Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-06-08
2002-11-26
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069100, C536S023720
Reexamination Certificate
active
06485965
ABSTRACT:
This invention relates to compositions and methods for the delivery of nucleic acids into cells, in vitro, ex vivo or in vivo. More preferably, the invention relates to compositions and methods for the delivery of nucleic acids into cells using replicating or semi-replicating viral constructs, in particular replicating or semi-replicating retroviral constructs. The invention also relates to the preparation of said constructs, genetically modified cells which contain said constructs, such as packaging cells, as well as the use of these compositions and methods for the delivery of any selected polynucleotide to a cell, for experimental, prophylactic, therapeutic or diagnostic applications.
The delivery of nucleic acids to cells finds applications in various biotechnology and pharmacology areas, such as experimental research, clinical research, therapeutic or prophylactic treatment as well as diagnostic. Gene delivery in vitro can be used to produce recombinant proteins or viruses, for instance, as well as to produce stable recombinant cells for screening purposes. In vivo or ex vivo, gene delivery enables the construction of transgenic animals, the study of gene regulation, as well as therapeutic and/or prophylactic treatments of mammals, including human beings. In this regard, increasing amounts of animal and clinical trials have been reported in the literature, designed at delivering to subjects nucleic acids encoding therapeutic or antigenic polypeptides. These approaches use different gene transfer strategies, which comprise for instance the use of (i) viral vectors (or cells producing viral vectors), (ii) non-viral vectors (such as plasmids in combination with chemical agents or physical treatments), (iii) naked DNA or (iv) genetically modified cells which are grafted into the patient, optionally upon encapsulation to limit immune reaction.
The use of recombinant retroviruses is one of the most successful methods to introduce a nucleic acid into dividing cells, in vitro, in vivo or ex vivo. This gene transfer strategy has proven to be useful for both basic research and clinical applications. In this regard, in humans, much progress has been made for the transduction of hematopoietic progenitors (Nolta et al., Exp. Hematol. 20 (1992) 1065-1071), mature lymphocytes (Bunnell et al., Proc. Natl. Sci. USA 92 (1995) 7739-7743), tumor cells (Klatzmann et al. Hum. Gen. Ther. 9 (1998) 2595-2604; Caruso et al.; PNAS 90 (1993) 7024-7028), etc. Retroviruses have thus been validated as a gene delivery vehicle in vivo, in human subjects, for clinical applications (treatment of cancers, genetic diseases (e.g., ADA-deficiency, SCID), viral infections (e.g., HIV treatment), immune disorders (e.g., graft versus host disease, autoimmune diseases, etc.), etc.
Recombinant retroviruses which have been used in the art for gene delivery applications, are genetically modified to be rendered defective, i.e., to avoid replication of their genome and/or propagation upon infection of competent cells, in the absence of trans-complementing functions. In this respect, most recombinant retroviruses are created by replacing, in the recombinant genome, the viral genes gag, pol and env with a nucleic acid of interest. The recombinant, defective retroviruses are prepared in a so-called packaging cell, which produces the complementing functions encoded by gag, pol and env. Examples of such packaging cell lines are, for instance PA317 (Miller et Buttimore, Mol. Cell. Biol. 6 (1986) 2895), PsiCRIP (Danos et Mulligan, PNAS 85 (1988) 6460), or GP+EnvAm12 (Markowitz et al. Virology 167 (1988) p. 400). Other examples of retrovirus packaging cells have been described for instance in EP 243 204, WO89/07150, WO90/02806, U.S. Pat. No. 5,766,945, EP476,953, WO93/04167 and WO93/10218. The recombinant retroviruses produced in said cells are infectious, but cannot replicate themselves upon infection. In this regard, they are considered replication-defective. Such replication-defective recombinant retroviruses have been constructed using different types of retroviruses, including MOMLV (Moloney Murine Leukemia Virus) ALV, BLV, MMTV or RSV for instance, or using lentiviruses such as HIV, SIV or CAEV, for instance.
It is generally admitted that the gene delivery vectors should be as deficient as possible in order to avoid adverse effects upon administration in vivo. For this reasons, the retroviral vectors which have been used to date essentially lack all of the viral proteins gag, pol and env. The same concern also exists for all other types of viral vectors which are currently being used, such as adenoviral vectors, AAV, herpes virus vectors and the like. For instance, adenoviruses generally comprise a deletion of the E1 region at least, and current efforts are being made to produce “gutless” adenovectors, i.e., adenoviral vectors devoid of all viral coding sequences. Similarly, most recombinant MV vectors are devoid of the rep and cap coding regions.
However, while much progress has now been made in gene delivery vectors, there is still a need for alternative strategies which may increase gene transfer efficacy, gene expression stability and/or facilitate gene vector production, especially for industrial uses.
In Patent Application n
o
PCT/FR 95/00208, Applicants have disclosed a novel gene delivery concept. This concept comprises delivering to a cell, in vitro, ex vivo or in vivo, nucleic acid construct(s) comprising all of the genetic elements allowing said cell to produce a recombinant virus. This method therefore uses nucleic acids that create in situ recombinant virus producing cells. This method has been used for instance to deliver therapeutic or toxic genes in vivo, or to prepare vaccine compositions (see PCT/FR 97/00619 Noguiez-Hellin et al., PNAS 93 (1996) 4175-4180). This method offers several advantages over prior gene delivery vectors, and in particular it avoids the need for packaging cells, allows the use of recombinant plasmids to deliver the viral genes in vivo, provides efficient gene transfer in vivo, etc.
The instant invention now provides a novel approach for gene delivery into cells. This approach is based on the use of replicating or semi-replicating viral constructs to deliver genes in vitro, ex vivo or in vivo. In contrast with all prior existing methods, which where based on the use of replication defective viral constructs, the instant invention now stems from a new and original concept of using replicating or semi-replicating viral constructs, in particular replicating or semi-replicating retroviral constructs, for in vivo, ex vivo or in vitro delivery of polynucleotides. The instant invention shows that such constructs can be made in high quantity and quality and efficiently deliver and express any gene or nucleic acid of interest into cells in vitro, ex vivo or in vivo.
The instant invention therefore relates to methods and compositions for polynucleotide delivery into cells, in vitro, ex vivo or in vivo. More particularly, the invention relates to compositions comprising replicating viral constructs and their use in delivering polynucleotides to cells. The invention also relates to compositions comprising semi-replicating recombinant viral constructs and their use in delivering polynucleotides to cells. In other aspects, the invention also resides in packaging cells which produce replicating or semi-replicating recombinant viruses, and uses thereof.
The Use of Replicating Viral Constructs
As indicated above, in one embodiment, the invention resides in replicating viral constructs and uses thereof, in particular replicating retroviral constructs and uses thereof.
Numerous systems of retroviral vectors have been developed for gene delivery and gene therapy in the past decade. The main advantage of these systems is seen in the ability of these vectors to stably integrate into the hosts genome. In this regard, retroviruses such as MoMLV, selectively infect dividing cells, and are therefore considered as promising vectors for the transduction of proliferating cells (i.e., tumor cells, prolife
Holzer Georg
Klatzmann David
Morel Arnaud
Salzmann Jean-Loup
Nixon & Vanderhye P.C.
Park Hankyel T.
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