JP4856694B2 - Auxiliary functions for use in recombinant AAV virion production - Google Patents

Description

TECHNICAL FIELD The present invention relates generally to an accessory function for use in adeno-associated virus (AAV) virion production. More particularly, the present invention relates to vectors that provide auxiliary functions that can support efficient recombinant AAV virion production in suitable host cells, and methods of use thereof.

Background of the Invention Gene delivery is a promising method for the treatment of acquired and inherited diseases. A number of virus-based systems for gene transfer purposes (eg, retrovirus systems, which are currently the most widely used viral vector systems for this purpose) have been described. For descriptions of various retroviral systems, see, eg, US Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7 : 980-990; Miller, AD (1990) Human Gene Therapy 1 : 5-14; Scarpa et al. (1991). Virology 180 : 849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90 : 8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3 : 102- See 109.

The adeno-associated virus (AAV) system has also been used for gene delivery. AAV is a helper-dependent DNA parvovirus that belongs to the genus Dependovirus. AAV requires infection with an unrelated helper virus, either adenovirus, herpesvirus, or vaccinia, to produce a productive infection. Helper viruses provide auxiliary functions necessary for many stages in AAV replication. In the absence of such infection, AAV establishes a latent state by insertion of its genome into the host cell chromosome. Subsequent infection with the helper virus rescues the integrated copy, which can then replicate to produce infectious virus progeny. AAV has a broad host range and can replicate in cells from any species so long as such cells are successfully infected with the appropriate helper virus. Thus, for example, human AAV replicates in canine cells co-infected with canine adenovirus. AAV is not associated with either human or animal disease and does not appear to alter the biological properties of the host cell upon integration. For a review of AAV, see, for example, Berns and Bohenzky (1987) Advances in Virus Research (Academic Press, Inc.) 32 : 243-307.

The AAV genome is composed of a linear single-stranded DNA molecule containing 4681 bases (Berns and Bohenzky, supra). The genome contains an inverted terminal repeat (ITR) that functions in cis at each end as a DNA replication origin and as a viral packaging signal. The ITR is approximately 145 bp long. The internal non-repetitive portion of the genome contains two large open reading frames known as AAVrep and cap regions, respectively. These regions encode viral proteins involved in virion replication and packaging. In particular, at least four families of viral proteins are synthesized from the AAVrep region, Rep78, Rep68, Rep52, and Rep40, which are named according to their apparent molecular weight. The AAV cap region encodes at least three parts (VP1, VP2, and VP3). For a detailed description of the AAV genome, see, for example, Muzyczka, N. (1992) Current Topics in Microbiol. And Immunol. 158 : 97-129.

The construction of recombinant AAV virions has been described. For example, US Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO92 / 01070 (published January 23, 1992) and WO93 / 03769 (published March 4, 1993); Lebkowski et al. (1988) Molec. Cell Biol. 8 : 3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, BJ (1992) Current Opinion in Biotechnology 3 : 533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158 : 97-129; and Kotin, RM (1994) HumanGene Therapy 5 : 793-801.

  Current production of recombinant AAV (rAAV) virions involves co-transfection into host cells with an AAV vector plasmid and a construct that provides an AAV helper function that complements the function lost from the AAV vector plasmid. In this manner, the host cell can express the AAV protein required for AAV replication and packaging. The host cell is then infected with a helper virus that provides accessory functions. The helper virus is generally an infectious adenovirus (type 2 or type 5) or a herpes virus.

  AAV helper function can be provided via an AAV helper plasmid that contains the AAVrep and / or cap coding region but lacks the AAV ITR. Thus, helper plasmids cannot themselves replicate or package. A number of vectors are known that contain the rep coding region, including the vectors described below: ATCC Deposit Numbers 53222, 53223, 53224, 53225, and 53226 US Pat. No. 5,139,941. Similarly, a method for obtaining a vector containing a homologue of AAVrepHHV-6 is described in Thomson et al. (1994) Virology 204: 304-311. A number of vectors containing cap coding regions including those described in US Pat. No. 5,139,941 have also been described.

  The AAV vector plasmid includes a functionally related nucleotide sequence of interest (eg, selected genes, antisense nucleic acid molecules, ribozymes, etc.) flanked by AAV ITRs that provide AAV replication and packaging functions. Can be designed. After an AAV helper plasmid and an AAV vector plasmid having a nucleotide sequence are introduced into a host cell by transient transfection, the transfected cell is present on a helper plasmid that directs the transcription and translation of the AAVrep and AAVcap regions It can be infected with a helper virus (most typically an adenovirus) that transactivates the AAV promoter from among other functions. During subsequent host cell culture, rAAV virions (having the nucleotide sequence of interest) and helper virus particles are produced.

  When host cells are harvested and a crude extract is produced, the resulting preparation contains, among other components, approximately equal amounts of rAAV virion particles and infectious helper virions. rAAV virion particles can be purified from many of the contaminating helper viruses, unassembled viral proteins (from helper virus and AAV capsids) and host cell proteins using known techniques. Purified rAAV virion preparations produced using infection with type 2 adenovirus contain high levels of contaminants. Specifically, over 50% of the total protein obtained in such rAAV virion preparations is free adenovirus fiber protein. There are also various amounts of some unidentified adenovirus and host cell proteins. In addition, significant levels of infectious adenovirus virions are obtained and require heat inactivation. Contaminating infectious adenovirus can be inactivated by heat treatment (1 hour at 56 ° C.) and rendered undetectable by a sensitive adenovirus proliferation assay (eg, by cytopathic effect (CPE) in permissive cell lines). obtain. However, heat treatment also results in an approximately 50% decrease in functional rAAV virion titer.

  Production of rAAV virions using infectious helper viruses that provide accessory functions (eg, type 2 adenovirus, or herpes virus) is undesirable for several reasons. AAV vector production methods using helper viruses require the use and manipulation of large amounts of high titer infectious helper viruses that present many health and safety concerns, particularly with respect to the use of herpes viruses. Also, the concomitant production of helper virus particles in rAAV virion producing cells diverts a large amount of cellular resources from rAAV virion production. Perhaps this results in a lower rAAV virion yield.

  More particularly, in methods where infection of cells with type 2 adenovirus is used to provide ancillary functions, over 95% of the contaminants found in purified rAAV virion preparations are derived from adenovirus. . The main contaminant, free adenovirus fiber protein, tends to co-purify with rAAV virions on a CsCl density gradient due to non-covalent binding between the protein and rAAV virions. This binding makes it particularly difficult to separate the two, which reduces the purification efficiency of rAAV virions. Many adenoviral proteins, including fiber proteins, can be cytotoxic (usually at high concentrations) and thus have been shown to adversely affect or kill target cells As such, such contaminants can be an important issue. Thus, methods that produce rAAV virions without the use of infectious helper viruses that provide the necessary auxiliary functions are advantageous.

  Many researchers have studied the genetic basis of auxiliary functions, particularly those derived from adenovirus. In general, the following two approaches have been used to attempt to identify adenoviral genes involved in AAV replication: testing the ability of various adenoviral variants to provide accessory functions; and absence of adenoviral infection Study of the effect of transfected adenoviral genes, or regions, on AAV replication below.

Studies using various adenovirus variants that can assist AAV replication (eg, by providing the necessary accessory functions) at or near the level obtained by infection with wild-type adenoviruses are specific adenoviruses It has been shown that a gene or gene region is not involved in AAV replication. However, because many of the adenoviral genes and control regions overlap and / or are incompletely mapped, loss-of-function data from such studies determines that a particular gene region is involved in AAV replication Information could not be provided.

In particular, adenoviral mutants with sufficiently well-characterized mutants in the following genes or gene regions have been tested for their ability to provide the accessory functions necessary for AAV viral replication: E1a (Laughlin et al. (1982) J. Virol. 41 : 868, Janik et al. (1981) Proc. Natl. Acad. Sci. USA 78 : 1925); E1b (Laughlin et al. (1982), supra, Janik et al. (1981). , Supra, Ostrove et al. (1980) Virology 104 : 502); E2a (Handa et al. (1975) J. Gen. Viorl. 29 : 239, Straus et al. (1976) J. Virol. 17 : 140, Myers et al. (1980) J. Virol. 35 : 665, Jay et al. (1981) Proc. Natl. Acad. Sci. USA 78 : 2927, Myers et al. (1981) J. Biol. Chem. 256 : 567); E2b ( Carter, BJ (1990) "Adeno-Associated Virus Helper Functions", in CRC Handbook of Parvoviruses, vol.I (P. Tijssen); E3 (Carter et al., (1983) Virology 126 : 505); and E4 (Carter et al., (1983), supra, Carter, BJ, (1995), supra), incompletely characterized as incapable of DNA replication and incapable of late gene synthesis. Adenovirus mutants have also been tested (Ito et al. (1970) J. Gen. Virol. 9 : 243, Ishibashi et al. (1971) Virology 45 : 317).

  Adenovirus mutants with defects in the E2b and E3 regions have been shown to assist AAV replication. This indicates that the E2b and E3 regions are probably not involved in providing ancillary functions (Carter et al. (1983), supra). Mutant adenoviruses that lack the E1a region or have a deleted E4 region, either directly or indirectly, cannot support AAV replication, indicating that E1a and E4 We show that the region is required for AAV replication (Laughlin et al., (1982), supra, Janik et al., (1981), supra, Carter et al., (1983), supra). Studies using E1b and E2a mutants have produced conflicting results. Furthermore, adenoviral mutants that are incapable of DNA replication and late gene synthesis have been shown to allow AAV replication (Ito et al., (1970), supra, Ishibashi et al., (1971), supra). . These results indicate that neither adenoviral DNA replication nor adenoviral late genes are required for AAV replication.

Whether transfection studies using selected adenoviral genes can provide a set of adenoviral genes that have been transfected can provide an auxiliary function for AAV replication at the same level as that provided by adenoviral infection Has been used in an attempt to establish In particular, in vitro AAV replication has been evaluated using human 293 cells transiently transfected with various combinations of adenoviral restriction fragments encoding a single adenoviral gene or group of genes. (Janik et al. (1981), supra). Since the above transfection studies were performed in cells stably expressing adenovirus E1a and E1b, the need for these regions could not be tested. However, it was postulated that the combination of three adenoviral gene regions (VAIRNA, E2a, and E4) could provide accessory functions (eg, supporting AAV replication) at levels substantially above background. However, it was still about 8,000 times lower than the level provided by infection with adenovirus. When all two combinations of the three genes were tested, the level of accessory function ranged from 10,000 to 100,000 times lower than the level provided by infection with adenovirus.

Transfection studies with selected type 1 herpes simplex virus (HSV-1) genes also show that a set of transfected HSV-1 genes are at the same level as provided by HSV-1 infection. Attempts have been made to establish whether an auxiliary function of AAV replication can be provided. Weindler et al. (1991) J. Virol. 65 : 2476-2483. However, such studies have been limited to identifying only the HSV-1 gene that is required to support wild-type AAV replication, not rAAV production. Furthermore, the identified HSV-1 accessory function was not significantly more effective than the function derived from adenovirus in supporting AAV replication.

  Thus, there remains a need in the art to identify a subset of an adenoviral genome that contains only the accessory functions required for rAAV virion production, or a functional homologue of an adenoviral genome. Thus, subsets are accessory function vectors that, when introduced into a suitable host cell, support the production of rAAV virions in an amount substantially equivalent to or greater than the amount produced using adenoviral infection. Or it may be included in the auxiliary function system. Furthermore, there remains a need to provide an accessory function system that can produce commercially significant levels of rAAV virion particles without producing significant levels of infectious adenovirus virions, or other contaminating by-products. .

