7 mu) in the Adenovirus 5 sequence makes the resulting viral genome too large to package.
  • An alternative construct comprises an adenovirus type 2 plasmid which contains all of the DNA sequence of the serotype 2 adenovirus but which contains an insertion of pBR322 at a similar location.
  • constructs comprising any other adenovirus serotype may be used, as long as the construct is rendered too large to be packaged.
  • Exemplary serotypes which can be employed include Adl, Ad6, Ad8, Ad9, AdlO, Adll, Adl2, Adl3, Adl5, Adl7, Adl9, Ad20, Ad22, Ad23, Ad24, Ad25, Ad26, Ad27, Ad28, Ad29, Ad30, Ad32, Ad33, Ad367, Ad37, Ad38, Ad39, Ad40, Ad41 and Ad42. See Fields Virology, 2 (Fields and Knipe, eds.), 1990.
  • the adenoplasmid accessory constructs can alternatively include one or more poly nucleotide homologues having substantially identical functions as the native sequence replacing the native adenoviral sequences.
  • Such homologues may be derived from a different adenovirus serotype (since the nucleotide sequence of the adenovirus type 5 genome is believed to be 99% homologous to the adenovirus type-2 genome), from another accessory virus or from another suitable source.
  • the adenoplasmid can be in the form of a circularized or linearlized
  • DNA fragment capable of replication when associated with appropriate control elements and which can be transcribed and expressed in a host cell can be engineered using conventional recombinant techniques.
  • the adenoplasmid can be assembled by inserting adenovirus nucleotide sequences (either derived from an adenovirus genome, from an adenovirus vector or chemically synthesized) having accessory functions into a vector construct in any desired order, for example, by ligating restriction fragments into the plasmid using poly linker oligonucleotides. The sequences can then be excised from the vector and inserted into an appropriate expression plasmid using techniques well known in the art.
  • plasmid pBHGlO is a bacterial plasmid that contains the Ad5 sequences required to produce infectious virus upon transfection of 293 cells but lacks the packaging signal, base pairs 194 through 358 needed to encapsidate viral DNA, since it contains a deletion of Ad5 sequences from bp 188 through bp 1339 (0.5 through 3.7 mu).
  • An ampicillin resistance gene and bacterial origin of replication substitute for the deleted Ad5 sequences and the plasmid also lacks the Ad5 E3 region, from 78.3 through 85.8 map units (mu). Details of its structure and its construction is described in Bett, Proc. Natl. Acad. Sci. 91 (1994) 8802-06. It is available from Microbix Biosystems, Inc., Ontario, Canada.
  • adenoplasmid constructs which can be employed include plasmid pBGl l, which is non-infectious when transfected into 293 cells, since it contains the same deletion of Ad5 packaging signal sequences as pBHGlO but lacks the Ad5 E3 region from 77.5 through 86.2 mu. Details of its structure and its construction is described in Bett, Proc. Natl. Acad. Sci. 91 (1994) 8802-06.
  • Another exemplary adenoplasmid construct contemplated for use in the invention is pJM17.
  • Construct pjM17 contains an insertion of a derivative of plasmid pBR322 at base pair (bp) 1339 (3.7 mu) in its Adenovirus 5 sequence, which makes the resulting viral genome too large to package. See Bett, Proc. Natl. Acad. Sci. 91 (1994) 8802-06 and McGrory, Virology 163 (1988) 614-17.
  • the construct pJM17 was derived from pFG140 (see Graham, EMBO J 3 (1984) 2917-22), such that vectors generated with pJM17 also contain the same deletion(s) and substitution(s) present in the E3 region of its Ad5 sequence as is present in the vector dl 309 (see Jones, Cell 17 (1979) 683-89).
  • the adenoviral gene regions in the adenoplasmid are operably linked to control sequences that direct their transcription or expression.
  • control sequences can comprise the adenoviral control sequences associated with the gene regions of the wild-type adenoviral genes or can comprise heterologous control sequences, such as heterologous promoters derived from mammalian or viral genes.
  • heterologous promoters include adenoviral promoters from homologous adenoviruses (i.e., from a different adenoviral serotype), the SV40 early promoter, the mouse mammary tumor virus LRT promoter; the adenovirus major late promoter; a herpes simplex virus promoter, a cytomegalovirus promoter, a rous sarcoma virus promoter, synthetic promoters or hybrid promoters. Such promoters are commercially available.
  • the adenoplasmid accessory construct can also include one or more selectable markers. Suitable markers are sequences that confer antibiotic resistance or sensitivity, impart color, or change the antigenic character of transfected cells when grown in suitable selective media. Exemplary selectable markers include the hygromycin B resistance gene, the ampicillin resistance gene and the kanamycin resistance gene. Other suitable markers are well known in the art.
