Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2000-05-02
2002-02-26
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S320100, C435S069100, C435S455000, C435S456000, C435S457000, C435S462000, C435S325000, C435S366000, C435S369000, C435S006120, C435S091420
Reexamination Certificate
active
06350575
ABSTRACT:
The present invention relates to novel helper vectors allowing defective recombinant viral vectors, which have the characteristic of being provided with recombination sequences recognized by a recombinase, to be complemented. It likewise relates to a complementation cell expressing the recombinase as well as a method of preparation of recombinant viral vectors in the form of infectious viral particles allowing the transfer and the expression of genes of interest in a cell or a host organism. The invention is of very particular interest for gene therapy prospects, especially in man.
The possibility of treating human diseases by gene therapy has passed in the course of a few years from the stage of theoretical considerations to that of clinical applications. The first protocol applied to man was initiated in the United States in September 1990 on a patient who was genetically immunodeficient because 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 especially viral diseases such as AIDS or cancers). The great majority of the protocols described until now employ viral vectors to transfer and express the therapeutic gene in the cells to be treated.
The interest in adenoviruses as gene therapy vectors has already been touched on in numerous documents of the prior art. In fact, the adenoviruses have a wide spectrum of hosts, are not very pathogenic and do not have the disadvantages connected with the retroviruses since they are nonintegrative and replicate equally in quiescent cells. By way of information, their genome is formed of a linear and double-stranded DNA molecule of approximately 36 kb carrying regions acting in cis (ITR 5′ and 3′ encapsidation region of the viral genome and ITR 3′) and additionally about thirty genes, at the same time early genes necessary for viral replication and late structure genes (see FIG.
1
).
The early genes are divided into 4 regions dispersed in the adenoviral genome (E1 to E4; E for early). They comprise 6 transcriptional units which have their own promoters. The late genes (L1 to L5; L for late) partly cover the early transcription units and are, for the majority, transcribed starting from the major late promoter MLP.
At the present time, all the adenoviral vectors used in gene therapy protocols are devoid of the major part of the E1 region essential for replication, in order to avoid their distribution in the environment and the host organism (first generation vectors). This deletion makes the viral genome deficient for replication. However, the E1 viruses can be propagated in a cell line which complements the E1 function to generate an infectious viral particle. The 293 line, established starting from human embryonic kidney cells, is currently used, in the genome of which is integrated the left 5′ end of the type 5 adenovirus (Graham et al., 1977, J. Gen. Virol. 36, 59-72).
The majority of adenoviral vectors of the prior art comprise supplementary deletiors. Certain of these have been introduced in the E3 region with the aim of increasing the cloning capacities but do not need to be complemented to the extent where the E3 region is nonessential for replication. More recently, second generation vectors have been proposed in the literature. They conserve the in cis regions (ITRs and encapsidation sequences) and comprise important internal deletions aimed at suppressing the main part of the viral genes whose expression can be responsible for inflammatory responses in the host. In this respect, a minimal vector which is deficient for all of the coding viral regions represents a choice alternative.
The techniques of preparation of adenoviral vectors are widely described in the literature. Firstly, the complete genome is formed by homologous recombination in the 293 line (see especially Graham et Prevect, 1991, Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols; Ed E. J. Murray, The Human Press Inc. Clinton, N.J.) or in
Escherichia coli
(technique described in the French Application No. 94 14470).
It is then necessary to propagate the vector in order to form a stock of viral particles containing it. This production step is critical and must allow high titers of infectious particles to be attained to be able to consider development on a large scale with a view to the preparation of clinical batches. If the first generation adenoviral vectors can be propagated relatively easily in the 293 cell line, the only complementation line described to date and capable of efficiently expressing E1, such is not the case for second generation vectors. In fact, according to the same basic principle, such a vector must be complemented for the essential functions which it cannot express.
The complementation can be provided “in trans” by the cell line employed (designated complementation cell line). It is then necessary to have new lines complementing several essential viral functions (E1 and E2, E1 and E4 or E1, E2 and E4). However, the various attempts carried out until now give the impression that the coexpression of several adenoviral regions is potentially toxic, such that the line risks not being optimal in terms of growth capacity and yield of viral particles, these two criteria being indispensable for industrial exploitation.
Another alternative is based on the use of a supplementary viral element, called “helper virus” introduced into the line at the same time as the adenoviral vector (two-component system) At the present time, an adenovirus from which the E1 region has been deleted and which is capable of synthesizing the expression products of other adenoviral regions is currently used. The co-transfection of such a helper virus and of an adenoviral vector in the 293 line allows the formation of viral particles.
However, a major disadvantage of this method is that the cells produce a mixed population of viral particles, one type comprising the recombinant vector and the other type the helper virus. In practice, the preparations mainly contain viral particles of helper virus, the contamination being able to reach and even exceed 90%. The presence of the helper virus is not desirable in the context of a therapy applied to man and therefore necessitates the employment of cumbersome and costly physical separation techniques, such as ultracentrifugation. In addition, this technology is not very well adapted to the production of vectors of complex structure, such as the second generation vectors, to the extent where, very often, the helper virus has a selective advantage (more rapid replication).
The unresolved problem to this day of the production of recombinant adenoviral vector particles with a high titer is an obstacle to the development of gene therapy.
A novel helper virus has now been constructed by insertion of direct repetitions on both sides of the encapsidation region. The action of a recombinase recognizing them involves the excision of the genetic material situated between them. This deletion does not have any notable consequence on the expression of the viral genes but limits the encapsidation of the helper virus in a viral particle. Thus, the employment of the two-component procedure described above in a cell line expressing the recombinase will allow preparations enriched in adenoviral vector particles of interest to be produced.
The present invention follows from the perfection of a genetic technique based on the use of recombination sequences and of a recombinase to produce mainly the recombinant viral vector and to limit the contamination by the helper virus. The aim of the present invention is to put at the disposal of the public a novel helper virus able to express the genes which it carries (that is to say capable of exercising its function of trans-complementation) but unable to be propagated in the presence of a recombinase. The solution provided by the present invention combines safety cf use (prepa
Lusky Monika
Mehtali Majid
Burns Doane Swecker & Mathis L.L.P.
Guzo David
Transgene S.A.
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