(Item 1) A nucleic acid molecule that provides one or more auxiliary functions to support recombinant AAV (rAAV) virion production in a suitable host cell and lacks at least one adenovirus late gene region. The molecule provides (i) a sequence that provides an adenovirus VARNA, (ii) an adenovirus E4 ORF6 coding region, (iii) a 72 kD coding region of adenovirus E2a, and (i), (ii), and (iii) A nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
(Item 2) A nucleic acid molecule that provides sufficient accessory functions to support efficient recombinant AAV (rAAV) virion production in a suitable host cell and lacks at least one adenovirus late gene region.
(Item 3) The nucleic acid molecule according to item 2, wherein the molecule lacks adenovirus early gene regions 2b and 3.
(Item 4) The nucleic acid molecule according to item 2 or 3, which provides an auxiliary function sufficient to efficiently support rAAV virion production in a human 293 host cell.
(Item 5) The nucleic acid molecule according to item 4, wherein:
A first nucleotide sequence providing an adenovirus VA RNA;
A nucleic acid molecule comprising a second nucleotide sequence comprising an adenovirus E4 coding region comprising ORF 6; and a third nucleotide sequence comprising an adenovirus E2a coding region.
(Item 6) A nucleic acid molecule according to item 2, wherein the molecule is infectious by adenovirus or in an appropriate host cell that is not capable of supporting adenovirus replication. rAAV) A nucleic acid molecule characterized in that it provides an auxiliary function to support virion production.
(Item 7) The nucleic acid molecule according to item 6, wherein:
A first nucleotide sequence providing an adenovirus VA RNA;
A second nucleotide sequence comprising an adenovirus E4 coding region comprising ORF 6;
A third nucleotide sequence comprising an adenovirus E2a coding region; and a fourth nucleotide sequence comprising an adenovirus E1a and E1b coding region;
A nucleic acid molecule comprising
(Item 8) The nucleic acid molecule according to item 4 or 6, comprising one or more nucleotide sequences derived from a type 2 or type 5 adenovirus genome.
(Item 9) An auxiliary function vector comprising the nucleic acid molecule according to any one of items 1, 2, or 6.
(Item 10) The auxiliary function vector according to item 9, wherein the vector is a plasmid.
(Item 11) The auxiliary function vector according to item 10, further comprising a selection gene marker.
(Item 12) The auxiliary function vector according to item 11, wherein the selection gene marker contains an antibiotic resistance gene.
(Item 13) The auxiliary function vector according to item 10, further comprising at least one heterologous promoter region.
(Item 14) The auxiliary function according to item 13, wherein the at least one heterologous promoter region comprises a nucleotide sequence that is substantially homologous to a cytomegalovirus (CMV) immediate early promoter region. vector.
(Item 15) An auxiliary functional system for recombinant AAV (rAAV) virion production comprising first, second, and third auxiliary function vectors, wherein:
(A) the first auxiliary function vector comprises a nucleotide sequence providing an adenovirus VA RNA;
(B) the second auxiliary function vector comprises an adenovirus E4 ORF6 coding region; and (c) the third accessory function vector comprises an adenovirus E2a 72 kD coding region.
(Item 16) The first auxiliary function vector is a plasmid pBSII-VA RNA (ATCC accession number 98233), and the second auxiliary function vector is p3) 3cE4ORF6 (ATCC accession number 98234), Item 16. The auxiliary function system according to Item 15, wherein the third auxiliary function vector is p3) 3cE2A (ATCC accession number 98235).
(Item 17) A host cell transfected with the auxiliary function vector according to item 9.
(Item 18) A cell capable of producing a recombinant AAV (rAAV) virion when transfected with an AAV vector, wherein the cell is expressed in the cell with an AAV helper construct capable of providing an AAV helper function. 18. A cell comprising the host cell of item 17, which is co-transfected.
(Item 19) A method for producing a recombinant AAV (rAAV) virion comprising the following:
(A) introducing an AAV vector into a suitable host cell;
(B) introducing an AAV helper construct into the host cell, the helper construct comprising an AAV coding region expressed in the host cell to complement the AAV helper function deleted from the AAV vector; Process;
(C) introducing the auxiliary function system according to item 15 into the host cell, wherein the auxiliary function system provides an auxiliary function for supporting efficient rAAV virion production in the host cell; And (d) culturing the host cell to produce rAAV virions,
Including the method.
(Item 20) A method for producing a recombinant AAV (rAAV) virion, comprising:
(A) introducing an AAV vector into a suitable host cell;
(B) introducing an AAV helper construct into the host cell, the helper construct comprising an AAV coding region expressed in the host cell to complement the AAV helper function deleted from the AAV vector. Process;
(C) introducing the auxiliary function system according to item 9 into the host cell, wherein the auxiliary function system provides the auxiliary function to support efficient rAAV virion production in the host cell; And (d) culturing the host cell to produce rAAV virions,
Including the method.
(Item 21) A recombinant AAV virion produced by the method according to item 19 or 20.

SUMMARY OF THE INVENTION The present invention is based on the identification of accessory functions required to support efficient AAV replication in a suitable host cell. The present invention provides a system that includes such functions and that allows the production of rAAV virions without the use of helper viruses.

  In certain embodiments, the present invention relates to nucleic acid molecules that encode accessory functions and lack at least one adenovirus late gene region. The molecule can be provided in one or more vectors comprising a nucleotide sequence derived from a type 2 or type 5 genomic adenovirus, or a functional homologue thereof. Accordingly, in one aspect, the invention provides (i) a sequence that provides an adenoviral VARNA, (ii) a coding region of adenovirus E4 ORF6, (iii) a 72 kD coding region of adenovirus E2a (72 kD of E2a And a vector comprising a nucleotide sequence selected from the group consisting of nucleotide sequences (i), (ii), any combination of (iii).

  In another embodiment, the present invention provides an accessory function that can support efficient recombinant AAV (rAAV) virion production in an appropriate host cell and lacks at least one adenovirus late gene region. Molecules and vectors containing the nucleic acid molecules.

  In another aspect, the invention relates to a vector comprising a number of nucleotide sequences, including sequences that provide adenoviral VA RNA, sequences that include an adenoviral E4 coding region, and sequences that include the coding region of adenoviral E2a. .

  In yet another aspect of the invention, comprising a sequence that provides an adenoviral VA RNA, a sequence that includes an adenoviral E4 coding region, a sequence that includes an adenoviral E2a coding region, and a sequence that includes an adenoviral E1a and E1b coding region. A vector is provided.

  In another embodiment of the invention, an accessory function system for rAAV virion production is provided, wherein the system provides suitable accessory function components to support efficient AAV virion production in a suitable host cell. A number of auxiliary function vectors.

In yet another embodiment of the invention, a method for producing rAAV virions is provided. This method generally involves (1) introducing an AAV vector into an appropriate host cell; (2) introducing an AAV helper construct into the cell, wherein the helper construct is deleted from the AAV vector. A step capable of being expressed in a host cell to complement the AAV helper function being performed; (3) introducing one or more auxiliary function vectors into the host cell, wherein the one or more auxiliary function vectors are Providing an auxiliary function capable of supporting efficient rAAV virion production in the host cell; and (4) culturing the cell producing the rAAV virion.

  In a further embodiment, recombinant AAV virions produced by the methods of the invention are also provided.

  These and other embodiments of the invention will be readily apparent to those skilled in the art in view of the disclosure herein.

Detailed Description of the Invention The practice of the present invention, unless otherwise indicated, uses conventional methods of virology, microbiology, molecular biology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. For example, Sambrook et al. Molecular Cloning: A Laboratory Manual (latest edition); DNACloning: A Practical Approach, Volumes I and II (Edited by D. Glover); Oligonucleotide Synthesis (Edited by N. Gait, updated edition); Nucleic Acid Hybridization (B. Hames and S. Higgins, latest edition); Transcriptionand Translation (B. Hames and S. Higgins, latest edition); CRC Handbook of Parvoviruses, Volumes I and II (P. Tijessen); Fundamental See Virology, 2nd Edition, Volume I and Volume II (BN Fields and DM Knipe).

  As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the content clearly dictates otherwise.

A. Definitions In describing the present invention, the following terms will be used, and are intended to be defined as indicated below.

“Gene transfer” or “gene delivery” refers to a method or system for reliably inserting foreign DNA into a host cell. Such methods include transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (eg, episomes), or transferred genetic material into the genomic DNA of the host cell. Can result in the incorporation of. Introgression provides a unique approach for the treatment of acquired and inherited diseases. A number of systems have been developed for gene transfer into mammalian cells. See, for example, US Pat. No. 5,399,346.

  By “vector” is meant any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., that is replicable when accompanied by the appropriate control elements, and cell Gene sequences can be transferred between. The term thus encompasses cloning and expression vehicles, as well as viral vectors.

  By “adeno-associated virus inverted terminal repeat” or “AAV ITR” is meant an art-recognized region found at each end of the AAV genome, together in cis, as a DNA origin of replication and Serves as a packaging signal for viruses. AAVITR, together with the AAV rep coding region, provides efficient excision and rescue from the mammalian cell genome and integration of the nucleotide sequence between two adjacent ITRs into the mammalian cell genome.

The nucleotide sequence of the AAV ITR region is known. For example, for the AAV-2 sequence, see Kotin, RM (1994) Human Gene Therapy 5 : 793-801; Berns, KI "Parvoviridae and its Replication" Fundamental Virology, 2nd Edition, (BNFields and DM Knipe). See As used herein, an “AAV ITR” need not have the wild-type nucleotide sequence shown, but can be modified, for example, by nucleotide insertions, deletions, or substitutions. Furthermore, AAVITR can be derived from any of several AAV serotypes, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7 and the like. In addition, in AAV vectors, the 5 ′ and 3 ′ ITRs adjacent to the selected nucleotide sequence will allow for the excision and rescue of the sequence of interest from the host cell genome or vector as they are intended. As long as it functions to allow integration of heterologous sequences into the recipient cell genome when the AAVRep gene product is present in the cell, or is necessarily identical to the same AAV serotype or isolate, or There is no need to derive from it.

"AAV rep coding region" encodes a viral replication protein that is collectively required to replicate the viral genome and for insertion of the viral genome into the host genome during potential infection A recognized region of the AAV genome, or a functional homologue thereof (eg, the human herpesvirus 6 (HHV-6) rep gene that is also known to mediate AAV-2 DNA replication (Thomson et al. (1994) Virology 204 : 304-311)). Thus, the rep coding region includes at least genes encoding AAVRep 78 and Rep 68 (“long form Rep”), and Rep52 and Rep 40 (“short form Rep”), or functional homologues thereof. For further description of the AAVrep coding region, see, eg, Muzyczka, N. (1992) Current Topics in Microbiol. And Immunol. 158 : 97-129; and Kotin, RM (1994) Human Gene Therapy 5 : 793-801. thing. As used herein, the rep coding region can be derived from any viral serotype, such as the AAV serotype described above. This region need not include all of the wild type gene, as long as the rep gene present provides sufficient integration function when expressed in an appropriate recipient cell, for example, nucleotide insertions, deletions, Or it can be modified by substitution.

By “AAV cap coding region” is meant an art-recognized region of the AAV genome that encodes the viral coat protein that is collectively required to package the viral genome. Accordingly, the cap coding region includes at least genes encoding the coat proteins VP1, VP2, and VP3. For further description of the cap coding region, see, eg, Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158 : 97-129; and Kotin, RM (1994) Human Gene Therapy 5 : 793-801. thing. As used herein, the AAV cap coding region can be derived from any AAV serotype, as described above. This region need not include all of the wild-type cap gene, as long as the gene provides sufficient packaging function when present in a host cell with an AAV vector, for example, by nucleotide insertion, deletion, or substitution. Can be modified.

  By “AAV vector” is meant a vector derived from an adeno-associated virus serotype, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, and the like. An AAV vector may have one or more AAV wild type genes, preferably rep and / or cap genes, deleted in whole or in part, but may retain functional flanking ITR sequences. Functional ITR sequences are required for AAV virion rescue, replication, and packaging. Thus, an AAV vector is defined herein to include at least those sequences that are required in cis for viral replication and packaging (eg, a functional ITR). The ITR need not be a wild-type nucleotide sequence and can be altered, for example, by nucleotide insertions, deletions, or substitutions, as long as the sequence provides functional rescue, replication, and packaging.

  “AAV helper function” refers to an AAV-derived coding sequence that can be expressed and then provide an AAV gene product that functions in trans for proliferative AAV replication. Thus, the AAV helper function includes both major AAV open reading frames (ORF) (rep and cap). Rep expression products have been shown to have many functions including, among others: AAV DNA origin of replication recognition, binding, and nicking; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters Has been. The Cap expression product provides the necessary packaging functions. AAV helper function is used herein to complement in trans the AAV function that is missing from the AAV vector.

