Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1997-03-31
2001-03-20
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S325000, C435S366000, C435S369000, C435S370000
Reexamination Certificate
active
06204060
ABSTRACT:
The present invention relates to new viral vectors permitting the transfer and expression of genes of interest in a host cell or body, the expression of the viral genes being regulated so as to be functional in a complementation cell and nonfunctional in the host cell or body. It also relates to the cells containing these new vectors, as well as to a method for preparing infectious viral particles intended for therapeutic use. The invention is of very special interest in relation to prospects for gene therapy, in particular in man.
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), cytomegaloviruses, 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 are nonintegrative and replicate also in resting cells. 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 and late structural genes (see FIG.
1
).
The early genes are divided into 4 regions dispersed in the adenoviral genome (E1 to E4; E standing for early). They contain 6 transcription units which possess their own promoters. The late genes (L1 to L5; L standing for late) partially overlap the early transcription units and are, for the most part, transcribed from the major late promoter (MLP).
At the present time, all the adenoviral vectors used in gene therapy protocols lack most of the E1 region essential for replication, in order to avoid their dissemination in the environment and the host body. Some of them contain additional deletions, in particular in the nonessential E3 region, enabling their cloning capacity to be increased. The genes of interest are introduced into the viral DNA in place of one or other deleted region. Deletion of the E1 region renders the viral genome deficient for replication. However, E1
−
viruses may be propagated in a complementation cell line, which supplies in trans the deleted viral functions to generate an infectious viral particle. Line 293, established from human embryonic kidney cells, which complements the E1 function effectively (Graham et al., 1977, J. Gen. Virol. 36, 59-72), is commonly used. The E3 region is nonessential and does not need to be complemented.
While the feasibility of gene transfer using these first generation vectors is now well established, the question of their safety remains unresolved. Apart from the safety aspects (risk of generating RCAs, that is to say replication competent particles), the problem of their toxicity arises. In effect, the first clinical trials have revealed the induction of inflammatory responses associated with the expression of the viral genes in the host.
Second generation adenoviral vectors have recently been proposed in the literature. They retain the in cis regions necessary for replication of the virus in the infected cell (ITRs and encapsidation sequences) and contain substantial internal deletions aimed at abolishing the bulk of the viral genes whose expression in vivo is not desirable. However, these vectors of the prior art have some drawbacks which limit their exploitation at an industrial level. It is, in effect, necessary to have at one's disposal new lines complementing the collective deleted functions and enabling viral particles to be produced at a high titer. In point of fact, such a line, in order to be optimal in terms of capacity for growth and yield of viral particles, is especially difficult to generate on account of the cytotoxicity of the adenoviral genes.
The present invention enables these drawbacks to be remedied. On the one hand, a new line derived from line 293 complementing the E1 and E2 or E4 adenoviral functions, for the amplification of conventional second generation adenoviral vectors, has now been constructed, in which line the expression of the E2 or E4 regions is directed by a promoter equipped at its 5′ end with so-called “operator” sequences of the bacterial tetracycline operon, these sequences hereinafter being designated tet O. The synthesis of the corresponding expression products will be activated only in the presence of an inducer which can be produced by the adenoviral vector or by the line itself. Similarly, a repressor may be added to the culture medium when complementation is no longer desired.
On the other hand, new adenoviral vectors from which the majority of the E1 and E3 regions have been deleted have now been generated, in which vectors the transcription units of the remaining viral regions (E2, E4 and/or L1-L5) are regulable with the object of permitting their expression when infectious viral particles are to be generated and of inhibiting it in the host cell. In the examples which follow, the regulation is effected by the tet O sequences. Their insertion on the 5′ side of the TATA box generates a promoter from which the baseline level of transcription is minimal but may be strongly stimulated in the presence of the inducer mentioned above. Thus, the production of viral proteins is activated in a 293 line expressing the inducer, which will enable infectious viral particles to be formed. In contrast, it is considerably reduced in the infected host cell which does not naturally produce the inducer of bacterial origin. The regulation of the viral genes has no effect on the expression of the exogenous nucleotide sequence placed under the control of a promoter that does not respond to tetracycline.
The adenoviral vectors of the present invention provide an advantageous approach to the drawbacks inherent in the use of the vectors of the prior art, since they combine safety of use and ease of production. On the one hand they may be propagated in a conventional complementation line with a high titer compatible with industrial requirements, and on the other hand they enable an exogenous nucleotide sequence to be transferred in vivo, and to be expressed stably while limiting the adverse effects (inflammatory responses in the host). They are most especially suitable for human gene therapy.
Accordingly, the subject of the present invention is a viral vector, characterized in that it comprises an expression unit containing one or more viral genes; said expression unit being functional in a complementation cell and nonfunctional in a host cell.
For the purposes of the present invention, a “viral vector” is obtained from a parent virus w
Lusky Monika
Mehtali Majid
Rittner Karola
Burns Doane Swecker & Mathis L.L.P.
Guzo David
Transgene S.A.
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