Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-05-06
2004-12-14
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S235100, C435S320100, C435S069100, C435S325000, C435S368000, C435S372000, C435S236000, C435S239000, C435S456000, C536S023100, C536S024100, C536S024500, C530S350000
Reexamination Certificate
active
06830929
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods of in vitro construction of SV40 viruses or pseudoviruses comprising exogenous nucleic acid or exogenous protein or peptide which are particularly suitable for use in gene therapy.
BACKGROUND OF THE INVENTION
Previous studies have shown that SV40 virions disrupted at pH 10.6 [Christensen, M. & Rachmeler, M. (1976)
Virology
75:433-41] or by reducing disulfide bonds [Colomar, M. C., et al. (1993) J. Virol. 67:2779-2788] may be reassociated to form infectious SV40 aggregates. The early attempts to package in vitro foreign DNA in these aggregates [Christensen & Rachmeler (1976) ibid] produced infectious products which did not resemble SV40 virions. Furthermore, their resistance to DNase has not been tested. Later, in vitro packaging experiments [Colomar et al. (1993) ibid.] did not yield particles with infectivity above the level of naked DNA.
Recently, pseudocapsids of the closely related murine polyoma virus, prepared from polyoma VP1, were used as carriers for heterologous DNA into mammalian cells [Forstova, J., et al. (1995) Hum. Gene Therapy 6:297-306]. The pseudo-capsid protected 2-30% of the input DNA from DNase I digestion. When a plasmid carrying the cat gene was tested, most of the DNA which was protected from DNase I appeared as a ~2 kb fragment, while the input plasmid was significantly larger (exact size was not reported), suggesting that each DNA molecule was only partially protected against DNases. Infectious units were not measured in those experiments. The DNA transferred into recipient cells was functional in gene expression, albeit at a very low efficiency. With a 1.6 kb DNA fragment which carries the polyoma middle T-antigen, <30 transformed foci were obtained per 1 &mgr;g of input DNA. Similarly, a low level of CAT activity was observed with the plasmid carrying the cat gene.
SV40 is a simian papovirus, with a small double-stranded circular DNA genome of 5.2 kb [reviewed in Tooze, J. (1981) DNA Tumor Viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.]. The viral capsid, surrounding the viral mini-chromosome, is composed of three viral-coded proteins, VP1, VP2, and VP3. Recent X-ray crystallographic studies on SV40 structure at 3.8 Å resolution [Liddington, R., et al. (1991) Nature 354:278-282] revealed that the outer shell of the virion particle is composed of 72 pentamers of VP1, 60 hexavalent and 12 pentavalent. The VP2 and VP3 appear to bridge between the VP1 outer shell and the chromatin core. The VP1 pentamers have identical conformations, except for the carboxy-terminal arms, which tie them together. Five arms extend from each pentamer and insert into the neighboring pentamers in three distinct kinds of interactions. It appears that this construction facilitates the use of identical building blocks in the formation of a structure that is sufficiently flexible as required for the variability in packing geometry [Liddington et al. (1991) ibid.].
Another protein encoded by the late regions of SV40 (which also encoded the three capsid proteins VP1, VP2 and VP3) is the agnoprotein, also called LP1. This is protein a small, 61 amino acid protein. Although the agnoprotein was not found in the viral capsid, it is thought to expedite viral assembly in vivo [Resnick, J & Shenk, T. (1986) J. Virol. 60:1098-1106; Ng, S. C., et al. (1985) J. Biol. Chem. 260:1127-1132; Carswell, S. & Alwine, J. C. (1986) J. Virol. 60:1055-1061].
The major hindrance in beginning to use the SV40 pseudovirions in preliminary experiments in humans is the present need for a viral helper for encapsidation. This results in pseudoviral stocks that contain also wild type SV40. Because of the similarity in properties (shape, size and density) between the pseudovirions and the helper, they cannot be separated by physical means. An ideal way to prepare pseudovirions for therapeutic purposes for human use would be by in vitro packaging. This would provide maximal safety, since all steps of the preparation can be well controlled. Ex vivo administration would circumvent problems associated with immune response.