The term “AAV helper construct” refers to a nucleic acid molecule comprising a nucleotide sequence that provides AAV function deleted from an AAV vector, generally used to produce a transduction vector for delivery of the nucleotide sequence of interest. . AAV helper constructs are generally used to provide transient expression of AAVrep and / or cap genes to complement the missing AAV function that is required for lytic AAV replication; The construct lacks AAVITR and cannot replicate or package itself. The AAV helper construct can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described (eg, commonly used plasmids pAAV / Ad and pIM29 + 45 encoding both Rep and Cap expression products). See, for example, Samulski et al. (1989) J. Virol. 63 : 3822-3828; and McCarty et al. (1991) J. Virol. 65 : 2936-2945. Many other vectors encoding Rep and / or Cap expression products have been described. See, for example, US Pat. No. 5,139,941.

  The term “accessory function” refers to non-AAV derived viral and / or cellular functions on which AAV depends for its replication. Thus, the term refers to the proteins and RNA required for AAV replication (parts involved in AAV gene transcription activation, time-specific AAV mRNA splicing, AAV DNA replication, Cap expression product synthesis and AAV capsid assembly). (Including). Virus-based auxiliary functions can be derived from any known helper virus such as adenovirus, herpes virus (other than herpes simplex virus type 1) and vaccinia virus.

For example, adenovirus-derived auxiliary functions have been extensively studied, and a number of adenoviral genes involved in auxiliary functions have been identified and partially characterized. For example, Carter, BJ (1990) “Adeno-associated virus helper function” CRC Handbook of Parvoviruses, Volume I (Edited by P. Tijssen), and Muzyczka, N. (1992) Curr. Topics. Microbiol. And Immun. 158 : 97 See -129. Specifically, the early adenovirus gene regions E1a, E2a, E4, VAIRNA and possibly E1b are thought to be involved in the auxiliary process. Janik et al. (1981) Proc. Natl. Acad. Sci. USA 78 : 1925-1929. Herpesvirus-derived auxiliary functions have been described. See, for example, Young et al. (1979) Prog. Med. Virol. 25 : 113. A vaccinia virus-derived auxiliary function has also been described. See, for example, Carter, BJ (1990), supra, Schlehofer et al. (1986) Virology 152 : 110-117.

  The term “auxiliary function vector” generally refers to a nucleic acid molecule comprising a nucleotide sequence that provides an auxiliary function. Auxiliary function vectors can be transfected into a suitable host cell, where the vector can then support AAV virion production in the host cell. Infectious viral particles that remain naturally occurring (eg, adenovirus, herpes virus or vaccinia virus particles) are expressly excluded from this term. Thus, the accessory function vector can be in the form of a plasmid, phage, transposon or cosmid.

  By “can support efficient rAAV virion production”, rAAV virion production in a particular host cell at a level substantially equivalent to or higher than that obtainable upon infection of the host cell with an adenovirus helper virus. Refers to the ability of an auxiliary function vector or system to provide an auxiliary function that is sufficient to complement. Thus, the ability of accessory function vectors or systems to support efficient rAAV virion production is obtained using rAAV virion titers obtained using accessory vectors or systems using infection with infectious adenoviruses. It can be determined by comparing with the titer. More particularly, the amount of virions obtained from an equivalent number of host cells is about 1/200 or more, more preferably about 1/100 or more, and most preferably the amount obtained using an adenovirus infection. When equal to or greater than the amount obtained using adenoviral infection, the accessory function vector or system is substantially the same or more efficient rAAV virion as obtained using infectious adenovirus. Support production.

  By “recombinant virus” is meant a virus that has been genetically modified, eg, by the addition or insertion of a heterologous nucleic acid construct into the particle.

  By “AAV virion” is meant a complete viral particle, such as a wild-type (wt) AAV viral particle (including a linear, single-stranded AAV nucleic acid genome combined with an AAV capsid protein coat). In this regard, any complementary sense single-stranded AAV nucleic acid molecule (eg, a “sense” strand or an “antisense” strand) can be packaged in any one AAV virion, and both strands are Equally infectious.

  A “recombinant AAV virion” or “rAAV virion” as used herein is an infectious, replication-deficient virus composed of an AAV protein coat enveloped by a heterologous nucleotide sequence of interest flanked by AAVITR on both sides Is defined as rAAV virions are produced in a suitable host cell having the AAV vector, AAV helper function and auxiliary functions introduced therein. In this method, the host cell encodes the AAV polypeptide required to package the AAV vector (containing the desired recombinant nucleotide sequence) into infectious recombinant virion particles for subsequent gene delivery. To be able to.

The term “transfection” is used to refer to the uptake of foreign DNA by a cell, and a cell is “transfected” when exogenous DNA has been introduced inside the cell membrane. Many transfection techniques are generally known in the art. For example, Graham et al. (1973) Virology, 52 : 456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene. See 13 : 197. Such techniques can be used to introduce one or more exogenous DNA moieties (eg, nucleotide integration vectors and other nucleic acid molecules) into suitable host cells.

  The term “host cell” can be used or used as a recipient of AAV helper constructs, AAV vector plasmids, accessory function vectors, or other transfer DNA, eg, microorganisms, yeast cells, insect cells, and mammals. Animal cells are shown. The term includes the progeny of the original cell that has been transfected. Thus, as used herein, “host cell” generally refers to a cell that has been transfected with an exogenous DNA sequence. Even if the progeny of a single parent cell are not necessarily completely identical in form or with genomic DNA complement or total DNA complementation, due to natural, accidental, or deliberate mutations It is understood that it is good.

  As used herein, the term “cell line” refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotypes during storage or transfer of such clonal populations. Thus, cells derived from the cell line referred to may not be exactly the same as the ancestral cell or culture, and the cell line referred to includes such variants.

  The term “heterologous” refers to sequences that are not normally linked together and / or are not normally associated with a particular cell, when referring to nucleic acid sequences such as coding and regulatory sequences. Thus, a “heterologous” region of a nucleic acid construct or vector is a segment of nucleic acid within or bound to another nucleic acid molecule that is not found in association with other molecules in nature. For example, a heterologous region of a nucleic acid construct can include a coding sequence that is flanked by sequences that are not found in nature linked to the coding sequence. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (eg, a synthetic sequence having codons different from the natural gene). Similarly, cells transformed with constructs not normally present in the cell are considered heterogeneous for the purposes of the present invention. Allelic variation or naturally-occurring mutational events as used herein do not give rise to heterologous DNA.

  A “coding sequence” or a sequence that “encodes” a particular protein is transcribed in vitro or in vivo (in the case of DNA) and translated into a polypeptide (in the case of mRNA) when placed under the control of appropriate regulatory sequences. ) Nucleic acid sequence. The boundaries of the coding sequence are determined by a start codon at the 5 ′ (amino) terminus and a translation stop codon at the 3 ′ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3 'to the coding sequence.

A “nucleic acid” sequence refers to a DNA or RNA sequence. This term captures sequences including any known DNA and RNA base analogs, including but not limited to: 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudo Isocytosine, 5- (carboxyhydroxymethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine 1-methyl adenine, 1-methyl pseudouracil, 1-methyl guanine, 1-methyl inosine, 2,2-dimethyl guanine, 2-methyl adenine, 2-methyl guanine, 3-methyl cytosine, 5-methyl cytosine, N6 -Methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy-amino Methyl-2-thiouracil, β-D-mannosylcuosin, 5′-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil-5- Oxyacetic acid, oxybutoxocin, pseudouracil, cueosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, uracil -5-oxyacetic acid, pseudouracil, cueosine, 2-thiocytosine, and 2,6-diaminopurine.

  The term DNA “regulatory sequence” refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, etc., that are present in recipient cells. Collectively provides replication, transcription, and translation of the coding sequence. All of these control sequences need not always be present so long as the chosen coding sequence can be replicated, transcribed and translated in a suitable host cell.

  The term “promoter region” is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, where the regulatory sequence is capable of binding RNA polymerase and downstream (3 ′ direction). ) From the gene capable of initiating transcription of the coding sequence.

  “Operably linked” refers to an arrangement of elements in which the components so described are arranged to perform their normal function. Thus, a control sequence operably linked to a coding sequence can result in the expression of the coding sequence. A control sequence need not be contiguous with the coding sequence, so long as it functions to direct its expression. Thus, for example, an intervening untranslated but transcribed sequence can exist between a promoter sequence and a coding sequence, and this promoter sequence is still considered “operably linked” to the coding sequence. Can be.

  When referring to a nucleotide sequence, “isolated” means that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. Thus, an “isolated nucleic acid molecule that encodes a particular polypeptide” refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode a polypeptide of interest; It may contain some additional bases or moieties that do not adversely affect the basic characteristics.

  For purposes of describing the relative position of a nucleotide sequence in a particular nucleic acid molecule throughout this application, for example, a particular nucleotide sequence may be “upstream”, “downstream”, “3 ′”, or “5 ′” with respect to another sequence. Should be understood to be the position of the sequence in the “sense” or “coding” strand of a DNA molecule, said to be customary in the art.

  “Homology” refers to the percent identity between two polynucleotide or two polypeptide moieties. The correspondence between a sequence from one part and a sequence from another part can be determined by techniques known in the art. For example, homology can be determined by direct comparison of sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology is defined as polynucleotide hybridization under conditions that allow the formation of stable duplexes between homologous regions, followed by digestion with single-stranded specific nuclease (s), And can be determined by sizing the digested fragment. As determined using the above method, if at least about 80%, preferably at least about 90%, and most preferably at least about 95% of the nucleotides or amino acids match over a defined length of molecule, 2 Two DNA or two polypeptide sequences are “substantially homologous” to each other.

  A “functional homolog” or “functional equivalent” of a given polypeptide is a set that functions in a manner similar to a molecule derived from the native polypeptide sequence and the reference molecule to achieve the desired result. It includes polypeptides that are produced by replacement or chemically synthesized. Thus, functional homologues of AAVRep68 or Rep78 are derivatives and analogs of these polypeptides (addition of any single or multiple amino acids that occur internally or at the amino or carboxy terminus) as long as the integration activity remains. , Substitutions, and / or deletions).

  A “functional homolog” or “functional equivalent” of a given adenoviral nucleotide region includes a similar region derived from a heterologous adenovirus serotype, a nucleotide region derived from another viral or cellular source, and the desired A polynucleotide that functions in a manner similar to the reference nucleotide region, is produced recombinantly, or is chemically synthesized to achieve the results of Thus, a functional homologue of the adenovirus VARNA gene region or adenovirus E2a gene region has the ability of the homolog to provide its own auxiliary function to support AAV virion production at a detectable level above the background. As long as they are retained, include derivatives and analogs of such gene regions, including any single or multiple nucleotide base additions, substitutions and / or deletions occurring within the region.

B. General Methods The core of the present invention is the development of an auxiliary function system that efficiently produces rAAV virions without infection with helper virus. Unlike conventional production methods, auxiliary functions are provided by introducing one or more vectors (eg, plasmids) containing the necessary genes to supplement rAAV virion production into the host cell. In this manner, the accessory function system supports the production of industrially significant levels of rAAV virions that are uncontaminated by significant levels of helper virus particles or other viral products (eg, adenovirus fiber proteins). obtain. Efficient production of rAAV virions is achieved when the yield of rAAV virions is obtained at a level not less than about 200 times that obtained when using a type 2 adenovirus infection to provide accessory functions. Is done.

  Auxiliary functions are provided on one or more vectors. The vector contains the adenovirus-derived nucleotide sequence necessary for the production of rAAV virions. As described further below, the sequences present on the accessory function construct are determined by the host cell used and may include the E1a, E1b, E2a, E4, and VARNA regions.

Without being bound by any particular theory, it is believed that the accessory functions provided by adenovirus E1b, E2a, and E4 early genes are required in AAV DNA replication. The accessory functions provided by the adenovirus E1b, E4, and VARNA gene regions appear to be involved in post-transcriptional or translational events in the AAV life cycle. Regarding the auxiliary functions provided by E4, only the E434kD protein encoded by the open reading frame 6 (ORF6) of the E4 coding region is clearly required for AAV replication. The auxiliary functions provided by the adenoviral gene region E1a are the other adenoviral gene regions (E1b, E2a
, Including E4 and VA RNA) are thought to be necessary as regulators that activate transcription or expression.

