Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-01-26
2002-11-19
Priebe, Scott D. (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S320100, C435S091100, C435S091410, C435S091420, C435S455000, C435S456000, C424S093200
Reexamination Certificate
active
06482617
ABSTRACT:
The present invention relates to new viral vectors, to their preparation and to their use in gene therapy. It also relates to pharmaceutical compositions containing said viral vectors. More especially, the present invention relates to recombinant adenoviruses as vectors for gene therapy.
Gene therapy consists in correcting a deficiency or an abnormality (mutation, aberrant expression, and the like) by introducing genetic information into the cell or organ affected. This genetic information may be introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the body, or directly in vivo into the appropriate tissue. In this second case, different techniques exist, including various techniques of transfection involving complexes of DNA and DEAE-dextran (Pagano et al., J.Virol. 1 (1967) 891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375) and of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al., J.Biol.Chem. 255 (1980) 10431), and the like. More recently, the use of viruses as vectors for gene transfer has been seen to be a promising alternative to these physical transfection techniques. In this connection, different viruses have been tested for their capacity to infect certain cell populations. This applies especially to retroviruses (RSV, EMS, MMS, and the like), the HSV virus, adeno-associated viruses and adenoviruses.
Among these viruses, the adenoviruses display certain properties which are advantageous for use in gene therapy. In particular, they have a fairly broad host range, are capable of infecting resting cells, do not integrate in the genome of the infected cell and have not been associated to date with pathologies of importance in man. Adenoviruses have thus been used to transfer genes of interest to muscle (Ragot et al., Nature 361 (1993) 647), the liver (Jaffe et al., Nature genetics 1 (1992) 372), the nervous system (Akli et al., Nature genetics 3 (1993) 224), and the like.
Adenoviruses are linear double-stranded DNA viruses approximately 36 kb in size. Their genome com prises, in particular, an inverted repeat sequence (ITR) at each end, an encapsidation sequence (Psi), early genes and late genes (see FIG.
1
). The main early genes are contained in the E1, E2, E3 and E4 regions. Among these, the genes contained in the E1 region (E1a and E1b in particular) are necessary for viral replication. The E4 and L5 regions, for example, are, for their part, involved in viral propagation. The main late genes are contained in the L1 to L5 regions. The genome of the Ad5 adenovirus has been sequenced completely, and is accessible on a database (see, in particular, Genebank M73260). Likewise, some portions, or even the whole, of the genome of adenoviruses of different serotypes (Ad2, Ad7, Ad12, and the like) have also been sequenced.
In view of the properties of the adenoviruses mentioned above, the latter have already been used for the transfer of genes in vivo. To this end, different vectors derived from adenoviruses have been prepared, incorporating different genes (&bgr;-gal, OTC, &agr;
1
-AT, cyto-kines, and the like). In each of these constructions, the adenovirus has been modified so as to make it incapable of replication in the infected cell. Thus, the constructions described in the prior art are adenoviruses from which the E1 (E1a and/or E1b) and possibly E3 regions have been deleted, in which regions, the heterologous DNA sequences are inserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161). Other constructions contain a deletion in the E1 region and of a non-essential portion of the E4 region (WO94/12649). Nevertheless, the vectors described in the prior art have some drawbacks which limit their use in gene therapy. In particular, the batches of recombinant viruses of the type described in the prior art may be contaminated with replicative particles, in particular of the wild type.
At the present time, the vectors derived from adenoviruses are, in effect, produced in a complementation line (line 293) in which a portion of the adenovirus genome has been integrated. More specifically, line 293 contains the left-hand end (approximately 11-12%) of the adenovirus serotype 5 (Ad5) genome, comprising the left-hand ITR, the encapsidation region and the E1 region, including E1a, E1b and a portion of the region coding for the pIX protein. This line is capable of trans-complementing recombinant adenoviruses which are defective for the E1 region, that is to say lacking all or part of the E1 region, necessary for replication. In effect, E1− recombinant adenoviruses may be prepared in 293 cells as a result of the good trans-complementation of the E1 region contained in this line. Nevertheless, there are zones of homology between the adenovirus region integrated in the genome of the line and the DNA of the recombinant virus which it is desired to produce. As a result, during production, different recombination events may take place, generating replicative viral particles, in particular adenoviruses of the E1+ type. As shown in
FIG. 2
, the outcome can be a single recombination event followed by chromosome breakage (FIG.
2
A), or a double recombination (FIG.
2
B). These two types of modification lead to a replacement of the left-hand portion of the recombinant DNA, lacking a functional E1 region, by the corresponding portion present in the genome of the cell, which carries a functional copy of the E1 region. Moreover, in view of the high titres of recombinant vector produced by line 293 (greater than 10
12
), the probability of these recombination events taking place is high. In fact, it has been found that many batches of defective recombinant adenoviral vectors were contaminated with replicative viral particles.
Contamination with replicative particles constitutes a major drawback. In effect, the presence of such particles in therapeutic compositions would induce in vivo a viral propagation and an uncontrolled dissemination, with risks of an inflammatory reaction, of recombination, and the like. Hence the contaminated batches cannot be used in human therapy.
The present invention enables these drawbacks to be remedied. The present invention describes, in effect, new constructions permitting the production of defective recombinant adenoviruses completely lacking contamination with replicative particles. The present invention also describes a method for the production of these recombinant adenoviruses. It thus provides new defective recombinant vectors derived from adenoviruses which are especially suitable for use in gene therapy, in particular for the transfer and expression of genes in vivo.
The present invention lies more especially in the construction of defective recombinant adenoviruses comprising an adenovirus genome whose genetic organization is modified, and the possible recombination of which with the genome of the producing line leads to the generation of non-replicative and/or non-viable viral particles. The Applicant has now shown that it is possible to modify the genomic organization of the adenovirus in order to avoid the production of replicative particles during the production of the stocks.
A first subject of the present invention hence relates to a recombinant adenovirus comprising an adenovirus genome (i) whose E1 region is inactivated, (ii) whose genomic organization is modified, and (iii) the possible recombination of which with the genome of the producing line leads to the generation of non-viable viral particles.
For the purposes of the present invention, genetic or genomic organization is understood to mean the arrangement of the different genes or functional regions present in the genome of the wild-type adenovirus as shown in
FIG. 1. A
modified genetic or genomic organization hence corresponds to a genome in which some genes or some regions are not in their original position. Thus, some genes or some regions may be moved from the genome and inserted at another site. It is also possible to insert
Orsini Cecile
Perricaudet Michel
Yeh Patrice
Aventis Pharma S.A.
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Priebe Scott D.
LandOfFree
Viable contaminant particle free adenoviruses, their... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Viable contaminant particle free adenoviruses, their..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Viable contaminant particle free adenoviruses, their... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2946871