Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-03-05
2003-01-21
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S235100, C435S320100, C435S325000, C435S006120, C435S007100, C435S455000, C435S456000, C435S457000, C435S465000, C435S366000, C435S239000, C435S373000
Reexamination Certificate
active
06509150
ABSTRACT:
The present invention relates to methods and compositions for the production of recombinant Adeno-Associated Viruses (rAAV). In particular, the invention discloses nucleic acid constructs and packaging cells having improved properties for rAAV production, as well as novel methods of titration and characterization of rAAV preparations. The invention also describes novel sequences which promote or increase the packaging of nucleic acids in rAAV, and their use for producing rAAV with high efficiency. The invention can be used for producing or testing high quality rAAV preparations, for biological, preclinical, clinical or pharmaceutical uses.
BACKGROUND AND INTRODUCTION
Wild type Adeno-Associated Virus (wtAAV) is a naturally defective parvovirus which requires co-infection with a helper virus, such as adenovirus or herpes virus, in order to establish a productive infection. The virus is not associated with any human disease and has been shown to have a broad host range of infection in vitro. AAV has a relatively simple genome organization composed of two major genes coding for the regulatory (rep) and structural (cap) proteins. Three viral promoters located at map unit 5 (p5), 19 (p19) and 40 (p40) control the synthesis of mRNA coding for the four Rep and the three Cap proteins. The viral genome is flanked by 145 bases inverted terminal repeats (ITRs) which contain palindromic sequences necessary in cis for replication of the viral genome (Leonard and Berns, 1994).
Recombinant AAV viruses (rAAV) are derived by deleting the rep and cap genes which are replaced by the transgene and the transcriptional control elements needed for its expression. The only viral sequences retained in cis are the viral ITRs (Muzyczka, 1992). The ability of rAAV to efficiently transduce tissues in mice such as the muscle, the retina or the liver (Fisher et al., 1997; Flannery et al, 1997; Kessler et al., 1996; Koeberl et al., 1997; Snyder et al., Herzog et al., 1997; 1997; Xiao et al., 1996; Zolotukhin et al, 1996) and to lead to a prolonged gene expression with little to no pathology makes this virus unique among the family of viral vectors. In this respect, rAAV can be used in vitro for recombinant protein production, gene regulation studies, AAV protein production (to be used in non-viral gene delivery systems), etc. rAAV can also be used ex vivo or in vivo, to deliver genes of interest for biological, toxicological, prophylactic or therapeutic indications for instance. In this regard, it should be noted that several clinical trials are currently ongoing using a rAAV gene delivery vector.
However, widespread use of rAAV is hampered by the relatively cumbersome and inefficient procedure needed to produce it at high titers and in sufficient amount and quality. The standard procedure relies upon the transfection of 293 cells with two plasmids: a plasmid providing in trans the rep and cap functions, and the rAAV vector plasmid itself. After subsequent infection with an adenovirus, rAAV particles are assembled in the nuclei of the cells concomitantly with adenoviral particles. rAAV stocks are obtained after purification from total cell lysates through CsCI gradients (Snyder et al., 1996).
However, these methods represent relatively long and complex procedures, which cannot be easily scaled-up. Furthermore, because of the number of constructs required, recombination events have been observed leading to rAAV preparations which are contaminated with replicating AAV particles and with adenoviruses. There is therefore a need for improved methods of producing rAAV, for biological, preclinical, clinical or pharmaceutical uses. In particular, there is a need for methods of producing rAAV preparations with high titers of infectious particles and which are essentially free of adenoviruses. There is also a need for methods to produce rAAV preparations with significantly reduced contamination by replication competent or recombined AAV. There is also a need for improved methods of titration of rAAV preparations, and for methods of characterization of such preparations, i.e., for use in Quality Control steps, as well as for detecting rAAV or contaminants in biological fluids, for instance.
The present invention now provides novel methods and compositions for producing and characterizing rAAV preparations. In particular, the invention provides methods of producing rAAV with very high yields of infectious particles and essentially free of detectable adenovirus contamination.
DESCRIPTION OF THE INVENTION
The present invention relates to compositions and methods for producing and/or characterizing rAAV preparations of improved quality. The invention relates more particularly to improved nucleic acid constructs that provide for an efficient production of rAAVs, as well as to compositions, cell lines and methods for characterizing rAAV preparations. The invention can be used to produce rAAV for biological, preclinical or clinical uses, e.g., pharmaceutically acceptable rAAV preparations.
Within the context of the present invention, a recombinant Adeno-Associated Virus (rAAV) designates an AAV virus which comprises at least a recombinant nucleic acid genome. More specifically, rAAVs generally comprise a recombinant genome lacking a functional rep and/or cap region, and comprising a heterologous nucleic acid. Most conventional rAAVs comprise a recombinant genome lacking the entire Rep and Cap regions, which are replaced by the heterologous nucleic acid. Such recombinant genomes thus usually comprise the heterologous nucleic acid flanked by the left and right Inverted Terminal Repeats of AAV. rAAVs may comprise additional modifications, such as artificial or heterologous capsid proteins, for instance. Furthermore, the recombinant genome may comprise, in replacement or in addition to ITR sequence, RES elements as disclosed in the present invention. rAAVs may be derived from different serotypes of AAV, such as for instance AAV-2, AAV-3, AAV-4 or AAV-6.
In the present invention, except otherwise indicated, all references to nucleotide positions of the AAV genome are made with respect to the sequence of wild-type AAV-2 available at Genebank under number AF043303.
A recombinant Adeno-Associated Virus vector plasmid (rAAV vector plasmid) designates a nucleic acid construct comprising a copy of the genetic information to be packaged into AAV capsids, to form the rAAV. The rAAV vector plasmid is therefore any nucleic acid construct comprising the recombinant genome of the rAAV as defined above, preferably a heterologous nucleic acid flanked by one or two AAV Inverted Terminal Repeats (ITR) and/or, optionally, one or several RES elements. The rAAV vector plasmid can be autonomously replicating, conditionally replicating, or stably integrated into the genome of the packaging cell.
A rep-cap plasmid designates any nucleic acid construct encoding the rep and/or cap proteins, which provide in trans the AAV complementing functions lacking in the rAAV vector plasmid. Generally, the rep-cap plasmid encodes Rep78, Rep68, Rep52, Rep40, VP1, VP2 and VP3. The rep-cap plasmid can be a single construct encoding all the required REP and CAP proteins, under the control of the same or separate promoters. The rep-cap plasmid can also be a mixture of distinct nucleic acid constructs encoding one or several REP and CAP proteins. The rep-cap plasmid can be autonomously replicating, conditionally replicating, or stably integrated into the genome of the packaging cell.
As indicated above, a first aspect of the invention resides in a method of characterizing rAAV preparations or stocks. Indeed, because of the complex methods and constructs needed to produce rAAV, it is important to have efficient and accurate methods of characterizing the rAAV preparations obtained, especially for biological uses. Previous methods known in the art essentially focus on the determination of the contamination by adenoviruses, and/or the number of infectious AAV particles. Also, in determining the number of infectious rAAV particles, most prior art methods rely on the detection
Chadeuf Gilliane
Moullier Philippe
Nony Pascale
Salvetti Anna
Guzo David
Universite de Nantes
Young & Thompson
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