  Alternatively, the accessory function vectors of the present invention can include one or more polynucleotide homologs that replace the adenovirus gene sequence, so long as each homolog retains the ability to provide the accessory function of the replaced adenovirus gene. . Thus, the homologous nucleotide sequence can be derived from another adenovirus serotype (eg, type 2 adenovirus), another helper virus portion (eg, herpes virus or vaccinia virus), or any other suitable supply. May come from the source.

Furthermore, accessory function vectors constructed in accordance with the present invention may be in the form of a plasmid, phage, transposon, or cosmid. Alternatively, the vector can be in the form of one or more linearized DNA or RNA fragments that, when combined with appropriate control elements and enzymes, can be transcribed or expressed to provide accessory functions in the host cell. . All of the above vectors can be readily introduced into suitable host cells using transfection techniques known in the art. Such transfection methods include calcium phosphate precipitation (Graham et al. (1973) Virol. 52 : 456-467), direct microinjection into cultured cells (Capecchi, MR (1980) Cell 22 : 479-488). Electroporation (Shigekawa et al. (1988) BioTechniques 6 : 742-751), liposome-mediated gene transfer (Mannino et al. (1988) BioTechniques 6 : 682-690), lipid-mediated transfection (Felgner et al. (1987) Proc Natl. Acad. Sci. USA 84 : 7413-7417) and nucleic acid delivery using high-speed microprojectiles (Klein et al. (1987) Nature 327 : 70-73) and are described in these documents.

  Accessory function vectors can be manipulated using conventional recombinant techniques. In particular, the nucleic acid molecule can be any desired by inserting one or more accessory function nucleotide sequences into the construct, for example, by linking a restriction enzyme fragment to a cloning vector using a polylinker oligonucleotide or the like. Can be easily assembled in the order in which they are made. The newly formed nucleic acid molecule can then be excised from the vector and placed in a suitable expression construct using restriction enzymes or other techniques well known in the art.

More particularly, selected adenoviral genes or gene regions (eg, E1a, E1b, E2a, E4, and VA RNA), or functional homologues thereof, are excised from the viral genome or from a vector containing the same. Standard ligation techniques (eg, as described in Sambrook et al., Supra) can be used and inserted into suitable vectors, individually or together, to provide auxiliary function constructs. With reference to FIG. 2, one such construct consists of four nucleic acid molecules derived from the type 5 adenovirus genome (region containing VARNA; region containing E2a; region containing E4, and region containing E1a, E1b). ). See Figure 2 for details.
Shows the following: A fragment containing VA RNA, a 1,724 bp SalI-HinDIII fragment (corresponding to nucleotides spanning from about 9,831 to about 11,555 positions of the type 2 adenovirus genome); E2a, a 5,962 bp SrfI-BamHI fragment A fragment comprising E4, a 3,669 bp HphI-HinDIII fragment (corresponding to about 32,172 to about 36,841 of the type 2 adenoviral genome). Corresponding to nucleotides spanning positions); and a fragment comprising E1a, E1b, which is a 4,102 bp BsrGI-Eco47III fragment (corresponding to nucleotides ranging from about 192 to about 4294 positions of the type 2 adenovirus genome), wherein the nucleic acid molecule is Linked together to provide complete complement of auxiliary functions in one auxiliary function construct. Ligation was performed using 20 mM Tris-Cl pH 7.5, 10 mM MgCl ₂ , 10 mM DTT, 33 μg / ml BSA, 10 mM to 50 mM NaCl, and 40 μMATP, 0.01 to 0.02 (Weiss) units of T4 DNA ligase (“sticky end” ligation). ), Or 1 mM ATP at 14 ° C. in 0.3-0.6 (Weiss) units of T4 DNA ligase (for “blunt end” ligation). Intramolecular “sticky end” ligations are usually performed at a total DNA concentration of 30-100 μg / ml (5-100 nM final concentration). The assembled molecule can then be readily inserted into an expression vector that can transfer the accessory function construct between cells.

Alternatively, nucleic acid molecules containing one or more auxiliary functions can be obtained synthetically using a combination of conventional solid phase direct oligonucleotide synthesis chemistry and enzyme ligation methods in the art. Synthetic sequences can be constructed to have features such as restriction enzyme sites and phosphoramidite methods on commercially available oligonucleotide synthesizers (eg, devices available from Applied Biosystems, Inc. (Foster City, Calif.)). Can be prepared using. See, for example, Beaucage et al. (1981) Tetrahedron Lett. 22 : 1859-1862. The nucleotide sequence of the type 2 adenovirus genome is generally known and publicly available (eg, GeneBank reference name: ADRCG, accession number: J01917; and NCBI identification number: 209811). The nucleotide sequence of the type 5 adenovirus genome is believed to be 99% homologous to the type 2 adenovirus genome. Preferred codons for expression of synthetic molecules in mammalian cells can also be readily synthesized. The complete nucleic acid molecule is then assembled from overlapping oligonucleotides prepared by the method described above. See, for example, Edge, Nature (1981) 292 : 756; Nambair et al., Science (1984) 223 : 1299; Jay et al., J. Biol. Chem. (1984) 259 : 6311.

When adenoviral gene regions are used in the vectors of the invention to provide accessory functions, these regions are operably linked to regulatory sequences that direct their transcription or expression. Such control sequences can usually include these adenovirus control sequences combined with a gene region in the wild type adenovirus genome. Alternatively, if a heterologous control sequence is desired, it can be used. Useful heterologous promoter sequences include promoter sequences derived from sequences encoding mammalian or viral genes. Examples include homologous adenovirus promoter, SV40 early promoter, mouse breast cancer virus LTR promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter (eg, CMV immediate early Promoter region), Rous sarcoma virus (RSV) promoter, synthetic promoter, hybrid promoter, etc., but are not limited thereto. In addition, sequences derived from non-viral genes such as the mouse metallothionein gene will also find use herein. Such promoter sequences are commercially available from, for example, Stratagene (San Diego, Calif.).

  Furthermore, the vectors of the present invention can also be constructed to include a selectable marker. Suitable markers include genes that confer antibiotic resistance, or susceptibility, or color, or change antigenic characteristics when cells transfected with the nucleic acid construct are grown in an appropriate selective medium. Including. Specific selectable marker genes useful in the practice of the present invention include the hygromycin B resistance gene (encoding aminoglycoside phosphotransferase (APH)), which allows selection in mammalian cells by conferring resistance to G418. Enable (available from Sigma, St. Louis, MO.). Other suitable markers are known to those skilled in the art.

  Auxiliary function vectors containing the complete complement of adenoviral accessory function genes or gene regions (eg, E1a, E1b, E2a, E4, VA RNA, and / or their functional homologs) are not tolerated by helper viruses. It can be used to provide accessory functions to a host cell containing cells (eg, not infectable by a helper virus such as adenovirus or unable to support helper virus replication). In this manner, rAAV virion production can be performed in a wide range of host cells, including host cells that have previously been refracted to support such production.

  Alternatively, auxiliary function vectors can be constructed to contain less than full complement of auxiliary functions. Such vectors may already be capable of providing one or more accessory functions, eg, one or more accessory functions genetically (eg, if the cell expresses a homologue of accessory functions) or by a transformation event. It can be used in a given cell. Auxiliary function vectors, including those that are less than full complement of auxiliary functions, can also be used in combination with other auxiliary auxiliary function constructs.

In particular, suitable host cells can be engineered using conventional recombinant techniques to produce cells that provide one or more accessory functions. For example, human cell line 293, type 5 adenovirus DNA fragments transformed human embryonic kidney cell line (Graham et al., (1977) J Gen.Virol 36: .. 59) in and, and adenovirus E1a and E1b The gene is expressed (Aiello et al. (1979) Virology 94 : 460). The 293 cell line is easily transfected and provides a particularly convenient platform for the production of rAAV virions. Accordingly, in one particularly preferred embodiment of the present invention, auxiliary function vectors having only adenovirus E2a, E4 and VARNA gene regions, or functional homologues thereof are provided.

  With reference to FIG. 1, one such construct consists of three nucleic acid molecules from the type 5 adenovirus genome: a fragment comprising VARNA, a 1,724 bp SalI-HinDIII fragment (from about 9,831 of the type 2 adenovirus genome). A fragment containing E2a, a SrfI-BamHI fragment of 5,962 bp (corresponding to nucleotides spanning from about 21,606 to about 27,568 of the type 2 adenovirus genome); and 3,669 bp of HphI -HinDIII fragment can be engineered to contain a fragment containing E4 (corresponding to nucleotides ranging from about 32,172 to about 36,841 positions of the type 2 adenovirus genome), where the nucleic acid molecules are ligated together to provide one auxiliary Provides a complementary complement of auxiliary functions in functional constructs.

  With reference to FIG. 3, another construct consists of three nucleic acid molecules from the type 2 adenovirus genome: a fragment containing VA RNA that is a 732 bp EcoRV-SacII fragment (from about 10,423 to about 11,155 of the type 2 adenovirus genome). A fragment containing E2, which is a 5,962 bp SrfI-KpnI fragment (corresponding to nucleotides spanning about 21,606 to about 27,568 of the type 2 adenovirus genome); and a 3,192 bp SrfI-SpeI modification It can be engineered to include a modified fragment comprising the fragment E4ORF6 (corresponding to nucleotides ranging from about 32,644 to about 34,120 of the type 2 adenovirus genome). The nucleic acid molecules are linked together to provide yet further truncated complements of auxiliary functions in one auxiliary function construct.

  These vectors can be constructed as described above using recombinant and / or synthetic techniques and can contain various auxiliary components such as heterologous promoter regions, selectable markers, and the like. Upon transfection into 293 host cells, the vector provides auxiliary functions that can support efficient production of rAAV virions.

  Once engineered, the accessory function vectors of the present invention can be used in a variety of systems for rAAV virion production. For example, when co-transfected with an AAV vector and an AAV helper construct that can be expressed in the cell to provide AAV helper function, suitable host cells transfected with one or more accessory function vectors thereby rAAV virions can be produced.

  AAV vectors, AAV helper constructs, and accessory function vectors can be introduced into host cells using the transfection techniques described above, either simultaneously or sequentially.

The AAV vector used to produce the rAAV virion to deliver the nucleotide sequence of interest has one or more heterologous nucleotides linked to a functional AAV ITR at both ends (5 ′ and 3 ′ ends). It can be constructed to contain a sequence. In the practice of the present invention, the AAV vector generally comprises at least one AAVITR and a suitable promoter sequence suitably located with respect to the heterologous nucleotide sequence, and at least one AAV ITR located downstream of the heterologous sequence. The 5 ′ and 3 ′ ITRs need not be identical to or derived from the same AAV isolate as long as they function as intended.

Suitable heterologous nucleotide sequences for use in AAV vectors include any functionally related nucleotide sequence. Thus, an AAV vector for use in the practice of the present invention may be any desired gene encoding a protein that is deleted or deleted from the genome of the recipient cell, or a desired biological or therapeutic effect (eg, antiviral function). Any desired gene encoding a non-natural protein having), or a sequence that may correspond to a molecule having antisense or ribozyme function. Suitable genes include, but are not limited to, genes that can be used for the following treatments: inflammatory diseases including diseases such as AIDS, cancer, neurological diseases, cardiovascular diseases, hypercholesterolemia, self Immune, chronic and infectious diseases; various blood diseases including various anemias, thalassemias and hemophilia; genetics such as capsular fibrosis, Gaucher disease, adenosine diaminase (ADA) deficiency, emphysema, etc. Deficiency. Many antisense oligonucleotides (eg, short oligonucleotides complementary to the sequence surrounding the translation start site (AUG codon) of mRNA) that are useful in antisense therapy of cancer, cardiovascular disease, and viral diseases are It has been described in the art. For example, Han et al. (1991) Proc. Natl. Acad. Sci. USA 88 : 4313-4317; Uhlmann et al. (1990) Chem. Rev. 90 : 543-584; Helene et al. (1990) Biochim. Biophys. Acta 1049 : 99-125; Agarwal et al. (1988) Proc. Natl. Acad. Sci. USA 85 : 7079-7083; and Heikkila et al. (1987) Nature 328 : 445-449. For a discussion of suitable ribozymes, see, for example, Cech et al. (1992) J. Biol. Chem. 267 : 17479-17482, and Goldberg et al. US Pat. No. 5,225,347.

