Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Reexamination Certificate
2001-07-30
2004-05-25
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
C435S440000, C435S235100, C530S300000, C530S350000
Reexamination Certificate
active
06740511
ABSTRACT:
This application is a U.S. national stage application of PCT International Application No. PCT/FR00/03377 filed Aug. 25, 2000.
The present invention relates, in particular, to an adenoviral fiber mutated in the regions involved in recognizing and binding to the natural cellular receptor for adenoviruses. It also relates to the adenoviral particles bearing, at their surface, such a fiber, optionally combined with a ligand which confers modified, or even targeted, host specificity on said particles. The invention is of most particular value in the context of the development of vectors which can be used in the context of gene therapy.
Adenoviral vectors are widely used in many gene therapy applications. They have been demonstrated in many animal species and are relatively nonpathogenic, and nonintegrating, and replicate both in dividing and in quiescent cells. In addition, they have a broad host spectrum and are capable of infecting a very great number of cell types, such as for example epithelial cells, endothelial cells, myocytes, hepatocytes, nerve cells and synoviocytes (Bramson et al., 1995, Curr. Op. Biotech. 6, 590-595).
The adenoviral genome consists of a double stranded, linear DNA molecule of approximately 36 kb containing two inverted repeat regions (referred to as ITRs for Inverted Terminal Repeat) framing the genes encoding the viral proteins. The early genes are divided into four regions dispersed in the adenoviral genome (E1 to E4; E for early), including 6 transcriptional units provided with their own promoters. The late genes (L1 to L5; L for late) cover, in part, the early transcription units and are, mostly, transcribed from the major late promoter (MLP).
Adenoviruses have been the subject of many studies and many scientific teams have developed adenoviral vectors which are replication-defective, i.e. in which the genome has been manipulated such that these adenoviral vectors are incapable of dividing or of proliferating in the cells which they infect. Defective adenoviral vectors are in particular obtained by deleting at least the El region (for examples of defective adenoviral vectors, see, in particular, patent applications WO 94/28152 and WO 94/12649).
More recently, other uses of adenoviral particles have been described, in particular in the context of implementing gene therapy protocols.
Thus, patent application WO 95/21259 describes a method for introducing a nucleic acid into a cell, which is based on combining adenoviral particles and nucleic acid, more particularly naked nucleic acid. This method is based mainly on the capacity of the adenoviral particle to transport molecules to the cell nucleus after endocytosis. Curiel et al. (1992 Hum. Gene Ther., 3: 147-154) and Wagner et al. (1992, Proc.
Natl. Acad. Sci., 89; 6099-6103), have, themselves also, shown that combining plasmid with inactivated adenoviral particles allows the endosome to be lysed before fusion with the lysosomes and, therefore, allows the plasmid to escape degradation. This ingenious device makes it possible to increase the efficiency of transfection of the plasmid 100-to 1000-fold in vitro. Preferably, in order for the cellular transfection to be independent of the adenoviral process and to indeed involve the use of a ligand chosen so as to allow targeting of the transfection, an antibody which neutralizes the adenoviral infection can be added to the complex (Michael et al., 1993, J. Biol. Chem., 268: 6866-6869). The contents of these publications and patent applications are incorporated by reference in their entirety, into the present application.
The infectious cycle of adenoviruses is based on two essential steps. The early phase precedes replication initiation and allows the production of the early proteins which regulate replication and transcription of the viral DNA. Replication of the genome is followed by the late phase during which the structural proteins which constitute the basis of the viral particles are synthesized. Assembly of the new virions takes place in the nucleus. Initially, the viral proteins assemble so as to form empty capsids of icosahedral structure, in which the newly formed genome is encapsidated. The adenoviruses released are capable of infecting other permissive cells.
During infection, the fiber and the penton base of the adenoviral particle, present at the surface of the capsids, play a critical role in the cellular attachment of the virions and their internalization (Wickham et al., 1993, Cell, 73, 309-319). Firstly, the adenovirus binds to a cellular receptor (the CAR) present at the surface of the permissive cells, via the fiber in its trimeric form (Philipson et al., 1968, J. Virol. 2, 1064-1075; Defer et al., 1990, J. Virol, 64, 3661-3673). The viral particle is then internalized by endocytosis, due to binding of the penton base to the &agr;
v
&bgr;
3
and &agr;
v
&bgr;
5
cellular integrins (Mathias et al., 1994, J. Virol. 68, 6811-6814).
The adenoviral fiber is composed of three distinct domains (Chroboczek et al., 1995, Current Top. Microbiol. Immunol. 199, 165-200):
(a) at its N-terminal end, is the tail, the sequence of which is very conserved from one adenoviral serotype to the other. It interacts with the penton base and ensures the anchoring of the molecule in the capsid;
(b) in the center, is the shaft. It is a rod-like structure composed of a certain number of pleated-sheet repeats, the number of which varies according to the serotypes under consideration;
(c) at its C-terminal end is the Knob, which has a spherical globular structure containing 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), and 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).
Several teams have already described adenoviral particles for which the native fiber has been modified so as to modify their natural tropism and change the binding specificity of this fiber such that it recognizes a different cellular receptor.
WO 94/10323 describes type 5 (Ad5) adenoviral particles in which the fiber has been mutated so as to comprise the sequence of a fragment of antibody specific for a given antigen (of scFv type), inserted at the end of one of the 22 repetitive units of the shaft. These mutants have a modified specificity of infection of the adenoviral particles and are capable of attaching to cells exhibiting the target antigen.
U.S. Pat. No. 5,543,328. describes a chimeric adenoviral fiber in which the Knob domain is replaced with the tumor necrosis factor (TNF) sequence, or that of the ApoE peptide, so as to redirect the attachment of the modified adenoviral particles toward cells expressing the cellular receptor for TNF or the LDL (low density lipoprotein) receptor, respectively, present at the surface of hepatic cells.
WO 95/26412 describes a fiber modified by incorporating a ligand at the C-terminal end.
WO 96/26281 describes a chimeric fiber obtained by replacing a portion of the native fiber, and in particular of the knob, with the equivalent portion of an adenoviral fiber of another serotype and, optionally, inserting a vitronectin-specific RGD peptide at the C-terminal end.
In addition, French patent application FR 2758821 (97 01005) has demonstrated the role of the class I major histocompatibility complex antigens and of the III modules of fibronectin as a primary receptor and a cofactor, respectively, for adenoviruses. In an identical way, Tomko et al. (1997, Proc. Natl. Acad. Sci 94, 3352-3356), Bergelson et al. (1997, Science 275, 1320-1323) and Roelvink et al. (1998, J. Virol. 72, 7909-7915) have described another receptor for the fiber of various adenoviral serotypes. It is a 46 kDa surface molecule, CAR (Coxsackie and Adenovirus Receptor).
Finally, Xia et al. (1994, Structure 2, 1259-1270) have determined the crystallographic three-dimensional structure of the adenoviral knob. Each monomer includes 8 antiparallel &bgr;-pleated she
Cusack Stephen
Legrand Valérie
Leissner Philippe
Mehtali Majid
Van Raaij Mark Johan
Burns Doane Swecker & Mathis L.L.P.
Foley Shanon
Housel James
Transgene S.A.
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