  • suitable host cells may provide one or more of the necessary accessory functions.
  • the human cell line 293 is a human embryonic kidney cell line that has been transformed with adenovirus type 5 DNA fragments so that it expresses adenovirus Ela and Elb genes.
  • an adenoplasmid is provided which lacks the packaging signal and the Ela and Elb gene regions. Upon transfection into a 293 hot cell, the adenoplasmid will provide the accessory functions supportive of rAAV virion production, without the formation of infectious adenovirus particles.
  • the adenoplasmids of the invention can be employed in methods for the production of rAAV virions.
  • One such method entails introducing into a suitable host cell an AAV vector, an AAV helper construct and an adenoplasmid accessory construct into the host cell.
  • the adenoplasmid accessory plasmid is composed of adenovirus plasmid DNA lacking packaging signal sequences as described above.
  • the host cell is cultured to produce crude rAAV virions and lysed.
  • the resulting cell lysate is applied to a chromatographic column or a cesium chloride density gradient and the purified rAAV virions are recovered from the column.
  • the heterologous nucleotide sequence(s) that are inserted into the replication-defective AAV vectors and virions of the present invention encode one or more therapeutic agents that include a therapeutic protein, polypeptide, antisense RNA or a ribozyme, or a combination thereof.
  • the vectors or virions contain from one to two therapeutic agents that are native or non-native to the recipient cell but which have a desired biological or therapeutic effect.
  • the heterologous nucleotide sequences that are introduced into the replication-defective AAV vectors and virions of the present invention include a gene that encodes a therapeutic protein or polypeptide, preferably a human protein or polypeptide.
  • therapeutic proteins and polypeptides that would be suitable for expression in the methods of the present invention include the LDL receptor, Factor VIII, Factor IX, phenylalanine hydroxylase, ornithine transcarbamylase, or ⁇ l-antitrypsin; a cytokine, such as interleukin (IL)-l, IL-2 IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14 and IL-15, ⁇ - interferon, ⁇ -interferon, the ⁇ -interferons, tumor necrosis factor CD3, ICAM-1, LFA- 1, or LFA-3, a chemokine including RANTES l ⁇ , or MlP-l ⁇ (see Cocci, Science 70 (1996) 1811-15); a colony stimulating factor, such as G-CSF, GM-CSF and M-CSF; growth factors such as IGF-1 and IGF
  • nucleotide coding sequences for these proteins and polypeptides are already known in the art. Even more sequences expressible in the methods and compositions of the invention include Protein S and Gas6, thrombin, Coagulation Factor Xa, acidic fibroblast growth factor (FGF-1), basic FGF (FGF-2), keratinocyte growth factor (KGF), TGF, platelet derived growth factor (PDGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and HGF activators, PSA, nerve cell growth factor (NCGF), glial cell derived nerve growth factor (GDNF), vascular endothelial growth factor (VEGF), Arg-vasopressin, thyroid hormones asoxymethane, triodothyronine, LIF, amphiregulin, soluble thrombomodulin, stem cell factor, osteogenic protein 1, the bone morphogenic proteins, MFG, MGSA, heregulins and melanotropin.
  • FGF-1 acidic fibroblast growth factor
  • Preferred proteins include but are not limited to erythropoietin, thrombopoietin (G-CSF), Factor VIII, Factor IX, Factor Xa, human growth hormone, leptin and IL-2, the DNA sequences of which are all known in the art, particularly the human DNA sequences.
  • the AAV vector may also include control sequences, such as promoters and poly adenylation sites, selectable markers, reporter genes, enhancers and other control elements permitting for transcription induction and/or selection.
  • control sequences such as promoters and poly adenylation sites, selectable markers, reporter genes, enhancers and other control elements permitting for transcription induction and/or selection.
  • Such AAV vectors can be constructed using techniques well known in the art.
  • the replication-defective AAV helper construct is used to complement the AAV vector by providing those genes, which are necessary for the production of AAV virions, particularly the cap structural genes. Suitable helper constructs having complementing functions are well known in the
  • the AAV vector, the replication-defective AAV helper construct and adenoplasmid accessory construct are introduced into the host cell either simultaneously or sequentially, using any of the well known, art recognized transfection techniques, for example, by calcium phosphate co-precipitation.
  • Culture conditions include incubating in the range of 35°- 40°C for approximately 48 to 120 hours.
  • the cells are collected and a lysate produced using three freeze/thaw cycles and/or sonication.
  • the lysates are then centrifuged to remove cell debris and the rAAV virions purified by cesium chloride equilibrium gradient centrifugation. Any residual adenoviral particles are inactivated by heating the purified rAAV preparation to at least 56°C for 20-30 minutes.