AAV vectors may also contain control sequences such as promoters and polyadenylation sites, as well as selectable marker or reporter genes, enhancer sequences, and other control elements that allow for the induction of transcription. Such AAV vectors can be constructed using techniques well known in the art. For example, US Pat. No. 5,173,414; International Publication Nos. WO92 / 01070 (published on January 23, 1992) and WO93 / 03769 (published on March 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8 : 3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, BJ (1992) CurrentOpinion in Biotechnology 3 : 533-539; Muzyczka, N (1992) Current Topics in Microbiol. And Immunol. 158 : 97-129; Kotin, RM (1994) Human Gene Therapy 5 : 793-801; Shelling and Smith (1994) GeneTherapy 1 : 165-169; and Zhou et al. (1994). ) See J. Exp. Med. 179 : 1867-1875.

In the methods of the invention, an AAV helper construct is used to complement the AAV function deleted from the AAV vector. A number of suitable AAV helper constructs have been described, including: plasmids pAAV / Ad and pIM29 + 45 encoding both rep and cap expression products (eg, Samulski et al. (1989) J. Virol. 63 : 3822-3828 and McCarty et al. (1991) J. Virol. 65 : 2936-2945). Complementary AAV helper functions that support rAAV virion production in this manner are art accepted techniques. However, due to the homologous recombination event between the AAVITR sequence present in the AAV vector and the AAV helper functional sequence present in the helper construct, such a technique also allows for the wild-type AAV virion in the rAAV virion stock. Causes contamination. The presence of wild-type AAV particles in an AAV-based vector system can potentially lead to unintended expansion of recombinant AAV virions and prevent efficient expression of foreign genes.

C. The following are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

  Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental error and deviation should, of course, be allowed for.

Plasmid Construction Plasmid JM17 (McGrory et al. (1988) Virology 163 : 614-617) (contains a circular isolate of type 5 adenovirus with plasmid vector pBR322X inserted into the unique XbaI site at the end of the adenovirus E1a gene) Was used as a source of adenoviral genes. Both type 5 adenovirus and pJM17 are not fully sequenced. Thus, the size of the type 5 adenovirus fragment described below is nearly accurate. Since the type 2 adenovirus has been fully sequenced and the type 2 and type 5 adenoviruses are believed to be approximately 99% homologous, the size of the type 5 adenovirus fragment is the corresponding type 2 adenovirus. Approximation based on viral fragments.

  Plasmid pBSII-VARNA (ATCC access number 98233) was constructed as follows. An approximately 5,324 bp HinDIII-HinDIII fragment (including the type 5 adenovirus VA RNAI and II coding regions) was obtained from plasmid pJM17. The 5,324 bp fragment was inserted into the HinDIII site of the plasmid vector pBIIs / k- (obtained from Stratagene). The 5,324 bp fragment extends from position 6,231 to position 11,555 (HinDIII site) of the type 2 adenovirus genome (eg, publicly available as GeneBank reference name: ADRCG, access number: J01917; and NCBI identification number: 209811) Corresponding to

Plasmid pBSII-E2a + E4 was constructed as follows. An approximately 15,667 bp BamHI-XbaI fragment containing a type 5 adenovirus E2a, E3, and E4 coding region, a head-to-head adenovirus terminal repeat, and a portion of a type 5 adenovirus E1a coding region Obtained from pJM17. The plasmid vector pBSIIs / k- was cut with BamHI and XbaI and the 15,667 bp fragment was cloned into the vector of interest. The 15,667 bp fragment consists of a nucleotide extending from position 21,606 (BamHI site) to position 35,937 (distal end of the 3 ′ end) and position 1 (first of 5 ′ end) to position 1,336 (XbaI site) of the type 2 adenovirus genome Corresponds to nucleotides extending to).

  Plasmid pBSII-E2a was constructed as follows. An approximately 5,935 bp BamHI-EcoRI fragment (including the type 5 adenovirus E2a coding region) was obtained from plasmid pJM17. The plasmid vector pBSIIs / k- was cut with BamHI and EcoRI and the 5,935 bp fragment was cloned into the vector of interest. The 5,935 bp fragment corresponds to a nucleotide extending from position 15,403 (BamHI site) to position 21,338 (EcoRI site) of the type 2 adenovirus genome.

  Plasmid pBSII-E4 was constructed as follows. An approximately 5,111 bp XhoI-XbaI fragment (containing the adenovirus E4 coding region, head-to-head adenovirus terminal repeat and part of the type 5 adenovirus E1 coding region) was obtained from plasmid pJA17. The plasmid vector pBSIIs / k- was cut with XhoI and XbaI and the 5,111 bp fragment was cloned into the vector of interest. The 5,111 bp fragment consists of nucleotides extending from position 29,788 (XhoI site) to position 35,937 (distal end of the 3 ′ end) and position 1 (first of 5 ′ end) to position 1,336 (XbaI site) of the type 2 adenovirus genome Corresponds to nucleotides extending to).

  Plasmid pWadhlacZ was constructed as follows. Plasmid pUC119 (GeneBank reference number: U07649, GeneBank accession number: U07649) is partially digested with AflIII and BspHI, modified to blunt ends using Klenow enzyme, and then ligated to the bacterial origin and amp gene only A circular 1732 bp plasmid containing (with the polylinker and F1 origin removed) was formed. The blunt-ended and ligated AflIII and BspHI junctions form a unique NspI site. The 1732 bp plasmid was cleaved with NspI, modified to blunt end using T4 polymerase, and the 20 bp HinDIII-HinCII fragment obtained from pUC119 polylinker (modified to blunt end using Klenow enzyme) Ligated to the activated NspI site. The blunted polylinker-derived HinDIII site was recreated and then placed adjacent to the bacterial origin of replication. The resulting plasmid was then cut at the unique PstI / Sse8387I site and the Sse8387I-PvuII-Sse8387I oligonucleotide (5′-GGCAGCTGCCTGCA-3 ′ (SEQ ID NO: 1)) ligated thereto. The only remaining BspHI site is cleaved, modified to blunt end using Klenow enzyme, and an oligonucleotide containing AscI linker (5′-GAAGGCGCGCCTTC-3 ′ (SEQ ID NO: 2)) is ligated thereto and BspHI The site was excluded. The resulting plasmid was called pWee.

  To create a pWadhlacZ construct, a CMVlacZ expression cassette (containing a nucleotide sequence flanked 5 ′ and 3 ′ by an AAV ITR, where the nucleotide sequence includes the following elements: CMV promoter, first intron of hGH, The adhlacZ fragment, and the SV40 early polyadenylation site) were inserted into the unique PvuII site of pWee using multiple steps, so that the CMV promoter was placed adjacent to the bacterial amp gene of pWee.

More specifically, the CMVlacZ expression cassette was derived from plasmid psub201CMV and was constructed as follows. Restriction enzyme sites: NotI-MluI-SnaBI-AgeI-BstBI-BssHII-NcoI-HpaI-BspEI-PmlI-RsrII-NotI
Was synthesized and cloned into the KasI-EarI site (partial) modified to the blunt end of pUC119, resulting in a 2757 bp vector fragment. A 653 bp SpeI-SacII fragment containing the nucleotide sequence encoding the CMV immediate early promoter was cloned into the SnaBI site of the 2,757 bp vector fragment. In addition, a 269 bp PCR BstBI-BstBI fragment containing a nucleotide sequence encoding the first intron of hGH (which was obtained using the following primer: 5′-AAAATTCGAACCTGGGGAGAAACCAGAG-3 ′ (SEQ ID NO: 4 ) And 3′-aaaattcgaacaggtaagcgcccctTTG-5 ′ (SEQ ID NO: 5)) were cloned into the BstBI site of the 2,757 bp vector fragment and contained the SV40 early polyadenylation site from the pCMV-β plasmid (obtained from Clonetech). A 135 bp HpaI-BamHI (modified to blunt end) fragment was cloned into the HpaI site of the vector fragment of interest to yield a plasmid called p1.1c. The p1.1c plasmid was then cut with NotI to obtain the first CMV expression cassette.

Plasmid psub201 (Samulski et al. (1987) J. Virol 61 : 3096-3101) was cut with XbaI, modified to blunt ends, and a NotI linker (5′-TTGCGGCCGCAA-3 ′ (SEQ ID NO: 6)) at its ends. To obtain a vector fragment containing the bacterial origin of replication and the amp gene. Here, the vector fragment is flanked on both sides by the NotI site. After cutting with NotI, the first CMV expression cassette was cloned into the psub201 vector fragment to generate psub201CMV. The expression cassette bound to the ITR from this plasmid was isolated by digestion with PvuII, and pWee was digested with PvuII and ligated to this plasmid to create pWCMV. The pWCMV was then cleaved with BssHII (partially) and a 3246 bp fragment containing the adhlacZ gene (AscI linker (5'-GAAGGCGCGCCTTC-3 '(SEQ ID NO: 5) at the end of the SmaI-DraI nucleotide fragment obtained from plasmid pCMV-β). 7)) was ligated to obtain a 3246 bp fragment) was ligated to the BssHII site of pWCMV to obtain the pWadhlacZ construct.

  Plasmid pW1909adhlacZ was constructed as follows. A 4723 bp SpeI-EcoRV fragment containing the AAVrep and cap coding regions was obtained from plasmid pGN1909 (ATCC accession number 69871). The pGN1909 plasmid is a highly efficient AAV helper plasmid with an AAVrep and cap gene, with an AAVp5 promoter region located in a construct that is downstream from its normal position (position in the wild type AAV genome) with respect to the rep coding region. The 4723 bp fragment was modified to blunt ends and an AscI linker (5′-GAAGGCGCGCCTTC-3 ′ (SEQ ID NO: 8)) was ligated to the blunt ends. The resulting fragment was then ligated to the unique AscI site of pWadhlacZ and oriented so that the AAV coding sequence was placed adjacent to the bacterial origin of replication in the construct.

Plasmid pW620adhlacZ was constructed as follows. A 4439 bp MscI fragment containing the AAVrep and cap coding regions was obtained from plasmid pSM620 (Samulski et al. (1982) Proc. Natl. Acad. Sci. USA 79 : 2077-2081). The 4439 bp fragment was modified to blunt ends and an AscI linker (5′-GAAGGCGCGCCTTC-3 ′ (SEQ ID NO: 9)) was ligated to the blunt ends. The resulting fragment was then ligated to the unique AscI site of pWadhlacZ and oriented so that the AAV coding sequence was placed adjacent to the bacterial origin of replication in the construct.

  Plasmid pW1909 was constructed as follows. The pW1909adhlacZ plasmid was cut with Sse8387 and the 6506 bp Sse8387I-Sse8387I fragment (containing the ampicillin resistance gene, the coli1 origin of replication, and the AAV helper sequence) was recirculated by intramolecular ligation to yield the pW1909 construct.

  The pVlacZ vector plasmid is a modified version of the Sse8387I-Sse8371I fragment obtained from pW1909adhlacZ cloned in the pUC119 plasmid. pVlacZ Sse8387I-Sse8371I differs from the Sse8387I-Sse8371I fragment of pW1909 in that all AAV sequences that are not derived from AAV inverted terminal repeats (ITR) are eliminated. Plasmid pVlacZ was constructed as follows. A synthetic fragment of the DNA formed by combining AAV serotype 2 base pairs 122-145 with downstream NotI compatible ends was constructed, resulting in an MscI-NotI fragment containing an AAVITR sequence containing all D-loops. Synthetic DNA was ligated to both ends of a 4384 bp NotI fragment derived from the pW1909adhlacZ plasmid. The 4384 bp fragment contains the CMVlacZ sequence. The resulting fragment was then ligated to the 6732 bp MscI fragment of pW1909adhlacZ to give an assembly construct. The assembly construct was cut with Sse8387I to give a 4666 bp Sse8387I-Sse8371I fragment (containing the CMVlacZ sequence), and the 4666 bp fragment was ligated into pUC119 at the Sse8387I site to give the pVlacZ vector plasmid.

