Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-07-07
2004-06-22
Winkler, Ulrike (Department: 1648)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C424S093200, C424S185100, C424S192100, C424S198100, C424S199100, C435S069100, C435S069700, C435S320100, C435S455000, C435S457000, C435S458000, C530S350000, C536S023100, C536S023500, C536S023510, C536S023720
Reexamination Certificate
active
06753419
ABSTRACT:
DESCRIPTION
1. Field of the Invention
The present invention refers to the field of molecular biology and particularly to the possibility of finely controlling important gene functions for the somatic gene therapy. More specifically, the present invention provides a system for generating forms of some Rep proteins of Adeno-associated virus (AAV) dependant on steroid hormones. The invention also refers to the regulation of the AAV Rep proteins activity to direct the integration of recombinant DNA sequences within specific regions of human host-cell genomic DNA.
2. State of the Art
The integration of therapeutic genes into specific DNA sites of actively dividing cells and of non dividing cells, accompanied by prolonged expression, is the optimal strategy for somatic gene therapy.
The Adeno-associated virus (AAV) has the unique capacity of preferentially integrating its viral DNA into defined regions of the cellular genome, thus reducing the risk of insertional mutagenesis associated with other viruses, e.g. retroviruses that integrate in totally random positions.
The Adeno-associated virus (AAV) is a non-pathogenic small virus, with a single-stranded DNA, not capsized, belonging to the parvovirus family (Balagué et al, 1997). AAV requires coinfection with a helper virus (adenovirus or herpes virus) or the host cells exposition to genotoxic agents (e.g. heat shock, hydroxyurea, UV light and X-rays) in order to undertake a productive infection. In the absence of a helper virus the AAV genome integrates into the chromosomal DNA to generate a latent infection. Analysis of flanking sequences of the provirus integrated genome, as the FISH analysis (in situ hybridisation with fluorescent probes) from latent infected cells, has revealed that the integration of the AAV genome is preferentially targeted to a specific site (called aavsl) localised in the q- terminus of the chromosome 19 (19q13-qter) (Kotin, R M, et al, 1990, PNAS USA, 87:2211-2215; Samulski. R J et al, 1991, EMBO J. 10:3941-3950]). The specificity of the integration refers to a spectrum of 60%-94% of the cases, depending on the cellular lines utilised and on the experimental conditions. This feature reduces the probability of insertional mutagenesis deriving from the random integration of the viral genome. Moreover, the absence of powerful transcription regulatory elements in the AAV genome makes it unlikely that the AAV integration in the aavsl site may be responsible of the transcription activation of endogenous chromosomal genes.
The integrated genome AAV can be rescued and replicated, if the cells containing an integrated provirus are superinfected with a helper virus like the Ad.
The AAV genome is a linear single-stranded DNA 4680 bp long, containing two sequences coding for proteins (ORFs=Open Reading Frames), three promoters (p5, p19 and p40), and an ITR sequence (Inverted Terminal Repeat sequence) of 145 bp, located at each end of the genome. The two ORFs code for non structural (Rep) and structural (Cap) proteins respectively (Kotin, R M, et al, 1990, PNAS USA, 87:2211-2215; Samulski. R J et al, 1991, EMBO J. 10:3941-3950).
The AAV ORF rep codes for four overlapping proteins. More specifically, the rep-coding ORF has two promoters located at map positions 5 (promoter 5, p5) and 19 (promoter 19, p19). The transcripts originated by each one of those two promoters share a common intron near to the 3′ terminus of the reading frame and to the polyadenilation site. The intron is utilised only in a subpopulation of the RNA messenger produced. Therefore, the ORF for rep generates four different mRNAs and the corresponding proteins: Rep78 and Rep68, expressed under control of p5 and Rep52 and Rep40, expressed under control of p19. Rep68 and Rep40 are coded by RNA transcripts that undergo a maturation process called “splicing” leading to the intron excision. Mutational analysis has shown the functional activities of the various Rep proteins.