  • the rAAV virions can be purified by sulfonated cellulose column chromatography following the protocol described in Tamayose, Human Gene Therapy 7 (1996) 507-513.
  • Vector pKm 201 CMV is a cloning vector in which an expression cassette containing a CMV immediate early enhancer, promoter and intron and a bovine growth hormone poly adenylation site is flanked by AAV-2 ITRs.
  • CMV was derived from pKm201, a modified AAV vector plasmid in which the ampicillin resistance gene of pEMBL-AAV-ITR (Srivastava, (1989)) has been replaced with the gene for kanamycin resistance.
  • pCMVAAV-lacZ the lacZ cDNA sequence was excised from the plasmid pCMV ⁇ (Clontech, Palo Alto, CA) and inserted into pKm201CMVLINK.
  • the plasmid pKm201CMVLINK has the backbone identical to vector pAAV-TK-MCSFa, which has been deposited with the ATCC, as Accession No. 98335.
  • the AAV helper plasmid, pKSrep/cap was constructed by cloning the AAV-2 genome without the ITRs, i.e., nucleotides 192 through 4493 of AAV-2 (see Srivastava, J. Virol. 45 (1983) 555-64) into pBluescript II KS+ (Strategene, La Jolla, CA).
  • Plasmid pJM17 is a non-infectious (replication-defective) adenovirus plasmid when transfected into human embryonic kidney cells (293 cells), since it contains an insertion of a derivative of plasmid pBR322 at base pair (bp) 1339 (3.7 mu) in its Adenovirus-5 sequence, which makes the resulting viral genome too large to package.
  • Plasmid pBHGlO is an Adenovirus-5 plasmid that is non- infectious when transfected into 293 cells, since it contains a deletion of Adenovirus type 5 (Ad5) sequences from bp 188 through bp 1339 (0.5 through 3.7 mu), which removes the packaging signals (psi) required to encapsidate the adenoviral DNA.
  • Ad5 Adenovirus type 5
  • An ampicillin resistance gene (Ap) and bacterial origin of replication (Ori) substitute for the deleted (bp 188 to 1339) Ad5 sequences.
  • This plasmid also lacks the Ad5 E3 region (from 78.3 through 85.8 mu).
  • Plasmid pBHGll is an Ad5 plasmid that is infectious when transfected into 293 cells, since it contains a deletion of Ad5 sequences from bp 188 through bp 1339 (0.5 through 3.7 mu) which removes the packaging signals (psi) required to encapsidate the adenoviral DNA.
  • An ampicillin resistance gene (Amp) and bacterial origin of replication (Ori) substitute for the deleted Ad5 sequences.
  • This plasmid also lacks the Ad5 E3 region (from 77.5 through 86.2 mu).
  • the cells were triple transfected with a mixture comprising the AAV vector, the replication-defective AAV helper plasmid and the adenoviral plasmid by the calcium phosphate co- precipitation method.
  • a mixture of lO ⁇ g of the AAV vector plasmid, lO ⁇ g of the replication-defective AAV helper plasmid and 20 ⁇ g of the adenoplasmid pBHGlO was added to 2.5 ml of 250 mM CaCl 2 and mixed with 2.5 ml of 2x HBS (Jordan, Nucleic Acids. Res. 24 (1996) 596-601).
  • the precipitate was left on the cells for eight hours and replaced with fresh IMDM medium containing 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • the cells were harvested with Hepes buffer (2.5 ml/dish) and lysed by three cycles of freezing and thawing.
  • the cell lysates were centrifuged at 12,000x g for twenty minutes to remove cell debris.
  • the packaged AAV particles were purified through two rounds of cesium chloride equilibrium gradient centrifugation and residual adenoviral particles were inactivated by heat treatment at 56°C for thirty minutes.
  • the packaged AAV particles are purified by sulfonated cellulose column chromatography as described in Tamayose, Human Gene Therapy 7 (1996) 507-13.
  • Adenovirus dl 312 has a deletion in the Ela gene and is propagated in 293 cells transformed with left-end of adenovirus sequences. It expresses Ela and Elb transcripts. See Moran Cell 48 (1987) 177-78. The transformed cells were harvested at 72 hours post-infection and purified as described above.
  • the purified vector stock was treated with DNAse I and the encapsidated DNA was extracted with phenol-chloroform, precipitated with ethanol, and subject to dot blot analysis as described in Nahreini and Srivastava, Interviriology 30 (1989) 74-85. 293 cells were then infected with serial dilutions of vector stock. The positive cells were counted and the functional titer was estimated.
