Methods for large-scale production of recombinant AAV vectors

Details Methods for large-scale production of recombinant AAV vectors Methods for large-scale production of recombinant AAV vectors

1999-09-22

2004-08-31

Leffers, Gerry (Department: 1636)

Chemistry: molecular biology and microbiology

Virus or bacteriophage, except for viral vector or...

C435S320100, C435S325000, C435S456000, C435S069100, C536S023100, C514S04400A

Reexamination Certificate

active

06783972

ABSTRACT:

1.0 BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns the replication and packaging of recombinant adeno-associated viral-based vectors, and a scaleable process for their large-scale production.
1.2 Description of Related Art
1.2.1 Adeno-associated Virus
Adeno-associated virus-2 (AAV)-2 is a human parvovirus that can be propagated both as a lytic virus and as a provirus (Cukor et al., 1984; Hoggan et al., 1972). The viral genome consists of linear single-stranded DNA (Rose et al., 1969), 4679 bases long (Srivastava et al., 1983), flanked by inverted terminal repeats of 145 bases (Lusby and Berns, 1982). For lytic growth AAV requires co-infection with a helper virus. Either adenovirus (Ad; Atchinson et al., 1965; Hoggan, 1965; Parks et al., 1967) or herpes simplex virus (HSV; Buller et al., 1981) can supply the requisite helper functions. Without helper, there is no evidence of AAV-specific replication or gene expression (Rose and Koczot, 1972; Carter et al., 1983). When no helper is available, AAV persists as an integrated provirus (Hoggan, 1965; Berns et al., 1975; Handa et al., 1977; Cheung et al., 1980; Berns et al., 1982).
Integration apparently involves recombination between AAV termini and host sequences and most of the AAV sequences remain intact in the provirus. The ability of AAV to integrate into host DNA is apparently an inherent strategy for insuring the survival of AAV sequences in the absence of the helper virus. When cells carrying an AAV provirus are subsequently superinfected with a helper, the integrated AAV genome is rescued and a productive lytic cycle occurs (Hoggan, 1965).
AAV sequences cloned into prokaryotic plasmids are infectious (Samulski et al., 1982). For example, when the wild type AAV/pBR322 plasmid, pSM620, is transfected into human cells in the presence of adenovirus, the AAV sequences are rescued from the plasmid and a normal AAV lytic cycle ensues (Samulski et al., 1982). This renders it possible to modify the AAV sequences in the recombinant plasmid and, then, to grow a viral stock of the mutant by transfecting the plasmid into human cells (Samulski et al., 1983; Hermonat and Muzyczka, 1984).
AAV contains at least three phenotypically distinct regions (Hermonat and Muzyczka, 1984). The rep region codes for one or more proteins that are required for DNA replication and for rescue from the recombinant plasmid, while the cap and lip regions appear to code for AAV capsid proteins and mutants within these regions are capable of DNA replication (Hermonat and Muzyczka, 1984). It has been shown that the AAV termini are required for DNA replication (Samulski et al., 1983).
The construction of two
E. coli
hybrid plasmids, each of which contains the entire DNA genome of AAV, and the transfection of the recombinant DNAs into human cell lines in the presence of helper adenovirus to successfully rescue and replicate the AAV genome has been described (Laughlin et al., 1983; Tratschin et al., 1984a; 1984b).
1.2.2 rAAV Vectors as Vehicles for Gene Therapy
Recombinant adeno-associated virus (rAAV) vectors have important utility as vehicles for the in vivo delivery of polynucleotides to target host cells (Kessler et al., 1996; Koeberl et al., 1997; Kotin, 1994; Xiao et al., 1996). rAAV vectors are useful vector for efficient and long-term gene transfer in a variety of mammalian tissues, e.g., lung (Flotte, 1993), muscle (Kessler, 1996; Xiao et al., 1996; Clark et al., 1997; Fisher et al., 1997), brain (Kaplitt, 1994; Klein, 1998) retina (Flannery, 1997; Lewin et al., 1998), and liver (Snyder, 1997).
It has also been shown that rAAV can evade the immune response of the host by failing to transduce dendritic cells (Jooss et al., 1998). Clinical trials have been initiated for several important mammalian diseases including hemophilia B, muscular dystrophy and cystic fibrosis (Flotte et al., 1996; Wagner et al., 1998).
