Compositions and methods for recombinant adeno-associated virus production
a technology of adenovirus and adenovirus, which is applied in the field of compositions and methods for recombinant adenovirus production, can solve the problems of affecting the widespread use of raav, cumbersome and inefficient procedures, and a large amount of raav, and achieves the effects of reducing the number of raavs, and reducing the number of r
Inactive Publication Date: 2002-05-30
UNIV DE NANTES
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Benefits of technology
[0033] After the contacting of the rAAV preparation with the cell cultures, the presence of viruses is determined. In this regard, the measuring comprises measuring the presence of rAAV viruses within each test a), b) and c). More preferably, the measuring of the presence of viruses in cultures a), b) and c) comprises measuring (i.e., detecting) the presence of rAAV replicating DNA in the cells. This measure is usually accomplished by using rAAV-specific probes, optionally after amplification of the cellular nucleic acids with rAAV-specific primers. In a particular embodiment, a probe is used which is complementary to all or part of the rAAV genome, in particular to all or part of the heterologous nucleic acid present in the rAAV genome. The probe comprises preferably at least 100 bp, to ensure higher selectivity. The probe can be labeled with any conventional technique (enzymatic, fluorescent, radioactive, etc.). In a preferred embodiment, the detection of the presence of rAAV replicating DNA in the cells of a), b) and c) is performed after cell lysis without amplification of the cellular nucleic acids. Detection without amplification allows a better quantitative evaluation of the number of rAAV genomes present within the cells. Of course, amplification can be performed, prior to the detection, by using primers specific for regions of the heterologous nucleic acid present in the rAAV genome.
[0040] The method is therefore very efficient and provides immediate information regarding the quality of a rAAV preparation, as illustrated in the Examples. Another object of the present invention therefore lies also in a method as described above, for detecting the presence of rAAV and / or rep-positive AAVs and / or adenoviral particles in biological fluids, in particular after in vivo administration of rAAV preparations in animals and / or human subjects. Such biological fluids are, more particularly, serum, urine, stool, saliva, broncho alveolar fluids, etc. The method is performed as described above, by contacting, in tests a), b) and c), samples of the biological fluids or dilutions or concentrates or derivatives thereof. It can thus be used to monitor safety issues during preclinical, clinical or pharmaceutical settings.
Problems solved by technology
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.
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.
Method used
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[0008] 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.
[0009] 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 geno...
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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.
Description
[0001] This a continuation of international application No. PCT / EP00 / 01854, filed Mar. 3, 2000, which designated the United States of America, and a continuation-in-part of copending application Ser. No. 09 / 263,093, filed Mar. 5, 1999.[0002] 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[0003] Wild type Adeno-Associated Virus (wtAAV) is a naturally defective parvovir...
Claims
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Login to View More IPC IPC(8): C12N7/01C12N15/35C12N15/864
CPCC12N2750/14143C12N15/86
InventorSALVETTI, ANNANONY, PASCALECHADEUF, GILLIANEMOULLIER, PHILIPPE
OwnerUNIV DE NANTES