Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1999-07-06
2002-11-05
Reynolds, Deborah J. (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C514S04400A, C435S320100, C435S235100, C435S440000, C435S455000, C435S456000
Reexamination Certificate
active
06475480
ABSTRACT:
This application claims priority under 35 U.S.C. §§119 and/or 365 to EP 98 40 1722,8 filed, in Europe on Jul. 7, 1998 and EP 98 40,2825,8 filed in Europe on Nov. 13, 1998; the entire contents of which are hereby incorporated by reference.
The present invention relates to a recombinant adenoviral vector deleted of all or part of the E1 region and a part of the E4 region but retaining sufficient E4 sequences to improve expression and/or persistence of expression of a recombinant gene in a host cell or organism. Furthermore, it relates to the use of adenoviral E4 open reading frames (ORFs) to improve expression or persistence of expression of a recombinant gene inserted in an expression vector. Finally, the invention relates to a method for preparing a viral particle, a cell, a pharmaceutical composition comprising such vectors as well as their therapeutic or prophylactic use. The invention is of very special interest in relation to prospect for gene therapy, in particular in men.
Gene therapy can be defined as the transfer of genetic material into a cell or an organism to treat or prevent a genetic or acquired disease. The possibility of treating human disorders 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 USA in September 1990 on a patient who was genetically immunodeficient as a result of a mutation affecting the gene encoding adenine deaminase (ADA). The relative success of this first experiment encouraged the development of this technology for various genetic and acquired diseases. The large majority of the current protocols employ vectors to carry the therapeutic gene to the cells to be treated. Numerous viral or synthetic vectors have been developed during these last years. Their structure, organization and biology are described in the literature available to a person skilled in the art.
Adenoviruses have been detected in many animal species, are nonintegrative and not very pathogenic. They are able to infect a variety of cell types, dividing as well as quiescient cells. They have a natural tropism for airway epithelia. In addition, they have been used as live enteric vaccines for many years with an excellent safety profile. Finally, they can be easily grown and purified in large quantities. These features have made adenoviruses particularly appropriate for use as gene therapy vectors for therapeutic and vaccine purposes. Their genome consists of a linear double-standed DNA molecule of approximately 36 kb carrying more than about thirty genes necessary to complete the viral cycle. The early genes are divided into 4 regions dispersed in the adenoviral genome (E1 to E4) which contain 6 transcription units directed by their own promoters. The E1, E2 and E4 regions are essential for viral replication whereas the E3 region, which is believed to modulate the anti-viral host immune response, is dispensable for viral growth in vitro. The late genes (L1 to L5) encode in their majority the structural proteins constituting the viral capsid. They overlap at least in part with the early transcription units and are transcribed from a unique promoter (MLP for Major Late Promoter). In addition, the adenoviral genome carries at both extremities cis-acting regions essential for DNA replication. These are the 5′ and 3′ ITR (Inverted Terminal Repeat) and a packaging sequence following 5′ ITR.
The E4 region is believed to be involved in viral DNA replication, late mRNA synthesis, viral assembly and the shut off of host protein synthesis. It is a complex transcription unit which encodes a variety of polypeptides. Those encoded by the open reading frames (ORFs) 6 and 7 are assumed to compete with the cellular RB protein for the binding to the E2F transcription factor, confering a function of transactivators. The expression product of ORF4 is able to bind and regulate the cellular phosphatase 2A to modulate the activity of viral (E1A) and cellular transcription factors. The polypeptides encoded by ORFs 3 and 6 are essential to viral growth because of their capability to maturate the primary 28 kb transcript derived from the adenoviral genome or its export into the cytoplasm. Their absence might be complemented in trans to allow the viral growth. In addition, the ORF6 polypeptide interacts with the E1B encoded 55K polypeptide to form a complex that facilitates the cytoplasmic accumulation of late messengers at the expense of cellular mRNA.
The adenoviral vectors presently used in gene therapy protocols lack most of the E1 region in order to avoid their dissemination in the environment and the host body. Additional deletions in the E3 region allow to increase the cloning capacity. The gene of interest is introduced into the viral DNA in place of a deleted region. The feasibility of gene transfer using these vectors designated “first generation” has been demonstrated in a number of cases. However, the question of their safety is still under evaluation. Indeed, the probability to generate replication-competent viruses during their propagation in conventional complementing cell lines, is not negligible. Furthermore, the potential immunogenicity of viral proteins still expressed by the viral backbone may reduce the persistence of transduced cells as well as the long term expression of the recombinant transgene and may be associated with inflammatory events.
These major drawbacks have led to the construction of vectors of second generation that retain the cis regions necessary for viral replication (ITRs and packaging sequences) and contain substantial genetic modifications aimed to abolish the residual synthesis of the viral antigens which is postulated to be responsible for the stimulation of inflammatory responses (see for example the international application WO94/28152 or U.S. Pat. No. 5,670,488 which discloses adenoviral vectors partially deleted of E4 sequences with the exception of ORF3 or ORF6/7 that do not need E4 complementation). A minimal vector deficient for the whole adenoviral functions can also be considered.
The persistence of transgene expression is a prerequisite before envisaging the general use of adenoviral vectors in human gene therapy protocols, in particular in view of treatment of chronic and genetic diseases. However, deletion of the E4 region has been recently shown to alter transgene expression conducted by a heterologous promoter (i.e. CMV promoter, RSV LTR). Coinfection studies indicated that E4 products could be supplied in trans to restore stable transgene expression (Armentano et al., J. Virol. 71 (1997) 2408-2416; Brough et al., J. Virol. 71 (1997), 9206-9213).
Thus, the technical problem underlying the present invention is the provision of expression vectors which do not show the instability of transgene expression as observed in E4-deleted adenovirus vectors and of means which allow to obtain long term expression of a transgene in cells.
This problem is solved by the provision of the embodiments characterized in the claims.
Accordingly, the present invention relates to a recombinant adenoviral vector derived from an adenovirus genome in which at least all or part of the E1 region is deleted or non-functional and a part of the E4 region has been deleted and which comprises a gene of interest operably linked to regulatory elements, wherein said adenoviral vector retains sufficient E4 sequences to improve expression and/or persistence of expression of said gene of interest in a host cell or organism. Preferably, the retained E4 sequences consist of:
(i) ORF3 and ORF6+ORF7;
(ii) ORF3 and ORF7;
(iii) ORF3 and ORF6;
(iv) ORF3 and ORF6/7;
(v) ORF3 and ORF4; or
(vi) ORFs 1, 2, 3 and 4.
It was surprisingly found that impaired transgene expression in E4-deleted adenoviral vectors could be fully restored by the presence and expression of certain E4 ORFs, in particular of the above-mentioned E4 ORFs.
Although the E4 region may vary between the different adenovirus strains, it can be identified on the basis of nucleotide sequ
Lusky Monica
Mehtali Majid
Burns Doane Swecker & Mathis L.L.P.
Reynolds Deborah J.
Sorbello Eleanor
Transgene S.A.
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