Rep78 and Rep68 are multifunctional proteins that play a crucial role in the AAV replication. Rep78 is 621 amino acids long while Rep68 is 536 amino acids long: Rep78 and Rep68 differ only in their carboxy-terminal end, while the first 529 amino acids are identical in the two peptides. Rep78 and Rep68 share similar biochemical properties: both perform activities that are required for the AAV DNA replication, including the capacity of binding the RBS (Rep Binding Site) in the ITRs, and of cleaving site-specifically in a single strand manner the trs (terminal resolution site) present in the ITRs. Furthermore, Rep78 and Rep68 act as DNA-DNA and DNA-RNA helicases, have an ATP-ase activity and are capable of regulating positively or negatively both AAV promoters and heterologous promoters. Up to today evident functional differences between Rep78 and Rep68 have not been observed. Rep52 and Rep40, which do not show binding activity or endonucleasic DNA activity, are nevertheless important for the AAV infective cycle, as they promote the accumulation of single-stranded capsized genome of AAV.
AAV integration mechanisms during the non productive infection were not entirely clarified. Anyhow, it was clearly established that, beside undetermined cellular factors, two viral elements are required: the ITRs and the Rep78/68 (Carter, B J. in “Handbook of Parvoviruses”, ed. P. Tijsser, CRC Press, pp. 155-168, Samulski, R W096/36364). This conclusion is the direct consequence of many observations: firstly, recombining AAV vectors lacking of the rep and cap coding sequences do not specifically integrate into chromosome 19. Secondly, a RBS and a potential flanking trs were identified into the preferential genomic integration site aavsl, and it was proved that Rep68/78 can simultaneously bind the RBS present in ITRs and in aavsl, thus bridging the two DNA sequences. Moreover, utilising an ex vivo assay it was proved that a 33 bp sequence comprising the RBS and the trs in aavsl is the shortest sequence required in order to obtain the targeted integration of AAV into a DNA propagated as episome. Even more interesting is the observation that the two elements required for the AAV site-specific integration, function with a rather high efficiency even when utilised outside of the viral genome context. Actually, it was proved that plasmids bearing a rep expressing cassette can promote, when transfected in cells, the site-specific integration of a transgene flanked by ITR (integration cassette) contained either in the same or in a cotransfected plasmid.
Those experiments overall prove that it is possible to utilise the AAV integration mechanism in a different context from that of the AAV genome. This is of great relevance in the field of somatic gene therapy, because the primary limitation of AAV use for the somatic gene therapy is the low packaging limit of the AAV virion, that cannot exceed 4.5 Kb. As a recombining vector AAV able to integrate specifically in the human chromosome 19 ought to contain Rep78 and/or 68 cDNA (about 2000 nt) as well, the wider DNA sequence (e.g. transcription regulating regions 5′ and 3′+transgene) transductable with the AAV vector could not be longer than 2-2.5 Kb.
These dimensional limitations could be overcome either by transducing the ITR-flanked transgene and the rep expression cassette with a non-viral system, or by the introduction of those elements into a viral vector with a wider cloning capacity (e.g. adenovirus, baculovirus, herpes virus, etc.) (as disclosed in the Italian patent application RM97A000200, priority date Apr. 8, 1997). Whatever the selected transduction system be, a tight regulation of the functioning Rep78/68 protein(s) is necessary.
In nonviral transduction systems, it would be necessary to have Rep proteins functioning only for the time required to obtain the integration, in order to avoid any undesired influence on the cell physiology. This is especially true in light of the observation that Rep exerts a cytotoxic-cytostatic effect on cell cultures (Yang et al, 1994). Moreover, a tight control of the Rep activity on targe
Ciliberto Gennaro
Rinaudo Cira
Toniatti Carlo
Cocuzzo Anna L.
Instituto di Ricerche di Biologia Molecolare P. Angeletti S.p.A.
Tribble Jack L.
Winkler Ulrike
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