Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-05-05
2001-03-27
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C536S023100, C536S024100
Reexamination Certificate
active
06207453
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a transduction system which comprises a rep-negative AAV vector and its use.
BACKGROUND OF THE INVENTION
The transfer of genes by means of viruses as a vector is referred to as transduction. Transduction is frequently used to integrate genes into the genome of cells. For example, adeno-associated viruses (AAVs) are used as viruses for this purpose.
AAVs are single-stranded DNA viruses belonging to the Parvorirus family. AAVs require helper viruses, particularly adenoviruses or herpesviruses, for their replication. In the absence of helper viruses AAVs integrate into the host cell genome, particularly at a specific site on 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 the rep 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 been determined that recombinant AAVs, i.e., AAVs containing foreign DNA, often do not integrate into the genome of cells, so that the foreign DNA is not transferred either. However, the latter is important and largely indispensable for manipulating cells, particularly for gene therapy.
It is an object of the present invention to provide a product by which a foreign DNA can be integrated into the genome of cells in an effective manner. According to the invention, this is achieved by the subject matter defined in the claims.
BRIEF SUMMARY OF THE INVENTION
Thus, the present invention provides a transduction system, comprising:
(a) a rep-negative AAV vector containing a foreign DNA, and
(b) a product providing an AAV Rep protein.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the discovery that AAVs lacking a rep gene do not integrate into the genome of cells.
The term “rep-negative AAV vector” relates to any AAV, i.e., virus particle, and the DNA thereof, which are rep-negative. This means that no rep gene or only a defective rep gene is present. Conventional methods can be used for providing a rep-negative AAV vector. For example, an AAV DNA can be modified by specific mutagenesis in the rep gene such that it becomes defective or the rep gene is deleted by special restriction cleavage and ligation. A rep-negative AAV DNA can then be transferred into cells which express an AAV-rep gene, and rep-negative AAVs, i.e., virus particules, are obtained after infection with a helper virus.
The term “foreign DNA” includes any DNA that can be integrated into a rep-negative AAV vector. The foreign DNA may be non-coding or coding. In the case of non-coding DNA, the foreign DNA can be a regulator element of the DNA replication and/or transcription. In the case of coding DNA, it is preferable for the foreign DNA to be expressible. It is especially preferred that the expression be controlled by an inducible promoter such as a tissue-specific promoter. In addition, the foreign DNA can code for a diagnostic and/or therapeutic protein. Examples of therapeutic proteins include tumor necrosis factor, interferons, interleukins, lymphokines, growth factors, plasma proteins and receptors. In addition, the foreign DNA can be inserted at any site on the rep-negative AAV vector. It may be advantageous for the foreign DNA to be present in or in place of the rep gene. Furthermore, in some embodiments, several foreign DNAs are present.
The term “a product providing an AAV-Rep protein” includes any product which can provide an AAV Rep protein, particularly Rep 78 or Rep 68, or a portion thereof. For example, the product may be a DNA (rep-DNA) adapted to express an AAV Rep protein and a portion thereof, respectively. It is preferable for the expression of the rep-DNA to be controlled by an inducible promoter such as an antibiotic-specific or tissue-specific promoter. The rep-DNA may be provided by the genome of an AAV virus particle. It is preferable for the genome to have a defective (deleted) cap gene and an inducible promoter for the cap gene, respectively, and/or one or more defective (deleted) ITR sequences. The genome and the corresponding AAV virus particle may also be a product within the meaning of the term. In addition, the product may be an AAV Rep protein, particularly Rep 78 or Rep 68, or a portion thereof and a fusion protein that contains an AAV Rep protein or a portion thereof. Such proteins can be provided by conventional methods.
Components (a) and (b) may be connected with each other in a transduction system according to the present invention. Such a connection may be made by conventional methods. For example, if the AAV vector of component (a) is present as a virus particle and the product of component (b) is available as rep-expressing DNA, it may be preferred to proceed in the following manner: The AAV vector may be modified chemically or enzymatically. For example, it may be biotinylated, i.e., biotin or a biotinylated anti-AAV antibody such as an antibody directed against the AAV proteins VP-1, VP-2 or VP-3 may be bound to an AAV vector. The rep-expressing DNA may be mixed with DNA-binding substances such as organic polycations, e.g., polylysine and/or polyornithine, and heterologous polycations having several differing, positively charged amino acids, respectively.
It is especially preferred to mix the rep-expressing DNA with polylysine and streptavidin so that polylysine binds to the DNA and streptavidine binds to the polylysine. The biotinylated AAV vector and the streptavidine-polylysine-modified DNA are then mixed, so that the bond between components (a) and (b) is formed.
Such a transduction system is suitable for the transduction of cells. The cells may be of any type or origin. Furthermore, the cells may be present separately or in aggregation such as in a tissue or an organ. The cells may also be present within or outside an organism. In the latter case, the cells may be held in culture. Moreover, the cells may be healthy cells, diseased cells such as virus-infected cells or cells affected by microorganisms or protozoa, or tumor cells.
The cells may be transduced by common methods. If a transduction system is used wherein components (a) and (b) are connected with each other and the AAV vector and/or the product is present as a virus particle, the cells can be infected with the transduction system. However, if the AAV vector and the product are present as DNA, the transduction system can be introduced into the cells, e.g., by transfection, lipofection or electroporation.
If a transduction system is used wherein components (a) and (b) are not connected with each other, and the AAV vector and the product are present as a virus particle, the cells can be infected with the virus particles. However, if the AAV vector is present as a virus particle and the product as DNA or vice versa, the cells may be infected with the AAV vector and the product. The DNA can be introduced into the cells as described above.
Furthermore, if the AAV vector and the product are each present as DNA, they can be introduced into the cells as described above. Moreover, if the product is present in the form of an AAV Rep protein or as a portion thereof or as a fusion protein containing an AAV Rep protein or a portion thereof, the product may be introduced into the cells by lipofection.
The present invention provides a means to integrate foreign DNA into the genome of cells. A specific site on chromosome 19 is frequently used as a site of integration. Additionally, the tissue-specific expression of the foreign DNA is possible. Moreover, the AAV Rep protein may be provided only temporarily to the transduced cell
Bogedain Christoph
Hallek Michael
Maass Gerd
Michl Doris
Wendtner Clemens
Davis Katharine F
Halluin Albert P.
Howrey Simon Arnold & White , LLP
Kung Viola T.
Medigene AG
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