Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Reexamination Certificate
2000-12-08
2003-02-18
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
C435S069100, C424S233100
Reexamination Certificate
active
06521426
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to viral vectors which may be used in gene delivery, such as in gene therapy, processes for viral replication, and viral vectors, cells and cell lines useful in preparation of viral vectors which may be used in gene delivery. The invention is not concerned with gene therapy itself, rather with the provision and production of vectors which may be used in gene delivery, such as in gene therapy.
The integration of therapeutic genes into specific locations of the DNA of dividing and non-dividing cells, accompanied by prolonged expression, is the optimal strategy for somatic gene therapy.
Adeno-associated virus (AAV) has the unique capacity of preferentially integrating its viral DNA within a defined region of the cellular genome, thus reducing the risks of insertional mutagenesis associated with other viruses such as retroviruses that integrate at random positions.
AAV is a non-pathogenic human parvovirus which usually requires Adenovirus (Ad) or Herpes virus as a helper to replicate efficiently. In the absence of helper virus the AAV genome integrates into host-cell genomic DNA at high frequency. Analysis of flanking sequences from latently infected cells of human origin have revealed integration of the AAV genome into a specific locus in 60-70% of cases. The integration locus (aavs1) has been sequenced and localised to human chromosome 19q13.3-qter (Kotin, R M, et al, 1990; Samulski. R J et al, 1991).
The integrated AAV genome can be rescued and replicated if cells containing an integrated provirus are superinfected with a helper virus such as Ad.
Parks et al., (1996) describe a Ad system in which a helper virus provides in trans all viral proteins required for propagation of the vector, which contains only the cis acting elements relevant for DNA replication and packaging (inverted terminal repeats and packaging signal). These two elements are contained within about 500 bp located at the end of viral genome. This allows in principle cloning into the vector of up to 37 kb of foreign DNA. Recently the complete (19 kb) human &agr;1 antitrypsin genomic DNA locus has been rescued in an Ad helper dependent vector (Ad HDV) (Schiedner et al., 1998) and its administration to mice resulted in long term expression of therapeutic levels of proteins.
The AAV DNA genome is a linear single-stranded DNA molecule having a molecular weight of about 1.5×10
6
daltons or approximately 4680 nucleotides long. The AAV2 genome has one copy of the 145 nucleotides long inverted terminal repeat (ITR) located at each end. The AAV ITR contain palindromic sequences that can fold over to form hairpin structures that function as primers during initiation of DNA replication. Additionally, the ITRs are needed for viral integration, rescue from the host genome, and encapsidation of viral nucleic acids into mature virions. Inserted in between the ITRs of AAV there is a unique region of about 4470 nucleotides that contains two main open reading frames (ORF). The right ORF encodes three capsid proteins VP1, VP2 and VP3. These three proteins form the viral particle and are produced from transcripts controlled by promoter P
40
located at map position 40. The left open reading frame of the AAV genome encodes the rep gene. Two promoters located at map positions 5 and 19 (promoters P
5
and P
19
, respectively) control the expression of the four polypeptides derived from this ORF. Rep proteins Rep 78 and Rep 68 are produced from the P
5
promoted transcripts, and Rep proteins Rep 52 and Rep 40 are synthesized from the P
19
promoted transcripts. Srivastava et al. (1983) and Berns, K. I. (1996) provide the nucleotide sequence and organisation of the Adeno-Associated Virus 2 genome.
In the development of AAV vectors, it has been shown that the entire rep and cap domain can be excised and replaced with a reporter or a therapeutic transgene, and that the ITRs are the minimal signal sequence required for rescue, replication, packaging and integration of the AAV genome (Carter, B J.; Samulski J., R WO96/36364).
The development of AAV vectors for gene therapy has faced several limitations: difficulties encountered in the large scale production of replication defective recombinants, and the packaging limit of the AAV virion that cannot exceed 4.5 kb. This limitation excludes several larger genes which may be considered as potential candidates for gene therapy programmes.
Production of replication defective recombinants resulting from excision of cap and rep domain requires the cotransfection of two different complementing plasmids, one containing the gene for delivery (e.g. reporter or therapeutic gene) sandwiched between the two cis acting AAV ITRs, the second encoding the virus ORF for rep and cap peptides. Moreover, the cotransfected cell must also be infected with a helper virus (usually adenovirus or herpesvirus). While laboratory low production scale can be effectively achieved by cotransfection and infection, reproducible large scale production required for preparation of a therapeutic product is very difficult.
Additionally, recombinant AAV vectors in which rep and cap genes have been deleted do not integrate into the AAVS1 locus but they do so in a random fashion.
Targeting of integration involves the AAV rep gene products. In particular, the larger polypeptides Rep 78 and Rep 68 have been shown to bind in vitro the AAV ITRs and the aavsl, and possess helicase and site-specific endonuclease activities which may be required for AAV replication as well as AAV integration. See for example Shelling, A. N. et al (1994); Balagué, C., M. et al. (1997); Surosky, R. T. et al. (1997).
A possible alternative strategy is to transfer the ability for site-specific integration of AAV by transferring the appropriate viral genes and cis-acting signals required for site-specific integration into other large capacity viral vectors. Relevant elements of the AAV genome, such as the rep and cap ORF or the AAV ITRs, can be introduced into adenoviral vectors. The resulting recombinant Ad/AAV vectors would have the adeno larger packaging capacity, and the adeno characteristic ability to infect a large variety of cell types in vivo and of most eukaryotic cell lines in vitro.
A major stumbling block in the amplification of chimeric Ad/AAV vectors became apparent when it was realised that the AAV rep gene's expression has a toxic effect on Ad (De La Maza L. M. et al., 1978; Berns, K. I., 1996). Thus, the expression of the Rep gene during virus replication in 293 cells hinders the amplification of the Ad vector, by a mechanism that is not fully understood but is associated with the disruption of the Ad replication centres, identified by immunofluorescence and in situ hybridization studies (Weitzman M. D. et al., 1996.).
To avoid this difficulty, alternative strategies have been developed where the rep peptides are provided separately and are not encoded by the Ad/AAV vector. For instance, in WO96/13598 is disclosed a hybrid Ad/AAV virus which comprises portions of an adenovirus, 5′ and 3′ ITR sequences from an AAV and a selected transgene. Additionally, the hybrid virus is linked via a polycation conjugate to an AAV rep gene (“hybrid virus conjugate” or “transinfection particle”). The major drawback of these procedures is in the low efficiency and reproducibility of the conjugation process.
Other elaborate approaches have been attempted, emphasizing that the problem with producing Ad/AAV vectors is a severe one.
SUMMARY OF THE INVENTION
The present invention is founded on the surprising realisation that a much simpler approach can be taken to produce the desired vectors in large amounts, as successfully demonstrated experimentally.
The present invention provides in various aspects new procedures for the efficient preparation of recombinant helper dependent adenoviral vectors comprising AAV rep genes, based on the finding that it is possible to avoid rep inhibition of replication of viral vectors by keeping on separate replicating units the viral replication function
Ciliberto Gennaro
Colloca Stefano
La Monica Nicola
Brown Stacy
Cocuzzo Anna L.
Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.p.A.
Park Hankyel T.
Tribble Jack L.
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