Example 1
Production of rAAV virions using transfected adenoviral genes to provide auxiliary functions Adenoviral genes introduced by transfecting into appropriate host cells are provided by adenivirus infection in the context of AAV replication The following experiment was conducted to determine whether a similar auxiliary function could be provided.

Cells from a stable human cell line, 293 (eg readily available through ATCC under access number CRL1573), were plated at 1 × 10 ⁶ cells / dish in a 10 cm tissue culture dish and washed 4 times. Duplicate experimental groups were provided. The cells were then grown at 37 ° C. and allowed to reach 90% confluence for approximately 24-48 hours prior to transfection. In each group, the plasmid pW1909adhlacZ was used as a source of rescueable AAVlacZ vector and AAVrep and cap coding regions.

Transfection was performed with a total of 20 μg of DNA for 5 hours using a modified calcium phosphate method. More specifically, media in tissue culture plates 1 to 4 hours prior to transfection with fresh DME / F12 culture media containing 10% FCS, 1% pen / strep, and 1% glutamine. Replaced. A total of 20 μg of DNA containing one or more vectors is added to 1 mL of sterile 300 mM CaCl ₂ , which is then added to 1 mL of sterile 2 × HBS solution (280 mM NaCl, 50 mM HEPES buffer, 1.5 mM Na ₂ HPO ₄ was mixed and added to (formed by adjusting to pH 7.1 with 10M NaOH) and mixed immediately by gentle inversion. The resulting mixture was immediately pipetted into 90% confluent cells (in 10 mL of the above culture medium) in a 10 cm plate and rotated to make a homogeneous solution. Plates were transferred to a 5% CO ₂ incubator and incubated at 37 ° C. for about 5 hours without agitation. After transfection, the medium was removed from the plate and the cells were washed once with sterile phosphate buffered saline (PBS).

Add adenovirus working stock, type 2 adenovirus master stock, 10% FCS, 1% pen / strep, 1% glutamine, and 25 mM sterile HEPES buffer (pH 7.4) Prepared in DME / F12 by dilution to a concentration of 10 ⁶ pfu / ml.

  Cell cultures from Group 1 were transfected with 10 μg each of plasmids pW1909adhlacZ and pBSII s / k−. After the transfection period, the medium was replaced, 10 mL of medium containing type 2 adenovirus at a multiplicity of infection (moi) was added, and the culture was incubated at 37 ° C. for about 72 hours.

  Cell cultures from group 2 were transfected with 10 μg each of plasmids pW1909adhlacZ and pJM17. After the transfection period, the medium was replaced and the culture was incubated at 37 ° C. for about 72 hours.

  Cell cultures from Group 3 were transfected with 10 μg each of pBSII s / k- and pJM17. After the transfection period, the medium was replaced and the culture was incubated at 37 ° C. for about 72 hours.

  Cell cultures from Group 4 were transfected with 10 μg each of pW1909adhlacZ and pBSII s / k−. After the transfection period, the medium was replaced and the culture was incubated at 37 ° C. for about 72 hours.

  Cells from each experimental group were collected, the medium was removed by centrifugation (1000 × g, 10 minutes), and 1 mL of lysate was removed from 3 freeze / thaw cycles (dry ice-ethanol and 37 ° C. Obtained by using a hot water bath). The lysate was free of debris by centrifugation (10,000 × g, 10 minutes). The rAAVlacZ virion product was evaluated by titrating the freeze / thaw extract on 293 cells and assaying for lacZ.

Specifically, 293 cells were plated in 12-well plates (1 × 10 ⁵ cells per well) and inoculated with the above frozen / thawed lysate volume range (10-0.01 μL) and 24 ° C. at 24 ° C. Incubated for hours. Then media removal, incubation of cells for 5 minutes in PBS containing 2% formaldehyde and 0.2% glutaraldehyde, one wash with PBS, then 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM Cells were fixed and stained by overnight incubation of the cells in PBS containing magnesium chloride and 1 mg / ml X-Gal (Sanes et al. (1986) EMBO 5 : 3133-3142). The rAAV virion titer was then calculated by quantifying the number of blue cells using a light microscope.

Contaminated infectious adenovirus products were assayed as follows. Samples from the cell lysate are added to 50% confluent 293 cells (cultured at 1 × 10 ⁵ cells / well in a 12-well dish) and the cultures for 30 days (eg, cultures every 3 days). 1-5), or the culture was allowed to elapse until it showed 100% CPE due to adenovirus infection. Cultures were tested daily for CPE, and the day on which each experimental culture exhibited 100% CPE was recorded. Reference 293 cell cultures infected with a range of known amounts of type 2 adenovirus (0-1 × 10 ⁷ pfu / culture) were also prepared and processed in the same manner. A standard curve was then prepared with the data obtained from the reference culture. Here, the pfu numbers of adenovirus were plotted against the day of 100% CPE. The titer of infectious type 2 adenovirus in each experimental culture was then easily obtained as determined from a standard curve. The detection limit in the assay was 100 pfu / mL.

  The results of the experiment are shown in Table 1 below.

  As can be seen by the results in Table 1, adenoviral genes introduced into host cells by transfection and expressed in the absence of adenoviral infection are at the level of accessory function provided by adenoviral infection. Auxiliary functions may be provided at about 20% (ie, 5 times less) levels of effectiveness.

Example 2
Identification of Adenovirus Gene Regions Responsible for Auxiliary Functions In order to determine which adenoviral genes or gene regions are necessary and sufficient for providing auxiliary functions, the following experiments were conducted.

293 cells were plated on 12 10-cm tissue culture dishes at 1 × 10 ⁶ cells / dish to provide six duplicate experimental groups. The cells were then grown to reach about 90% confluency at 37 ° C. for a period of about 24-48 hours prior to transfection. In each group, plasmid pW1909adhlacZ was used as a source of rescueable AAVlacZ vector and AAVrep and cap coding regions. Transfection was performed as described in Example 1 above. An adenovirus working stock was also prepared as described above.

  Each 293 cell culture was transfected with either plasmid pW1909adhlacZ and plasmid pBSIIs / k- (as a control) or various combinations of isolated adenoviral genes.

  More specifically, cells from the first experimental group were co-transfected with 5 μg of plasmid pW1909adhlacZ (providing AAV auxiliary function) and 15 μg of plasmid pBSII s / k−. After the transfection period, the medium was changed and the cells were infected with 10 mL medium containing type 2 adenovirus (moi = 1). The culture was then incubated at 37 ° C. for about 72 hours.

  Cells from the second experimental group were co-transfected with 5 μg of plasmid pW1909adhlacZ (providing AAV accessory function) and 15 μg of plasmid pJM17. After transfection, the medium was changed and the culture was incubated at 37 ° C. for about 72 hours.

  Cells from the third experimental group were co-transfected with 5 μg plasmid pW1909adhlacZ (providing AAV accessory function), 10 μg plasmid pBSII-E2a + E4, and 5 μg plasmid pBSIIs / k−. After transfection, the medium was changed and the culture was incubated at 37 ° C. for about 72 hours.

  Cells from the fourth experimental group were cotransfected with 5 μg of plasmid pW1909adhlacZ (providing AAV accessory function), 10 μg of plasmid pBSII-E2a + E4, and 5 μg of plasmid pBSII-VARNA. After transfection, the medium was changed and the culture was incubated at 37 ° C. for about 72 hours.

  Cells from the fifth experimental group were co-transfected with 5 μg of plasmid pW1909adhlacZ (providing AAV accessory function), 5 μg of plasmid pBSII-E2a, 5 μg of plasmid pBSII-E4 and 5 μg of plasmid pBSIIs / k−. After transfection, the medium was changed and the culture was incubated at 37 ° C. for about 72 hours.

  Cells from the sixth experimental group were co-transfected with 5 μg plasmid pW1909adhlacZ (providing AAV accessory function), 5 μg plasmid pBSII-E2a, 5 μg plasmid pBSII + E4 and 5 μg plasmid pBSII-VARNAs. After transfection, the medium was changed and the culture was incubated at 37 ° C. for about 72 hours.

  Cells from each experimental group were then collected, the medium was removed by centrifugation (1000 × g, 10 minutes), and 1 mL of lysate was removed in 3 freeze / thaw cycles (dry ice-ethanol bath and 37 ° C. In place of bath). Centrifugation (10,000 × g, 10 minutes) produced a lysate free of debris. The production of rAAVlacZ virions was then assessed using the technique described in Example 1 and the amount of rAVV genome was quantified by the following assay.

Lysates from each experimental group of 50λ were added to 100λ aliquots of DMEM medium (available from Sigma, St. Louis, MO) containing 50 U / mL DNAseI to make assay samples. After incubating the sample for about 1 hour at 37 ° C., 100 λ proteinase K (1 mg / mL) in 2 × proteinase K buffer (20 mM Tris Cl, 20 mM EDTA, 1% SDS, pH adjusted to 8.0) was added to each sample. In addition to the sample, it was then incubated for an additional hour at 37 ° C. DNA from each sample was phenol / chloroform extracted, precipitated in ethanol, and then recovered by centrifugation at 5 ° C. for 15 minutes. The DNA pellet was then redissolved in 200λ TE to provide a DNA sample. A dot plot assay was then performed as follows. A ζ probe (ZetaProbe®) membrane (BioRad, Richmond, Calif.) was cut to size and assembled into a dot plot device. The DNA sample was denatured with 200λ 2 × alkaline solution (0.8 M NaOH, 20 mM EDTA) and after 5 minutes, the membrane was rinsed in 2 × SSC solution for 1 minute, dried on filter paper, then And baking at 80 ° C. for about 30 minutes under vacuum. Hybridization was performed at 65 ° C. for 30 minutes in a hybridization buffer (1 mM EDTA, 40 mM Na ₂ HPO ₄ (pH 7.2), 7% SDS). Filters were then washed and autoradiographed for about 20 hours and radioactivity was determined using scintillation counting.

  The results of the experiment are shown in Table 2 below.

  As can be seen from the results shown in Table 2, the isolated adenoviral gene regions can be successfully transfected into host cells to provide the necessary and sufficient accessory functions for rAAV virion replication. Furthermore, the results obtained in Groups 3 and 5 indicate that the adenovirus VARNA region is not essential for rAAV virion replication. However, this region is necessary to obtain rAAV titers comparable to those obtained using adenovirus infection.

Example 3
Correlation of Adenovirus VARNA Dosage for rAAV Virion Production Between the amount of adenovirus VA RNA gene region transfected into the host cell and the level of accessory function provided to complement rAAV replication in the host cell In order to investigate whether a correlation exists, the following experiment was conducted.

293 cells are plated at 1 × 10 ⁶ cells / dish in 10-cm tissue culture dishes and cultured at 37 ° C. until reaching about 90% confluency for a period of about 24-48 hours prior to transfection. did. Transfection was performed as described in Example 1 above.

  Specifically, 293 cells were transfected with 5 μg of plasmid pW1909adhlacZ, 10 μg of plasmid pBSII-E2a + E4 and 0-25 μg of plasmid pBSII-VARNA to determine the molar ratio of VARNA with plasmid (relative to other plasmids). It varied over a range of 0-5. After the transfection period, the medium was changed and the cells were incubated at 37 ° C. for about 72 hours.

Cells from each experimental group were then collected, the medium was removed by centrifugation (1000 × g, 10 minutes), and 1 mL of lysate was removed in 3 freeze / thaw cycles (dry ice-ethanol bath and 37 ° C. In place of bath). Centrifugation (10,000 × g, 10 minutes) produced a lysate free of debris. The production of AAVlacZ vector was then evaluated using the technique described in Example 1. The results of the experiment are shown in Table 3 below.

  As can be seen from Table 3, the adenoviral VA RNA was prepared in spite of being required to obtain rAAV virion production at a level substantially equivalent to that obtained with adenoviral infection. Variation in the ratio of VARNA / other adenovirus gene regions in rAAV does not significantly affect the production of rAAV virions.

Example 4
Adenovirus E2a, E4 and VA RNA gene regions in auxiliary functions
The demand for
The following experiments were performed with the aim of probing the relative contribution of adenovirus E2a, E4 and VA RNA gene regions in providing accessory functions for rAAV virion production.

293 cells are plated at 1 × 10 ⁶ cells / dish in 10-cm tissue culture dishes and reach about 90% confluency at 37 ° C. for a period of about 24-48 hours prior to co-transfection. Cultured. All transfections were performed as described in Example 1 above.

