Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1999-12-21
2001-08-28
Bui, Phuong T. (Department: 1638)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C424S184100, C424S199100, C424S200100, C435S007200, C435S069100, C435S069300, C435S252300, C435S320100, C514S04400A, C530S300000, C530S350000, C536S023100, C536S023700, C536S023720
Reexamination Certificate
active
06281000
ABSTRACT:
The present invention relates to a method for preparing a viral vector in vitro in a prokaryotic cell and to its application for producing an infectious viral particle intended for therapeutic use, and especially for use in gene therapy.
The possibility of treating human diseases by gene therapy has changed in a few years from the stage of theoretical considerations to that of clinical applications. The first protocol applied to man was initiated in the U.S. in September 1990 on a patient who was genetically immunodeficient as a result of a mutation affecting the gene coding for adenine deaminase (ADA). The relative success of this first experiment encouraged the development of new gene therapy protocols for various genetic or acquired diseases (infectious diseases, and viral diseases in particular, such as AIDS, or cancers). The large majority of the protocols described hitherto employ viral vectors to transfer the therapeutic gene to the cells to be treated and to express it therein.
To date, retroviral vectors are among the ones most widely used on account of the simplicity of their genome. However, apart from their restricted capacity for cloning, they present two major drawbacks which limit their systematic use: on the one hand they chiefly infect dividing cells, and on the other hand, as a result of their integration at random in the genome of the host cell, the risk of insertional mutagenesis is not insignificant. For this reason, many scientific teams have endeavored to develop other types of vector, among which those originating from adenoviruses, adeno-associated viruses (AAV), poxviruses and herpesviruses may be mentioned. Generally speaking, their organization and their infection cycle are amply described in the literature available to a person skilled in the art.
In this connection, the use of adenoviral vectors has been seen to be a promising alternative. Adenoviruses have been demonstrated in many animal species, have a broad host range, have little pathogenicity and do not present the drawbacks associated with retroviruses since they replicate in resting cells and are nonintegrative. As a guide, their genome consists of a linear, double-stranded DNA molecule of approximately 36 kb carrying more than about thirty genes, both early genes necessary for viral replication (E1 to E4) and late structural genes (L1 to L5) (see FIG.
1
).
Generally speaking, the adenoviral vectors are obtained by deletion of at least one portion of the viral genes (in particular of the E1 region essential for viral replication), which are replaced by the therapeutic genes. Consequently, they are propagated in a cell line, termed complementation line, which supplies in trans the deleted viral functions to generate a viral vector particle which is defective for replication but capable of infecting a host cell. The line 293, established from human embryonic kidney cells, which complements adenoviruses that are defective for the E1 function (Graham et al., 1977, J. Gen. Virol., 36, 59-72), is commonly used.
The techniques of preparation of adenoviral vectors are amply described in the literature (see, in particular, Graham and Prevec, Methods in Molecular Biology, Vol. 7; Gene Transfer and Expression Protocols; Ed: E. J. Murray, 1991, The Human Press Inc., Clinton, N.J.). One of the methods most often employed consists in generating the recombinant adenoviral vector in complementation cells transfected with a bacterial plasmid carrying the gene of interest subcloned within its adenoviral insertion region and the adenoviral genome. Generally, the latter is cleaved with a suitable restriction enzyme so as to reduce the infectivity of the parent viral DNA and to increase the efficiency of isolation of the recombinant adenoviruses. However, a substantial background of infectious viral particles of parenteral origin is nevertheless observed, which necessitates an especially arduous task of analysis of the plaques obtained (arduous from a time and cost standpoint, since each plaque has to be amplified and analyzed individually). This is problematical when the parent virus displays a selective advantage over the recombinant adenovirus, for example when the latter replicates more slowly than the parent virus as a result of the insertion of a large-sized gene of interest (factor VIII, dystrophin), reducing proportionately the probability of obtaining it.
Ligation between two DNA fragments generated by the standard techniques of molecular biology and carrying, respectively, the 5′ and 3′ portions of the recombinant adenoviral vector may also be employed. Transfection of the ligation mixture into the complementation cells makes it possible in theory to encapsidate the genome of the recombinant adenovirus to form an infectious particle. This technology is of low efficiency and its application limited by the restricted number of suitable and unique restriction sites.
It has now been shown to be possible to generate a recombinant adenoviral vector in
Escherichia coli
(
E. coli
) by intermolecular homologous recombination between a plasmid containing the genome of a type 5 adenovirus and an insert carrying an exogenous DNA sequence surrounded by adenoviral sequences A and B (FIG.
2
). This method leads to the replacement in the adenoviral genome of the targeted region located between A and B by the exogenous sequence. Transfection of the recombinant adenoviral vector thus generated into an appropriate complementation line gives rise to an infectious viral particle which may be used without a prior purification step to infect a host cell. The background (contamination with parent viral particles) is reduced or even abolished. In addition, it was found, surprisingly, that the use of
E. coli
recBC sbcBC strains is especially advantageous for promoting intermolecular recombination between any two DNA fragments.
The method of the present invention is based on exploitation of the endogenous enzymatic activities of the prokaryotic cells involved in homologous recombination. This intermolecular recombination technique had already been described for the cloning of small inserts into bacterial vectors (Bubeck et al., 1993, Nucleic Acids Research, 21, 3601-3602) or for generating hybrid genes by intramolecular recombination (Calogero et al., 1992, FEMS Microbiology Letters, 97, 41-44; Caramori et al., 1991, Gene, 98, 37-44). However, this recombination technology had never been employed in prokaryotic cells to generate infectious viral vectors (capable of being encapsidated into infectious viral particles).
The method of the invention has the advantage over the previous techniques of being rapid and especially efficient, and of requiring only a small number of manipulations in vitro. Furthermore, it may be employed with DNA fragments generated by PCR (polymerase chain reaction), thereby avoiding the steps of subcloning of the exogenous DNA sequence into the insertion region of the viral genome. Lastly, it provides an advantageous solution to the problem of background which is clearly identified in the literature. Consequently, it proves especially efficacious for vectors originating from large-sized viruses or viruses into which the insertion of a large-sized exogenous DNA sequence is envisaged.
Accordingly, the subject of the present invention is a method for preparing, in a prokaryotic cell, a recombinant viral vector derived from a parent virus into the genome of which an exogenous DNA sequence is inserted, by intermolecular recombination between (i) a first DNA fragment comprising all or part of said genome of the parent virus and (ii) a second DNA fragment comprising said exogenous DNA sequence surrounded by flanking sequences A and B which are homologous to (i).
For the purposes of the present invention, a recombinant viral vector is obtained from a parent virus modified so as to carry at a suitable site of the viral genome and to express an exogenous DNA sequence. The parent viruses capable of being used in the context of the invention are, for example, alphaviruses, retroviruses, poxviruses
Chartier Cecile
Degryse Eric
Bui Phuong T.
Burns Doane Swecker & Mathis L.L.P.
Transgene S.A.
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