Viral packaging in vivo occurs by gradual addition and organization of capsid proteins around the SV40 chromatin [Garber, E. A., et al. (1980) Virology 107: 389-401; Bina, M. (1986) Comments Mol. Cell Biophys. 4:55]. The three capsid proteins VP1, VP2 and VP3 bind to DNA non-specifically [Soussi, T. (1986) J. Virol. 59:740-742; Clever, J., et al. (1993) J. Biol. Chem. 268:20877-20883]. How the specific recognition between the viral capsid proteins and its DNA is achieved remains unclear. The packaging of SV40 using pseudovirions, in which most of the viral DNA is replaced by other sequences has been investigated [Oppenheim, A., et al. (1986) Proc. Natl. Acad. Sci. USA 83:6925-6929]. The pseudoviral particles are prepared by encapsidating plasmids that carry the SV40 origin of replication (ori) and the packaging signal (ses) [Oppenheim, A., et al. (1992) J. Virol. 66:5320-5328]. The model suggests that ses serves several functions in SV40 packaging: as a sensor for the level of the late viral proteins in the transition from replication and/or transcription to packaging, in nucleosomal reorganization and the initiation of viral assembly [Oppenheim, A., et al. (1994). J. Mol. Biol. 238:501-513] and probably also as a nucleation center for viral assembly [Dalyot-Herman, N. et al. (1996) J. Mol. Biol. 259:69-80].
The pseudovirions, carrying various genes of therapeutic interest, are very efficient in DNA transfer into a wide range of cells, including human bone marrow cells, and are therefore potential vectors for gene therapy [Oppenheim et al. (1986) ibid.; Oppenheim A., et al. (1987) Ann. New York Acad. Sci. 511:418-427; Dalyot, N. & Oppenheim, A. (1989) Efficient transfer of the complete human beta-globin gene into human and mouse hemopoietic cells via SV40 pseudovirions. In: Gene Transfer and Gene Therapy (Beaudet, A. L., Mulligan R, I. M. Verma, eds), pp. 47-56, Alan R. Liss, Inc., New York; Oppenheim, A., et al. (1992) Development of somatic gene therapy: A simian virus 40 pseudoviral vector for hemopoietic cells. In Genetic Among Jews (Bonne-Tamir, B., A. Adams, eds), pp. 365-373, Oxford University Press, Oxford]. The ideal way to prepare pseudovirions for therapeutic purposes for human use is by in vitro packaging. This would provide maximal safety, since all steps of the preparation can be well controlled.
SUMMARY OF THE INVENTION
The present invention relates to construct capable of infecting a mammalian cell comprising at least one semi-purified or pure SV40 capsid protein and a constituent selected from the group consisting of an exogenous DNA encoding an exogenous protein or peptide product, or encoding therapeutic RNA, or itself a therapeutic product, a vector comprising an exogenous DNA encoding an exogenous protein or peptide product, or encoding therapeutic RNA, or itself a therapeutic product, an exogenous RNA encoding an exogenous protein or peptide product or itself a therapeutic product, a vector comprising an exogenous RNA encoding an exogenous protein or peptide product or itself a therapeutic product, an exogenous protein or peptide product, and antisense RNA, ribozyme RNA or any RNA or DNA which inhibits or prevents the expression of undesired protein/s in said mammalian cell; and optionally further comprising operatively linked regulatory elements sufficient for the expression and/or replication of said exogenous protein in a mammalian cell.
The construct of the invention may optionally further comprise additional SV40 protein or proteins, preferably SV40 agnoprotein.
Constructs according to the invention may comprise as said constituent exogenous circular or linear DNA encoding an exogenous protein or peptide product, or is itself a therapeutic product, or a vector comprising exogenous DNA encoding a therapeutic RNA, or encoding an e
Oppenheim Amos
Oppenheim Ariella
Sandalon Ziv
Cooper & Dunham LLP
Guzo David
White John P.
Yissum Research & Development Company of the Hebrew University
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