  Each adenovirus encoding E2a, E4 and VARNA was co-transfected with 5 μg of plasmid pW1909adhlacZ and either 15 μg (total) of any of the following plasmid combinations or combinations thereof: Cultures were provided that excluded gene regions from co-transfection: pBSII-E2a; pBSII-E4; and pBSII-VARNA. After the transfection period, the medium was changed and the cells were incubated at 37 ° C. for about 72 hours.

  Cells from each co-transfection group were then collected, the medium was removed by centrifugation (1000 × g, 10 minutes), and 1 mL of lysate was removed in 3 freeze / thaw cycles (dry ice-ethanol bath and Using 37 ° C bath). Centrifugation (10,000 × g, 10 minutes) produced a lysate free of debris. The production of rAAVlacZ virions was then evaluated using the technique described in Example 1. The results of the experiment are shown in Table 4 below.

As can be seen from the results shown in Table 4, each adenoviral gene region E2a, E4 and VA RNA need not provide any auxiliary functions necessary to support rAAV virion production. However, all gene regions are necessary to produce rAAV virion levels comparable to those obtained with adenovirus infection. More specifically, omitting plasmid-containing VARNA from co-transfection resulted in a 7-fold reduction in rAAV virion production (7 × 10 ⁸ functional units / 10 cm dish). Production of rAAV virions was further significantly affected by omission from co-transfection of E4 and E2a containing plasmids. Omission of the E4 construct resulted in an 83-fold reduction in production (6 × 10 ⁷ functional units / 10 cm dish), and omission of the E2 construct resulted in a 10,000-fold reduction in rAAV virion production (5 × 10 ⁵ functional units) / 10cm dish).

Example 5
Using AAV help based on pW1909adhlacZ or pW620adhlacZ
Comparison of rAAV virion production Compare the efficiency of rAAV virion production in host cells using a non-viral accessory function system with an AAV helper construct containing either wild-type or modified AAV help functions (AAV rep and cap coding regions) The following experiment was conducted for the purpose of:

293 cells are plated at 1 × 10 ⁶ cells / dish in 10-cm tissue culture dishes and reach about 90% confluency at 37 ° C. for a period of about 24-48 hours prior to co-transfection. Cultured. All transfections were performed as described in Example 1 above.

  A first set of 293 cell cultures were co-transfected with 5 μg of plasmid pW620adhlacZ (including wild type AAV help function), 10 μg of plasmid pBSII-E2a + E4 and 5 μg of plasmid pBSII-VARNA. A second set of 293 cultures was co-transfected with 5 μg of plasmid pW1909adhlacZ (including the modified AAV help function), 10 μg of plasmid pBSII-E2a + E4 and 5 μg of plasmid pBSII-VARNA. Following transfection, the medium was changed and the culture was incubated at 37 ° C. for about 72 hours.

Cells from each co-transfection group are then collected and the medium is centrifuged (1000 × g
, 10 minutes) and 1 mL of lysate was produced using 3 freeze / thaw cycles (alternating dry ice / ethanol bath and 37 ° C. bath). Centrifugation (10,000 × g, 10 minutes) produced a lysate free of debris. The production of rAAVlacZ virions was then evaluated using the technique described in Example 1. The results of the experiment are shown in Table 5 below.

  As can be seen from the results shown in Table 5, both wild-type and modified forms of AAV help supported rAAV virion production at approximately the same level.

Example 6
Comparison of plasmid- based auxiliary function rAAV virion production efficiency Isolated plasmid-based auxiliary functions, combinations thereof, and a single containing adenovirus VA RNA, E4 and E2a gene regions (pladeno1 plasmid above) In order to compare the efficiency of rAAV virion production in host cells with the construct and the efficiency of rAAV virion production obtained using type 2 adenovirus (ad-2) infection, the following experiment was performed.

1. Construction of pladeno1 and pladeno1E1:
The pladeno1 plasmid (including the adenovirus VA RNA, E4 and E2a gene regions) was assembled by cloning the type 5 adenovirus gene into a conventional polylinker inserted between the PvuII sites of pBSII s / k-. A map of the pladeno1 construct is shown in FIG. More specifically, a double-stranded oligonucleotide polylinker encoding the restriction enzyme site SalI-XbaI-EcoRV-SrfI-BamHI (5′-GTCGACAAATCTAGATATCGCCCGGGCGGATCC-3 ′: SEQ ID NO: 10) Ligated to the vector fragment to provide an assembly plasmid. The following fragment containing the type 5 adenovirus gene or gene region was then obtained from the pJM17 plasmid: a 1,724 bp SalI-HinDIIIVA RNA-containing fragment (corresponding to nucleotides spanning about positions 9,831 to 11,555 of the type 2 adenovirus genome) ); A 5,962 bp SrfI-BamHIE2a containing fragment (corresponding to nucleotides spanning about positions 21,606 to about 27,568 of the type 2 adenovirus genome); and a 3,669 bp HphI-HinDIIIE4 containing fragment (about 32,172 to about 2 of the type 2 adenovirus genome) Corresponding to nucleotides spanning 36,841). The XbaI site was added by cloning a 3,669 bp HphI-HinDIII fragment into the HpaI site of the cloning vector at the HphI end of the fragment containing E4 and then excising the fragment with XbaI and HinDIII (partial cleavage). The fragment containing 5,962E2a was cloned between the SrfI and BamHI sites of the assembly plasmid, and the 1,724 bp VARNA containing fragment and the modified 3,669 bp E4 containing fragment were joined by their common HinDIII ends and assembled Plasmids SalI and XbaI
Ligated between the sites, a pladeno1 construct was obtained.

As shown in FIG. 2, the pladeno1E1 plasmid was assembled as follows. A 4,102 bp BsrGI-Eco47III fragment (containing the Ela and Elb coding regions of type 5 adenovirus) was obtained from the pJK17 plasmid. The subject fragment corresponds to a nucleotide spanning about positions 192 to about 4,294 of the type 2 adenovirus genome. The 4,102 bp fragment was blunt end modified and then inserted into the HpaI site in the VA RNA fragment of pladenol 1 plasmid, resulting in pladenol 1 El plasmid.

2. rAAV virion production assay:
293 cells are plated at 1 × 10 ⁶ cells / dish in 10-cm tissue culture dishes and reach about 90% confluency at 37 ° C. for a period of about 24-48 hours prior to co-transfection. Cultured. All transfections were performed as described in Example 1 above.

  All 293 cell cultures were transfected with 5 μg of plasmid pW1909adhlacZ. Also, experimental groups of cultures were co-transfected with various combinations of 5 μg of each of the accessory function containing plasmids or control plasmids (pBSIIs / k−). Following transfection, the medium was changed and the culture was incubated at 37 ° C. for about 72 hours. For comparison, 10 mL of medium containing type 2 adenovirus (moi = 1) was added to 293 cells transfected with 5 μg of plasmid pW1909adhlacZ and incubated at 37 ° C. for about 72 hours.

Next, cells were collected from each experimental group, and the medium was removed by centrifugation (1000 × g for 10 minutes). Three freeze / thaw cycles (alternating dry ice-ethanol bath and 37 ° C. bath) were then used to produce 1 mL of lysate. Debris was removed from the lysate by centrifugation (10,000 × g for 10 minutes). The techniques described in Example 1 were then used to assess rAAV lacZ virion production. The results of the experiment are shown in Table 6 below.

  As the results in Table 6 can show, the pladeno 1 construct can support efficient rAAV virion production (substantially the same level as obtained using type 2 adenovirus infection). The combination of accessory function constructs pBSII-E2a, pBSII-E4, and pBSII-VARNA could also support efficient rAAV virion production at a level substantially equivalent to ad-2 infection. The combination of accessory function constructs pBSII-E2a + E4 and pBSII-VARNA could support rAAV production (10-fold below ad-2 infection levels); and the construct containing E2a (pBSII-E2a) Supporting rAAV production at a level approximately 200-fold below that obtained using two infections.

Example 7
Comparison of large-scale rAAV virion production obtained using adenovirus-based or plasmid-based auxiliary functions Use either type 2 adenovirus (ad-2) -based or pladeno 1-based auxiliary functions In order to compare the large-scale preparation of rAAV virions produced by

Using a transfection method described in Example 1, were transfected with about ¹⁰⁹ 293 cells in AAV vector containing the human erythropoietin gene. As a source of accessory function, one preparation was cotransfected with pladenol construct and the other preparation was infected with type 2 adenovirus.

  After an appropriate incubation period, cells were then harvested from each experimental group and the growth medium was removed by centrifugation (1000 × g for 10 minutes). The lysate was then generated using three freeze / thaw cycles (alternating dry ice-ethanol bath and 37 ° C. bath). Debris was removed from the lysate by centrifugation (10,000 × g for 10 minutes) to obtain a crude lysate. The crude lysate was subjected to density gradient centrifugation to obtain a purified sample.

  The amount of rAAV genome produced by each preparation was quantified by the dot blot assay described in Example 2. The results of the experiment are shown in Table 7 below.

  As the results in Table 7 show, the preparation using the auxiliary function based on pladeno 1 provides a rAAV virion yield 2.4 times greater than that obtained from the preparation using the auxiliary function based on type 2 adenovirus. did.

Example 8
Determination of the relative contribution of individual adenovirus accessory functions in rAAV virion production
In order to determine the relative contribution of individual adenovirus accessory functions in rAAV virion production, the following experiment was performed. Individual adenovirus accessory functions (either alone or in combination) were used to assist rAAV virion production in the host cells. In addition, constructs that include the entire E2a and E4ORF6 regions and are driven by homologous promoters (pBSII-E2a and pBSII-E4 constructs) are replaced by CMV-driven E2a or E4ORF6 constructs (p3.3cE2A and p3.3cE4ORF6 constructs; ) Was evaluated.

1. Construction of p3.3cE2A and p3.3cE4ORF6 :
The structural genes encoding the type 5 adenovirus E2a 72kD DNA binding protein and the type 5 adenovirus E4 open reading frame 6 (ORF6) protein were each cloned into a CMV-driven expression construct (p3.3c), respectively. A p3.3cE2A plasmid construct and a p3.3E4ORF6 plasmid construct were provided.

  Plasmid p3.3c was constructed as follows. The p1.1c-derived 2732 bp NotI fragment containing the pUC119 vector sequence was ligated to a synthetic DNA fragment containing the following restriction sites: NotI; MluI; Ecl136II; SacII; BstBI; BssHII; SrfI; BssHII; BglII; SnaBI; BstEII; PmlI ; RsrII; and NotI. The sequence of the synthetic DNA fragment is: 5′-GCGGCCGCACGCGTGAGCTCCGCGGTTCGAAGCGCGCAAAGCCCGGGCAAAGCGCGCAGATCTACGTAGGTAACCACGTGCGGACCGGCGGCCGC-3 ′ (SEQ ID NO: 11). Using the following primers 5'-AAAATTCGAACAGGTAAGCGCCCCTTTG-3 '(SEQ ID NO: 12) and 3'-AAAATTCGAATCCTGGGGAGAAACCAGAG-5' (SEQ ID NO: 13) containing the cytomegalovirus immediate early (CMVIE) promoter The resulting 269 bp PCR-generated BstBI-BstBI fragment encoding the hGH first intron; and a 213 bp BamHI-BamHI (blunted) fragment containing the SV40 late polyadenylation site from pCMV-β (obtained from Clonetech), respectively, Cloning into the synthetic linker Ecl136II, BstBI, and SnaBI sites yielded the p3.3c expression construct.

  Plasmid p3.3cE4ORF6 (ATCC accession number 98234) was prepared as follows. A pBSII-E4 derived 1024 bp BglII-SmaI fragment containing the sequence encoding type 5 adenovirus E4 ORF6 was obtained and modified to blunt ends. This fragment corresponds to positions 33,309 (SmaI site) to 34,115 (BglII site) of the type 2 adenovirus genome. The modified fragment was then cloned into the SrfI site of p3.3c, providing the p3.3cE4ORF6 plasmid.

