The present invention relates to an AAV DNA having helper virus sequences which are necessary for developing AAV viral particles, a system containing such a DNA and the use of both.

FIELD OF THE INVENTION

The present invention relates to AAV DNA having helper virus sequences, a system containing such a DNA and its use.

BACKGROUND OF THE INVENTION

AAVs (adeno-associated viruses) are single stranded DNA viruses belonging to the Parvovirus family. For their replication, i.e. for forming viral particles, AAVs require helper viruses, particularly adenoviruses or herpesviruses. In the absence of helper viruses, AAVs may incorporate into the host cell genome, particularly at a specific site of chromosome 19.

The genome of AAVs is linear and has a length of about 4680 nucleotides. It comprises two reading frames which code for a structural gene and a non-structural gene. The structural gene is referred to as cap gene. It is controlled by the P40 promoter and codes for three capsid proteins. The non-structural gene is referred to as rap gene and codes for the Rep proteins Rep 78, Rep 68, Rep 52 and Rep 40. The two former proteins are expressed under the control of the P5 promoter, while the expression of Rep 52 and Rep 40 is controlled by the P19 promoter. The functions of the Rep proteins are represented inter alia by the control of replication and transcription of the AAV genome.

It has now turned out that preparations of recombinant (r)AAV viral particles are frequently contaminated with helper viruses, e.g., adenoviruses or herpesviruses. This contamination considerably limits the use of rAAV viral particles for gene therapy. Efforts made to remove the helper viruses by CsC1 density gradient centrifugation or filtration methods have produced little success so far, in particular these methods comprise steps which manifest themselves negatively as regards costs and yield.

Therefore, it is the object of the present invention to provide a product by which rAAV viral particles can be provided without contamination with helper viruses.

SUMMARY OF THE INVENTION

The present invention relates to an AAV DNA having helper virus sequences which are necessary for developing AAV viral particles, a system containing such a DNA and the use of both.

DETAILED DESCRIPTION OF THE INVENTION

It is the object of the present invention to provide a product by which rAAV viral particles can be provided without contamination with helper viruses. According to the invention this is achieved by the subject matters defined in the claims.

Thus, the subject matter of the present invention relates to an AAV DNA having helper virus sequences which are necessary for developing AAV viral particles.

The present invention is based on the applicant's finding that an AAV DNA according to the invention serves for inducing an rAAV vector being present together in the celss with an AAV DNA and containing a foreign DNA to develop rAAV viral particles without having to add helper virus for this purpose.

The expression "AAV DNA" comprises any AAV DNA which may contain helper virus sequences necessary to develop AAV viral particles.

The expression "helper virus sequences" concerns any sequences of a helper virus necessary to develop AAV viral particles. Such sequences originate particularly from herpesviruses and/or adenoviruses, more particularly from adenovirus 5. The sequences may comprise the entire virus genome or fragments thereof.

The expression "rAAV vector" comprises any AAV viral particle and its DNA, which may contain a foreign DNA, except for that of a helper virus, which is necessary to develop AAV viral particles.

With the above exception, the expression "foreign DNA" relates to any DNA which can be incorporated in an AAV vector. The foreign DNA can be non-coding or coding. In the former case, it may be a control element of DNA replication and/or transcription. In the latter case, it is favorable for the foreign DNA to be expressible, it being particularly advantageous for the expression to be controlled by an inducible promoter such as a tissue-specific promoter. In addition, the foreign DNA may code for a diagnostic and/or therapeutic protein. Examples of a therapeutic protein are tumor necrosis factor, plasma proteins and receptors. Moreover, the foreign DNA may be inserted at any site of the AAV vector.

An AAV DNA according to the invention can be prepared by common methods. By way of supplement, reference is made to Sambrook, J. et al., Molecular Cloning, A Laboratory Handbook (Vols. 1 3), Cold spring Habour, New York, (1989). Furthermore, reference is made, in Example 1, to the preparation of the pTG9585 AAV DNA according to the invention. This AAV DNA comprises the complete adenovirus 5 sequence with the exception of the E1 region, as helper virus sequences. PTG9585 is preferred. It was deposited with the DSM [German-type collection of micro-organisms and cell cultures], Braunschweig, as plasmid pTG9585 under number DSM 11248 on Oct. 18, 1996. Also, an AAV DNA according to the invention is preferred which differs from pTG 9585 in that it has a deletion in the structural gene L1 of the adenovirus 5 sequence, particularly in the region of nucleotides 16614 18669. This AAV DNA is referred to as pTG9585 .DELTA. 16614 18669. Besides, an AAV DNA according to the invention is preferred which differs from pTG 9585 in that it comprises two deletions from a total of 18323 base pairs, one deletion relating to great portions of the adenovirus capsid genes and the other deletion relating to the E3 region of adenovirus. This AAV DNA is referred to as pDG and was deposited with the DSM as plasmid pDG under number DSM 11817 on Oct. 15, 1997.

A further subject matter of the present invention relates to a system comprising the above elements, i.e. an AAV DNA, an rAAV vector and optionally a cell. The AAV DNA and/or the cell represent a complementation as regards the AAV sequences of the rAAV vector. The expression "cell" concerns any cell, particularly mammalian cell, which permit the absorption and multiplication of AAV.

By means of the present invention it is possible to provide rAAV viral particle preparations without having to use helper viruses. Therefore, the rAAV viral particle preparations are also free from helper viruses. This is shown particularly when the pDG AAV DNA according to the invention is used. Moreover, the rAAV viral particle preparations distinguish themselves in that they contain no AAV wild-type. They also represent a subject matter of the present invention.

rAAV viral particle preparations according to the invention are perfectly suited for the transduction of cells. It may be favorable for the preparations to be treated with a Dnase prior to their use, so that free AAV DNA is degraded. The cells in consideration are any cells which are present in a body or isolated from a body. Hence it is possible by the present invention to take measures for an ex vivo and in vivo gene therapy.
 

Claim 1 of 15 Claims

1. A nucleic acid sequence comprising an adeno-associated virus (AAV) nucleic acid sequence, wherein said AAV nucleic acid sequence comprises a rep gene or a cap gene or a rep gene and a cap gene, and an AAV helper virus nucleic acid sequence, wherein said AAV helper virus sequence comprises the complete adenovirus 5 sequence with exception of the E1 region.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.