  • the rAAV stock was diluted serially and 293 cells (2xl0 5 ) were infected along with Ad at an MOI of 2.
  • the low molecular weight DNA was isolated following the method of Hirt, J. Mol. Biol. 26 (1967) 365-69, fractionated on an agarose gel and transferred to a nylon membrane. The blot was hybridyzed to a biotinylated oligonucleotide probe specific for the AAV capsid region.
  • the titer was reported base on the highest dilution of the vector stock showing positive signal for AAV capsid DNA.
  • the blots were probed with either a monoclonal antibody against AAV capsid protein (ARP Inc., Belmont, MA) and a goat anti-mouse (IgG HRP conjugate (Bo-Rad Laboratories, Richmond, CA) or a polyclonal antibody against adenovirus type 5 (DAKO, Denmark) and a goat anti-rabbit IgG HRP conjugate.
  • ARP Inc. Belmont, MA
  • IgG HRP conjugate Bo-Rad Laboratories, Richmond, CA
  • DAKO polyclonal antibody against adenovirus type 5
  • DAKO goat anti-rabbit IgG HRP conjugate
  • Lane 1 which contained the crude preparation (0.008% of the total) from the standard protocol, showed an extensive smear of protein extending from about 29 kD to over 140 kD.
  • lane 2 which contained the crude preparation (0.008%) from the triple transfection protocol, showed very low levels of adenoproteins.
  • the purified AAV products when analyzed for adenovirus protein contamination, showed still some adenoprotein contamination in standard protocol (lane 3), whereas the triple transfection protocol of the present invention had no detectable adenoprotein contamination (lane 4).
  • the samples were diluted and added to 50% confluent 293 cells (plated on 12 well dishes with lxlO 5 cells).
  • the cultures were passaged for at least three weeks or until the cultures exhibited cytopathic effect.
  • the Control included 293 cells infected with known amount of adenovirus stock.
  • the limit of detection in the assay was 100 pfu/ml.

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Abstract

L'invention concerne un procédé relatif à la production de virions AAV de recombinaison purifiés à déficience de réplication, qui consiste à introduire dans une cellule hôte appropriée un vecteur AAV, un produit de synthèse assistant AAV et un produit de synthèse adénoplasmide accessoire. Le plasmide adénoplasmide accessoire est composé d'ADN de plasmide d'adénovirus impossible à empaqueter dans des particules adénovirales car ne possédant pas de séquence(s) de signaux d'empaquetage ou parce que ces particules contiennent des séquences additionnelles leur conférant une taille trop importante pour l'empaquetage. On met en culture la cellule hôte pour produire des virions rAAV bruts, et la cellule est ensuite soumis à une lyse. Le lysat de cellule résultant est appliqué à une colonne de chromatographie renfermant de la cellulose sulfonée ou soumis à une centrifugation à gradient d'équilibre au chlorure de césium, et on peut ainsi récupérer les virions rAAV purifiés.
PCT/US1999/022789 1999-09-30 1999-09-30 Materiaux et procedes pour la production simplifiee d'aav Ceased WO2001025464A1 (fr)

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AU14414/00A AU1441400A (en) 1999-09-30 1999-09-30 Materials and methods for simplified aav production
PCT/US1999/022789 WO2001025464A1 (fr) 1999-09-30 1999-09-30 Materiaux et procedes pour la production simplifiee d'aav
JP2001528615A JP2003511038A (ja) 1999-09-30 1999-09-30 単純化したａａｖ産生のための材料および方法
EP99974094A EP1220938A1 (fr) 1999-09-30 1999-09-30 Materiaux et procedes pour la production simplifiee d'aav
CA002386722A CA2386722A1 (fr) 1999-09-30 1999-09-30 Materiaux et procedes pour la production simplifiee d'aav

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EP3219313A3 (fr) * 2011-04-18 2017-12-06 National Center of Neurology and Psychiatry Particule de libération de médicament et son procédé de fabrication
WO2024206396A1 (fr) * 2023-03-28 2024-10-03 Biogen Ma Inc. Procédés de production de virus adéno-associé recombiné (raav)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3219313A3 (fr) * 2011-04-18 2017-12-06 National Center of Neurology and Psychiatry Particule de libération de médicament et son procédé de fabrication
EP3777843A1 (fr) * 2011-04-18 2021-02-17 National Center of Neurology and Psychiatry Particule de libération de médicament et son procédé de fabrication
US11191733B2 (en) 2011-04-18 2021-12-07 National Center Of Neurology And Psychiatry Drug delivery particle and method for producing the same
WO2024206396A1 (fr) * 2023-03-28 2024-10-03 Biogen Ma Inc. Procédés de production de virus adéno-associé recombiné (raav)

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