1.2.3 Contemporary Methods for Preparing rAAV Vectors
Currently, rAAV is most often produced by co-transfection of rAAV vector plasmid and wt AAV helper plasmid into Ad-infected 293 cells (Hermonat and Muzyczka, 1984). Recent improvements in AAV helper design (Li et al., 1997) as well as construction of non-infectious mini-Ad plasmid helper (Grimm et al., 1998; Xiao et al., 1998; Salvetti, 1998) have eliminated the need for Ad infection, and made it possible to increase the yield of rAAV up to 10
5
particles per transfected cell in a crude lysate. Scalable methods of rAAV production that do not rely on DNA transfection have also been developed (Chiorini et al., 1995; Inoue and Russell, 1998; Clark et al., 1995). These methods, which generally involve the construction of producer cell lines and helper virus infection, are suitable for high-volume production.
The conventional protocol for downstream purification of rAAV involves the stepwise precipitation of rAAV using ammonium sulfate, followed by two or preferably, three rounds of CsCl density gradient centrifugation. Each round of CsCl centrifugation involves fractionation of the gradient and probing fractions for rAAV by dot-blot hybridization or by PCR™ analysis.
1.3 Deficiencies in the Prior Art
A major problem associated with the use of rAAV vectors has been the difficulty in producing large quantities of high-titer vector stocks (Clark et al., 1995, Clark et al., 1996). The standard production protocol involves low-efficiency transfection of plasmid DNA containing the rep and cap genes and a plasmid containing the rAAV provirus with inverted terminal repeats. Cells are then superinfected with adenovirus to provide essential helper functions required for rAAV production.
Alternative procedures have been developed to improve the efficiency of rAAV production by delivering rep, cap and the adenovirus helper genes. These technologies have included the generation of rep and cap inducible cell lines and plasmids expressing the essential adenovis helper genes (Clark et al., 1995; Clark et al., 1996; Vincent et al., 1990; Xiao et al., 1998; Grimm et al., 1998). Although these techniques have improved the yield of rAAV production, they have not been entirely satisfactory. Procedures employing transfection methods are not efficient, and tend to be extremely variable in yield from preparation to preparation. Moreover, such procedures are difficult to scale up to produce the large quantity of rAAV vector needed for clinical trials.
The production of rep and cap inducible cell lines is a particular challenge because the yield of rAAV produced from different clones is variable and does not exceed the efficiency of transfection methods (Clark et al., 1995; Clark et al., 1996, Vincent et al., 1990). Production procedures for rAAV that utilize adenovirus and transfection of rep and cap containing plasmids have the potential to generate wild type AAV (wt AAV) through illegitimate recombination of the ITRs with rep and cap sequences. This leads to preferential amplification of the wt AAV genome over the rAAV genome.
A major drawback in the use of rAAV vectors for gene transfer studies in vivo and their application to clinical procedures, such as that of gene therapy, has been the difficulty in producing large quantities of rAAV vector. For the therapeutic correction of some diseases, it is estimated that 1×10
14
rAAV particles must be administered per patient. This will require the culture of greater than 1×10
12
cells to produce the quantity of rAAV vector that will be needed to therapeutically treat each patient. The use of contemporary transfection methods on this scale of rAAV production is extremely problematic, costly and time consuming.
The development of a packaging system that provides all the helper functions needed for rAAV production from a rAAV producer cell line would greatly facilitate the large-scale production of rAAV. Transfection procedures would not be required and the producer cell line could be grown in large quantities at high densities in commercially available l

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