Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
2000-09-11
2002-06-25
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S005000, C435S006120, C435S069100, C435S325000, C536S023100
Reexamination Certificate
active
06410298
ABSTRACT:
The present invention relates to a method for reducing recombination events between nucleic acids. It also relates to the use of this method in processes for preparing nucleic acids such as plasmids or viral vectors. The invention also relates to novel viral constructs.
Recombination between nucleic acids is a well known phenomenon of molecular biology. Recombination is a molecular mechanism by which novel combinations of genetic material are generated, which contribute to Darwinian evolution by providing a source of material for natural selection. Genetic recombination requiring strong sequence homology between the participating nucleic acids is generally referred to as homologous recombination. During homologous recombination events, an exchange of genetic information occurs between two regions of a nucleic acid, this exchange possibly being reciprocal (“crossing over”) or nonreciprocal (conversion).
During meiosis, homologous recombination is responsible for the rearrangement of the genetic information and plays an important role in the correct segregation of the chromosomes. During mitosis, homologous recombination participates in DNA repair. It can introduce genomic rearrangements, such as deletions and duplications, when it involves dispersed homologous regions, or also contractions or expansions when it involves tandem repeat sequences.
The mechanism by which homologous recombination occurs has been partly elucidated. Thus, in bacteria, homologous recombination begins with a step which involves a single-stranded end (Holliday, 1964; Meselson, 1975). In eukaryotes, on the other hand, most results suggest a mechanism of double-strand break (DSB) (Szostak et al., 1983). DSBs appear to be at the origin of two principal mechanisms of homologous recombination: one conservative, according to which all the nucleic acid sequences participating in the recombination are present in the recombination products (Szostak et al.), the other nonconservative, during which certain sequences are lost. In mammalian somatic cells the majority of homologous recombination events by DSB appear to take place according to a nonconservative process (Lin et al., 1990, Jeong-Yu, 1992).
With the constant-development of biotechnology, an ever-increasing exploitation of DNA is being carried out: production of recombinant proteins, creation of transgenic animals, gene therapy and cellular therapy, etc. In these different domains, the occurrence of uncontrolled recombination events can constitute, in certain cases, a drawback.
Thus, during the production of recombinant proteins, recombination events in the expression plasmid (intramolecular recombination) can for example lead to the excision of the expression cassette for the transgene and thus to a loss of the expression. Recombination events can also be at the origin of the excision of an expression cassette which is stably integrated into the genome of a host producer cell and thus can induce a loss of stability.
Another example of adverse effects linked to the occurrence of homologous recombination events is liable to take place during the construction and production of vectors, in particular of viral vectors. Viral vectors (adenovirus, retrovirus, adeno-associated virus, herpes virus, etc.) constitute particularly efficient means for transferring nucleic acids into cells in vitro, ex vivo or in vivo. For constructing defective viral vectors the regions which are essential for the replication of the wild-type virus are generally deleted from the genome and replaced with the nucleic acid of interest. To produce and amplify these viruses it is therefore necessary to supply, in trans, the complementing functions (either in a plasmid, or in a form which is integrated into the genome of the producer cell, or via a helper virus). However, in certain cases, homologous recombination events occur between the defective viral genome and the complementing functions, which reconstitute replicating viral particles. Thus, the vectors derived from adenoviruses are generally produced in a complementing line (293 line or derivative) into which part of the adenovirus genome has been integrated. More specifically, the 293 line contains the left-hand end (about 11-12%) of the genome of the adenovirus serotype 5 (Ad5), comprising the left ITR, the encapsidation region and the E1 region, including E1a, E1b, and part of the regions encoding the protein pIX and IVa2. This line is capable of transcomplementing recombinant adenoviruses which are defective for the E1 region, i.e. lacking in all or part of the E1 region, which is required for replication. However, there exist zones of homology between the region of the adenovirus which is integrated into the genome of the line and the DNA of the recombinant virus whose production is desired. For this reason, various recombination events can take place during production, generating replicating viral particles, in particular adenoviruses of type E1+. As indicated in
FIG. 1
, it can be a single recombination event followed by a break of the chromosome (FIG.
1
A), or a double recombination (FIG.
1
B). These two types of modification lead to the replacement of the left-hand portion of the recombinant DNA, lacking in a functional E1 region, with the corresponding portion present in the cell's genome, which carries a functional copy of the E1 region. Moreover, taking into account the high titres of recombinant vector produced by the 293 line, the probability of these recombination events taking place is high. In fact, it has been found that many batches of defective recombinant adenoviral vectors are contaminated with replicating viral particles.
The presence of replicating particles in the batches of virus constitutes a considerable drawback for applications for transferring genes in vitro or in vivo (risks of viral propagation and uncontrolled dissemination).
The same type of problematics exist for generating defective retroviruses. Thus, constructed defective retroviruses are generally deleted of the viral coding regions (gag, pol and env), which are provided in trans by the production line. Here again, for certain lines described, overlapping zones exist between the genome of the defective retrovirus and the complementing functions carried by the cells. It is the case in particular for the cells PA317, Psi2, etc. Homologous recombination events can therefore take place in these zones, generating replicating particles.
The present invention relates to a method for reducing the frequency of the recombination events between two given nucleic acids and thus for minimizing the impact of such events on a biological process.
More particularly, the present invention relates to a method for reducing the frequency of the homologous recombination events between two given nucleic acids or two regions of a nucleic acid. To reduce homologous recombination events, the prior art teaches various approaches which are all based on the same principle: replacing or deleting the regions of homology. Thus, certain plasmids which allow the expression of genes of interest carry regions which are homologous to the genome of the host cell. It can be, in particular, a promoter region, a marker gene or an origin of replication. To reduce the risks of recombination, it has hitherto been proposed to substitute these regions with others, which are nonhomologous (different promoter, etc.). Moreover, to limit the risks of recombination in the processes for producing viral vectors, it has been suggested to remove the sequences which are homologous between the complementing genes and the defective viral genome. Thus, patent application WO 97/00326 describes a cell line for producing adenovirus, designated PER, comprising a restricted unit of the adenoviral genome carrying the E1 region. With this line, the flanking sequences which are homologous to the genome of the defective virus are reduced, which makes it possible to limit the risks of homologous recombination between them. Similarly, application WO 95/11984 describes the construction of a recombinan
Crouzet Joel
Robert Jean-Jacques
Vigne Emmanuelle
Yeh Patrice
Aventis Pharma S.A.
Park Hankyel T.
Wiley Rein & Fielding LLP
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