Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2001-10-04
2004-02-17
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C424S233100
Reexamination Certificate
active
06692956
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns a recombinant adenoviral vector derived from an adenovirus genome in which at least a part of the E3 region is deleted or is non functional, wherein said adenoviral vector retains E3 sequences encoding a functional 14.7K protein, a functional 14.5K protein, and/or a functional 10.4K protein. The present invention further relates to the use of a polynucleotide comprising at least one or more gene(s) of an E3 region of an adenovirus, taken individually or in combination, to protect from an inflammatory reaction in a host cell, tissue or organism. The present invention additionally concerns a viral particle, a host cell and a composition comprising said recombinant adenoviral vector or said polynucleotide, as well as their use for therapeutic or prophylactic purpose. The invention is of very special interest in gene therapy applications and in the protection from TNF (Tumor necrosis factor) or Fas-mediated inflammatory conditions.
BACKGROUND OF THE INVENTION
Gene therapy can be defined as the transfer of genetic material into a cell or an organism. The possibility of treating human disorders by gene therapy has changed in the last 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 suffering from adenine deaminase (ADA) deficiency. This first encouraging experiment has been followed by numerous new applications and promising clinical trials based on gene therapy are currently ongoing. The large majority of the current protocols employ vectors to carry the therapeutic gene to the cells to be treated.
There are two main types of gene-delivery vectors, viral and non-viral. Viral vectors are derived from naturally-occuring viruses and use the diverse and highly sophisticated mechanisms that wild-type viruses have developed to cross the cellular membrane, escape from lysosomal degradation and deliver their genome to the nucleus. Many different viruses are being adapted as vectors, but the most advanced are retrovirus, adenovirus and adeno-associated virus (AAV) (Robbins et al., 1998, Trends Biotechnol. 16, 35-40; Miller, 1997, Human Gene Therapy 8, 803-815; Montain et al., 2000, Tibtech 18, 119-128). Substantial effort has also gone into developing poxviruses (especially vaccinia) and herpes simplex virus (HPV). Non-viral approaches include naked DNA (i.e., plasmidic DNA; Wolff et al., 1990, Science 247, 1465-1468), DNA complexed with cationic lipids (for a review, see for example Rolland, 1998, Critical reviews in Therapeutic Drug Carrier Systems 15, 143-198) and particles comprising DNA condensed with cationic polymers (Wagner et al., 1990, Proc. Natl. Acad. Sci. USA 87, 3410-3414 and Gottschalk et al., 1996, Gene Ther. 3, 448-457). At the present stage of development, the viral vectors generally give the most efficient transfection but their main disadvantages include their limited cloning capacity, their tendency to elicit immune and inflammatory responses and their manufacturing difficulties. Non-viral vectors achieve less efficient transfection but have no insert-size limitation, are less immunogenic and easier to manufacture.
Adenoviruses have been detected in many animal species, are non-integrative and of low pathogenicity. They are able to infect a variety of cell types, dividing as well as quiescent 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). The E1, E2 and E4 regions are essential for viral replication. Early region 3 (E3) has been termed a “non essential region” based on the observation that naturally occuring mutants or hybrid viruses deleted within the E3 region still replicate like wild-type viruses in cultured cells (Kelly and Lewis, 1973, J. Virol. 12, 643-652). 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, respectively the 5′ and 3′ ITRs (Inverted Terminal Repeats) and a packaging sequence.
The E3 region spans map units (MU) 76.6-86.2 (nucleotides 27329 to 31103 in Ad5) and is controlled by its own promoter (E3 promoter) that is quite stringently dependent on the presence of E1 transcription factors for expression. Transcription occurs from left to right with regards to the adenoviral genome (sense orientation) and produces a variety of different mRNA species which differ both in their splicing patterns and in poly A site utilization. Among the nine proteins which are potentially encoded by the mRNAs which initiate from the E3 promoter, seven have been clearly identified. They have been named according to their estimated molecular weight, respectively 19, 14.7, 14.5, 12.5, 11.6, 10.4 and 6.7 kDa. To date, the function of only five of them can be assigned. The E3 11.6K protein is involved in the lysis of adenovirus-infected cells (Tollefson et al., 1996, J. Virol. 70, 2296-2306) whereas the E3-gp19K, 10.4K, 14.5K and 14.7K proteins are immunomodulatory proteins allowing an attenuation of the host immune response against adenovirus-infected cells.
The best characterized of the E3 protein, E3-gp19K, is an integral membrane protein anchored in the membrane of the endoplasmic reticulum (ER). In vitro studies have established that the E3 gp19K protein blocks cytolysis by CTLs (Cytotoxic T lymphocytes) by binding major histocompatibility complex (MHC) class I antigens (Signas et al., 1982, Nature 299, 175-178). This interaction results in the retention of class I molecules in the ER, thus preventing their cell-surface expression (Burgert et al., 1985, Cell 41, 987-997) and, ultimately, recognition of adenovirus-infected cells by CTLs (Andersson et al., 1987, J. Immunol. 138, 3960-3966).
Tumor Necrosis Factor &agr; (TNF&agr;) has been shown to be important for adenovirus clearance during infection. TNF&agr; is a potent cytokine responsible for a wide variety of physiologic and immunologic effects. It is secreted by activated macrophages and lymphocytes in response to virus infections, tissue damages, bacterial endotoxins and other cytokines. TNF&agr; binds to specific receptors, leading to activation of signal transduction pathways, transcription factors and protein kinases. In addition, TNF&agr; is cytotoxic to a wide variety of primary tumors and transformed cell lines (Browning and Ribolini, 1989, J. Immunol. 143, 1859-1967). TNF&agr; can also suppress the replication of both DNA and RNA viruses in infected cells (Mestan et al., 1986, Nature 323, 816-819). TNF activates phospholipase A2 (PLA2), resulting in the release of arachidonic acid (AA) which are responsible for the establishment of an inflammmatory status.
A number of experimental evidences suggest that three E3-encoded proteins, respectively 14.7K, 10.4K and 14.5K inhibit TNF&agr;-induced cytolysis and TNF-induced release of AA (Krajcsi et al., 1996, J. Virol. 70, 4904-4913). The 14.7K protein is a hydrophilic protein found in the soluble fractions of both cytosol and nucleus of adenovirus-infected cells. The mechanism by which the 14.7K protein inhibits TNF&agr;-mediated cytolysis, is not fully defined but it probably interferes with the TNF&agr; receptor signaling pathway.
E3 10.4K and E3 14.5K proteins are integral membrane proteins that act as a comp
Burns Doane Swecker & Mathis L.L.P.
Chen Stacy B.
Housel James
Transgene S.A.
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