Plasmid p3.3cE2A (ATCC accession number 98235) was prepared as follows. A 2467 bp MscI (partial) -BamHI fragment from pBSII-E2a was obtained. This fragment contains the type 5 adenovirus E2a coding sequence and corresponds to positions 24,073 (MscI site) to 21,606 (BamHI site) of the type 2 adenovirus genome. The 2467 bp fragment was subcloned between the MscI and BamHI sites of pCITE2A (obtained from Novagene) to provide the pCITE2AE2A construct. The 1636 bp NcoI (partial) -BsrGI fragment was then excised from pCITE2AE2A. This fragment contains the sequence encoding the E2a 72kD protein and corresponds to positions 24,076 (MscI / NcoI sites) to 22,440 (BsrGI sites) of the type 2 adenovirus genome. The 1636 bp fragment was modified to blunt ends and cloned into p3.3c to provide the p3.3cE2A plasmid.

2. rAAV virion production assay :
293 cells were plated at 2 × 10 ⁶ cells / dish in a 10 cm tissue culture dish and cultured at 37 ° C. until reaching about 90% confluency for a period of about 48 hours prior to co-transfection. All transfections were performed using the CaPO ₄ method with the following DNA combinations: All 293 cell cultures were transfected with 5 μg of plasmid pW620adhlacZ. 5 μg of various auxiliary function constructs (as shown in Table 8 below) were used to provide a total amount of 20 μg of DNA per dish. For samples containing less than 4 constructs, the total amount of DNA transfected using pBSII DNA was 20 μg. Following transfection, cultures that changed media and used adenovirus for accessory function received type 2 adenovirus at a MOI of 5. All cultures were then incubated at 37 ° C. for approximately 72 hours before harvesting.

Next, cells were collected from each dish, and the medium was removed by centrifugation (1000 × g for 10 minutes). Three freeze / thaw cycles (alternating dry ice-ethanol bath and 37 ° C. bath) were then used to produce 1 mL of lysate. Debris was removed from the lysate by centrifugation (10,000 × g for 10 minutes). The techniques described in Example 1 were then used to assess rAAV lacZ virion production. The titration of lac Z was performed in the presence of adenovirus. All samples were assayed in duplicate. The results of the experiment are shown in Table 8 below.

As the results shown in Table 8 can show, the E2a, E4, and VA RNA regions of adenovirus are:
All are necessary to provide efficient rAAV virion production (a level substantially equivalent to that obtained using adenoviral infection to provide accessory function). However, one of these regions is not absolutely necessary for rAAV virion production.

The E2a and E4 regions subcloned into the pBSII-E2a and pBSII-E4 constructs contain several open reading frames (ORFs) in addition to the ORF for the 72kDE2a DNA binding protein and the E4 ORF6 protein. In the above study, by replacing the pBSII-E2a and pBSII-E4 constructs with CMV-driven p3.3cE2A and p3.3cE4ORF6 constructs, the E2a72kD DNA-binding protein and the E4 ORF6 protein all other open reading frames It has been established that complete E2a or E4 auxiliary functions can be provided when are not present in their respective coding regions.

Example 9
Comparison of pladeno 1 plasmid reconstruction and rAAV virion production efficiency
Instead of the pJM17-derived adenovirus gene used for the construction of pladeno1, the pladeno1 plasmid was reconstructed using purified type 2 adenovirus DNA as the source of the adenovirus gene. The reconstructed plasmid is named pladeno 5 and is described in detail below. This reconstruction was done to reduce the overall size of the plasmid. Furthermore, the reconstructed plasmid (pladeno5) does not encode an adenoviral protease, whereas pladeno1 encodes an adenoviral protease.

1. Construction of pladeno5 :
The pladeno5 plasmid was constructed as follows. DNA fragments encoding E2a, E4, and VARNA regions isolated from purified type 2 adenovirus DNA (obtained from Gibco / BRL) were ligated into a plasmid called pAmpscript. The pAmpscript plasmid was assembled as follows: 623 bp containing a polylinker and an alpha complement expression cassette from pBSII s / k + (obtained from Stratagene) using oligonucleotide-directed mutagenesis. A region was removed and replaced with an EcoRV site. The sequence of the mutagenic oligo (oligo) used for oligonucleotide-directed mutagenesis was 5′-CCGCTACAGGGCGCGATATCAGCTCACTCAA-3 ′ (SEQ ID NO: 14). A polylinker (including the following restriction sites: BamHI; KpnI; SrfI; XbaI; ClaI; Bst1107I; SalI; PmeI; and NdeI) was synthesized and inserted into the EcoRV site created above, resulting in the linker BamHI The site was proximal to the f1 origin in the modified plasmid to provide the pAmpscript plasmid. The sequence of the polylinker was 5′-GGATCCGGTACCGCCCGGGCTCTAGAATCGATGTATACGTCGACGTTTAAACCATATG-3 ′ (SEQ ID NO: 15).

  A DNA fragment containing the type 2 adenovirus E2a and VA RNA sequences was cloned directly into pAmpscript. In particular, a 5962 bp SrfI-KpnI (partial) fragment containing the E2a region was cloned between the SrfI and KpnI sites of pAmpscript. The 5962 bp fragment contains 21,606 to 27,568 base pairs of the type 2 adenovirus genome. A 732 bp EcoRV-SacII (blunted) fragment containing VARNA was cloned into the Bst1107I site of pAmpscript. The 732 bp fragment is equivalent to 10,423-11,155 base pairs of the type 2 adenovirus genome.

  Before it could be inserted into the pAmpscript polylinker, the DNA containing the type 2 adenovirus E4 sequence had to be modified. In particular, PCR mutagenesis was used to replace the proximal adenoviral terminal repeat of E4 with a SrfI site. The location of this SrfI site is equivalent to 35,836-35,844 base pairs of the type 2 adenovirus genome. The oligonucleotide sequences used in the mutagenesis were: 5′-AGAGGCCCGGGCGTTTTAGGGCGGAGTAACTTGC-3 ′ (SEQ ID NO: 16); and 5′-ACATACCCGCAGGCGTAGAGAC-3 ′ (SEQ ID NO: 17). The 3,192 bp E4 fragment generated by cleaving the above modified E4 gene with SrfI and SpeI was ligated between the SrfI and XbaI sites of pAmpscript already containing E2a and VARNA sequences, and pladeno 5 plasmid was occured. The 3,192 bp fragment is equivalent to 32,644-35,836 base pairs of the type 2 adenovirus genome.

2. rAAV virion production assay :
293 cells were plated at 1 × 10 ⁶ cells / dish in a 10 cm tissue culture dish and cultured at 37 ° C. until reaching about 90% confluency for a period of about 24-48 hours prior to co-transfection. All transfections were performed as described in Example 1 above.

  293 cell cultures were transfected with 5 μg of pVlacZ vector plasmid, 5 μg of plasmid pW1909, and 5 μg of either pladeno1 or pladeno 5. Following transfection, the medium was changed and the cultures were incubated at 37 ° C. for approximately 72 hours before harvesting.

  Next, cells were collected from each experimental group, and the medium was removed by centrifugation (1000 × g for 10 minutes). Three freeze / thaw cycles (alternating dry ice-ethanol bath and 37 ° C. bath) were then used to produce 1 mL of lysate. Debris was removed from the lysate by centrifugation (10,000 × g for 10 minutes). The technique described in Example 1 was then used to assess rAAVlacZ virion production. The results of the experiment are shown in Table 9 below.

  As can be seen from the results reported in Table 9, greater rAAV virion production efficiency was seen when pladeno 5 was used (pladeno 5 production resulted in 2.5 times more rAAV virions than pladeno 1 production). ).

  Thus, a novel accessory function has been described that can support efficient recombinant AAV virion production. Although the preferred embodiment of the present invention has been described in some detail, it will be understood that obvious variations can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Deposits of strains useful in the practice of the invention Deposits of biologically pure cultures of the following strains were made at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, under the provisions of the Budapest Treaty. The indicated accession number was issued after a successful survival test and paid the necessary fee. Access to this culture is available while the patent application is pending, as determined by the Commissioner of the Patent Office entitled to the provisions under 37 CFR 1.14 and USC 122. When a patent is granted for this application, all restrictions imposed by the depositor on the public availability of the deposited biological material are specified in § 1.808 (b) As such, with one exception: The exception is that during the life of the issued patent, the sample of the deposit is required to be distributed only if the claim for the sample meets any one or all of the following three conditions: The depositor reserves the right to make a contract with the depositary institution: (1) the request is in writing or in a specific form and is dated; and / or (2) the request is the party who requests it And / or (3) the request will be notified in writing from the depository to the depositor with the date the sample was distributed and the name and address of the party receiving the sample distribution. Is done. In addition, the deposit shown is maintained for 30 years from the date of deposit, or 5 years after the final claim of the deposit; or whether the US patent has a longer enablement period. When a culture becomes non-viable or inadvertently destroyed, or in the case of a plasmid-containing strain, the plasmid is lost, the culture becomes viable with the same taxonomic description. Replaced with one or more cultures.

  These deposits are provided only for the convenience of those skilled in the art and are not admitted that deposits are required. The nucleic acid sequences of these plasmids, as well as the amino acid sequences of the polypeptides encoded thereby, control where there is any discrepancy with the description herein. A license may be required to make, use, or sell the deposit, and such license is not accepted by this.

FIG. 1 shows a plasmid construct pladeno 1 containing VARNA derived from type 5 adenovirus, E4 (including ORF 6) and E2a adenovirus gene regions inserted into a pBSIIs / k-vector plasmid. FIG. 2 shows the plasmid construct pladeno 1 E1 formed by inserting a 4,102 bp BsrGI-Eco47III fragment (including type 5 adenovirus E1a and E1b coding regions) into the pladeno 1 construct. FIG. 3 shows the plasmid construct pladeno 5, which contains the VA RNA from the type 2 adenovirus genome, E4 ORF 6, and the E2a adenovirus gene region.

  (Sequence Listing)

Claims (10)

A method for producing a recombinant AAV (rAAV) virion preparation free of helper virus, the method comprising:
(A) introducing an AAV vector into a suitable host cell;
(B) introducing an AAV helper construct into the host cell, the helper construct comprising an AAV coding region expressed in the host cell to complement the AAV helper function deleted from the AAV vector. Process;
(C) a step of introducing an auxiliary function vector into the host cell, wherein the auxiliary function vector and the host cell do not contain a sequence encoding the adenovirus gene region E1, and are used for adenovirus production. Together with a sequence encoding at least one required adenoviral gene, wherein the accessory function vector comprises (i) a sequence providing adenoviral VA RNA, (ii) a sequence encoding adenoviral E4 ORF6, and (Iii) a sequence encoding at least two nucleotides selected from the group consisting of a sequence encoding 72 kD of adenovirus E2a;
(D) culturing the host cell to produce a preparation of rAAV virions, wherein a cell lysate from the preparation is cultured with 293 cells for 30 days, and cytopathology in the 293 cells Determining that the rAAV is not contaminated with a helper virus by determining the above effect. The method of claim 1, wherein the auxiliary function vector comprises an E2a 72 kd region. The method of claim 1, wherein the auxiliary function vector comprises an E4 orf6 region. 2. The method of claim 1, wherein the accessory function vector comprises an E2a 72kd region and provides adenoviral VA RNA. The method of claim 1, wherein the auxiliary function vector comprises an E4 orf6 region. The method of claim 1, wherein the auxiliary function vector comprises an E2a 72 kd region and an E4 orf6 region. The method of claim 1, wherein the auxiliary function vector comprises a sequence providing an E2a 72kd region, an E4 orf6 region and VA RNA. The method according to claim 1, wherein the auxiliary function vector is a plasmid. 9. The method of claim 8, wherein the plasmid further comprises at least one heterologous promoter region operably linked to the nucleotide sequence. A host cell for producing a recombinant adeno-associated virus (rAAV) virion, said host cell;
(A) an AAV vector;
(B) an AAV helper construct; and (c) an auxiliary function vector, the auxiliary function vector being a plasmid, (i) a sequence providing adenovirus VA RNA, (ii) a sequence encoding adenovirus E4 ORF6 And (iii) a sequence encoding 72 kD of adenovirus E2a, and comprising at least two sequences selected from the group consisting of the adenovirus vector and the host cell as a whole A host cell that does not contain a sequence encoding region E1 and does not contain a sequence encoding at least one adenovirus gene required for adenovirus production.

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