Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-10-04
2003-05-27
Priebe, Scott D. (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C435S091400, C424S093200, C514S04400A
Reexamination Certificate
active
06569677
ABSTRACT:
The subject of the present invention is an adenoviral fiber mutated in the regions involved in the recognition and the binding of the natural cell receptor for adenoviruses. It also relates to the recombinant viruses carrying at their surface such a fiber and a ligand which confers on them a modified or targeted host specificity towards a particular cell type, the cells containing these adenoviruses as well as a method for preparing infectious viral particles thereof intended for therapeutic use. The invention is most particularly of interest for gene therapy perspectives, in particular in humans.
By virtue of their particular properties, adenoviruses are used in an increasing number of applications in gene therapy. Having been identified in numerous animal species, they are not very pathogenic, are nonintegrative and replicate both in dividing and quiescent cells. Furthermore, they exhibit a broad host spectrum and are capable of infecting a very large number of cell types such as epithelial cells, endothelial cells, myocytes, hepatocytes, nerve cells and synoviocytes (Bramson et al., 1995, Curr. Op. Biotech. 6, 590-595). However, this absence of specificity of infection could constitute a limit to the use of recombinant adenoviruses, on the one hand, from a safety point of view since there may be dissemination of the recombinant gene in the host organism and, on the other hand, from the efficiency point of view since the virus does not infect specifically the cell type which it is desired to treat.
In general, the adenoviral genome consists of a double-stranded linear DNA molecule of about 36 kb containing the genes encoding the viral proteins and, at its ends two inverted repeats (designated ITR for Inverted Terminal Repeat) involved in the replication and the encapsidation region. The early genes are distributed in 4 regions dispersed in the adenoviral genome (E1 to E4; E for early), containing 6 transcriptional units equipped with their own promoters. The late genes (L1 to L5; L for late) partly cover the early transcription units and are, for the most part, transcribed from the major late promoter MLP.
As a guide, all the adenoviruses used in gene therapy protocols are deficient for replication by deletion of at least the E1 region and are propagated in a complementation cell line which provides in trans the deleted viral functions. The 293 line, established from human embryonic kidney cells, which efficiently complements the E1 function (Graham et al., 1977, J.
Gen. Virol. 36, 59-72), is commonly used. Second-generation vectors have recently been proposed in the literature. They conserve the regions in cis which are necessary for the replication of the virus in the infected cell (ITRs and encapsidation sequences) and contain substantial internal deletions designed to eliminate most of the viral genes whose expression in vivo can lead to the establishment of inflammatory or immune responses in the host. The adenoviral vectors and the technique for their preparation have been the subject of numerous publications which are accessible to persons skilled in the art.
The infectious cycle for adenoviruses occurs in 2 steps. The early phase precedes the initiation of replication and makes it possible to produce the early proteins regulating the replication and transcription of the viral DNA. The replication of the genome is followed by the late phase during which the structural proteins which constitute the viral particles are synthesized. The assembly of the new virions takes place in the nucleus. In a first stage, the viral proteins assemble so as to form empty capsids of icosahedral structure into which the genome is encapsidated. The adenoviruses liberated are capable of infecting other permissive cells. In this regard, the fiber and the penton base present at the surface of the capsids play a critical role in the cellular attachment of the virions and their internalization.
The adenovirus binds to a cellular receptor present at the surface of the permissive cells via the trimeric fiber (Philipson et al., 1968, J. Virol. 2, 1064-1075; Defer et al., 1990, J. Virol. 64, 3661-3673). The particle is then internalized by endocytosis through the binding of the penton base to the cellular integrins &agr;
v
&bgr;
3
and &agr;
v
&bgr;
5
(Mathias et al., 1994, J. Virol. 68, 6811-6814). The capacity of the soluble fiber or of anti-fiber antibodies to inhibit infection demonstrates its role in the cellular attachment of the virus.
The fiber is composed of 3 domains (Chroboczek et al., 1995, Current Top. Microbiol. Immunol. 199, 165-200):
(1) At the N-terminus, the tail, which is highly conserved from one serotype to another, interacts with the penton base and ensures the anchorage of the molecule in the capsid.
(2) The stem is a structure in the form of a rod, composed of a number of repeats of &bgr; sheets, this number varying depending on the serotypes.
(3) Finally, at the distal end of the stem, the head is a spherical globular structure which contains the trimerization signals (Hong and Engler, 1996, J. Virol. 70, 7071-7078; Novelli and Boulanger, 1991, J. Biol. Chem. 266, 9299-9303; Novelli and Boulanger, 1991, Virology 185, 365-376). Furthermore, most of the experimental data show that the head domain is responsible for the binding to permissive cells (Henry et al., 1994, J. Virol. 68, 5239-5246; Louis et al., 1994, J. Virol. 68, 4104-4106).
“Targeted” adenoviruses whose native fiber is modified so as to recognize a different cellular receptor have already been proposed in the literature. Thus, WO94/10323 describes mutants of the fiber of Ad5, into which a sequence encoding an antibody fragment (of the scFv type) is inserted at the end of one of the 22 repetitive units of the stem with the aim of modifying the specificity of infection towards cells having the target antigen. U.S. Pat. No. 5,543,328 describes an Ad5 chimeric fiber in which the head domain is replaced by tumor necrosis factor (TNF) so as to interact with the cellular receptor for TNF. In another construct, the Ad5 native fiber is fused at its C-terminal end with the peptide ApoE allowing binding to the LDL (for low density lipoprotein) receptor present at the surface of hepatic cells. WO95/26412 describes a fiber modified by incorporation of a ligand at the C-terminal end which conserves its trimerization capacities. WO96/26281 describes a chimeric fiber obtained by replacing part of the native fiber and, in particular, the head, with the equivalent part of an adenoviral fiber of another serotype and, optionally, by inserting at the C-terminal end a peptide RGD which is specific for vitronectin.
As indicated above, the specificity of infection of an adenovirus is determined by the attachment of the adenoviral fiber to a cellular receptor situated at the surface of permissive cells.
French patent application 97 01005 has identified the role of the antigens of the class I major histocompatibility complex and of the III modules of fibronectin as primary receptor and as cofactor, respectively, for adenoviruses. However, other proteins may be involved. In this regard, recent studies have presumed the use of the cellular receptor for the coxsackie viruses by the types 2 and 5 adenoviruses to penetrate into their target cells (Bergelson et al., 1997, Science 275, 1320-1323). The problem which the present invention proposes to solve is to modify the region for interaction of the adenoviral fiber with the cellular receptor(s) in order to alter the natural host specificity of the adenoviruses carrying the mutated fiber. For ease of understanding, the term “cellular receptor” for adenoviruses will be used hereinafter to designate the cellular polypeptide(s) involved directly or otherwise in the binding of adenoviruses to their natural target cells or in the penetration into the latter. Of course, said receptor may be different depending on the serotypes. The addition of a ligand makes it possible to confer a new tropism toward one or more specific cell types carrying at their surface a target molecule recognized by the liga
Boulanger Pierre
Legrand Valérie
Mehtali Majid
Priebe Scott D.
Transgene S.A.
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