Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Recovery or purification
Reexamination Certificate
2000-07-21
2003-12-09
Zeman, Mary K. (Department: 1631)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Recovery or purification
C435S005000, C435S235100, C435S320100, C435S803000, C435S948000, C530S412000, C530S826000
Reexamination Certificate
active
06660514
ABSTRACT:
BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates generally to the field of virology, and in particular, to methods for preparing highly-purified, high-titer recombinant adeno-associated virus compositions. In certain embodiments, the invention concerns the use of equilibrium density centrifugation techniques, affinity chromatographic media, and in certain embodiments anion- and cation-exchange resins, to remove rAAV particles from solution and to prepare highly purified viral stocks for use in a variety of investigative, diagnostic and therapeutic regimens. Methods are also provided for purifying rAAVs from solution and for reducing the concentration of adenovirus in rAAV stocks.
1.2 Description of Related Art
1.2.1 Adeno-Associated Virus
Adeno-associated virus-2 (AAV) is a human parvovirus which 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 et al., 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 helper function. Without helper, there is no evidence of AAV-specific replication or gene expression (Rose et al., 1972; Carter et al., 1983; Carter et al., 1983). When no helper is available, AAV can persist 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 et al., 1984). AAV contains at least three phenotypically distinct regions (Herrnonat et al., 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 et al., 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 Conventional Methods for Preparing Recombinant AAV
Recombinant adeno-associated virus (rAAV) has been demonstrated to be a useful vector for efficient and long-term gene transfer in a variety of tissues, including lung (Flotte, 1993), muscle (Kessler, 1996; Xiao and Samulski, 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 demonstrated to evade the immune response of the host by failing to transduce dendritic cells (Jooss et al., 1998). Phase I clinical trails are underway for cystic fibrosis rAAV-mediated gene therapy (Flotte et al., 1996; Wagner et al., 1998). Yet in spite of these promising developments one of the problems that remains to be solved is that vector production remains very laborious.
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 etal., 1995; Conway etal., 1997; Inoue and Russell, 1998; Clark etal., 1995). These methods, which generally involve the construction of producer cell lines and helper virus infection, are suitable for high-volume production.
However, little progress has been made on the downstream purification of rAAV. The conventional protocol 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. No only does it require two weeks to complete, but the current protocol often results in poor recovery of the vector and poor virus quality. The growing demand for different rAAV stocks often strains the limited capacities of vector production facilities. There is, therefore, a clear need for a protocol that will reduce the preparation time substantially without sacrificing the quality and/or purity of the final product.
2.0 SUMMARY OF THE INVENTION
In a first embodiment, the invention concerns a method of purifying a recombinant adeno-associated virus. In general, the method comprises centrifuging a sample containing or suspected of containing recombinant adeno-associated virus through at least a first iodixanol gradient, and collecting the purified virus or at least a first fraction comprising the recombinant adeno-associated virus, from the gradient. Preferably the gradient is a discontinuous gradient, although the inventors contemplate the formulation of continuous iodixanol gradients that also provide purification of rAAV compositions. In certain aspects of the invention, multiple iodixanol gradients, for example at least a second, at least a third and/or at least a fourth iodixanol gradient, are used to purify the recombinant adeno-associated virus.
In an exemplary discontinuous iodixanol gradient, the gradient comprises an about 15% iodixanol step, an about 25% iodixanol step, an about 40% iodixanol step, and an about 60% iodixanol step. Optionally, the gradient may contain steps having lower concentrations of iodixanol, and likewise, the gradient may contain steps that have higher concentrations of iodixanol. Naturally, the concentrations of each step do not need to be exact, but can vary slightly depending upon the particular formulation and preparation of each step. The inventors have shown that most rAAV particles will band in an iodixanol gradient at a level corresponding to a percentage of iodixanol approximately equal to 52%, although depending upon the number of viral particles loaded on the gradient and the volume and capacity of the gradient, the range of concentrations at which purified rAAV particles may be found may range on the order of from about 50% to about 53%, or from about 50% to about 54%, 55%, 56%, 57%, 58%, 59% and even up to and including about 60% iodixanol. Likewise, the range of concentrations at which the rAAV particles may be isolated following centrifugation may be on the order of from about 55% down to and including about 49%, about 48%, about 47%, about 46%, about 45%
Byrne Barry J.
Muzyczka Nicholas
Zolotukhin Sergei
University of Florida Research Foundation
Williams Morgan & Amerson P.C.
Zeman Mary K.
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