Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1998-02-27
2001-10-02
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S235100, C435S320100
Reexamination Certificate
active
06297004
ABSTRACT:
FIELD OF INVENTION
This invention relates to recombinant viruses, also referred to as recombinant viral particles. By “recombinant virus”, we mean a virus in which at least one of the components of the virion particle is altered or derived by recombinant DNA technology.
This invention also relates to the field of therapeutic gene transfer and concerns the teleological design and use of vectors to derive recombinant proteins or protein components suitable for display on the surface of a gene delivery vehicle which, when displayed on the surface of the gene delivery vehicle, through their interaction with components of the surface of a eukaryotic target cell, are capable of influencing the efficiency with which the gene delivery vehicle delivers its nucleic acid into the target cell, or of transmitting a signal to the target cell which influences the subsequent fate of the delivered nucleic acid, and which are thereby capable of enhancing the suitability of the gene delivery vehicle for an intended application.
BACKGROUND TO THE INVENTION
Display of a Functional Nonviral Polypeptide on a Virus which can Infect Eukaryotic Cells
Recombinant viruses have been widely used as vectors for the delivery of foreign genes into eukaryotic cells. Recombinant viruses which are used for delivery of foreign genes to animal cells include members of several virus families, including Adenovirus, Herpesvirus, Togavirus, and Retrovirus families. Viruses which infect eukaryotic cells comprise a protein shell or shells (the capsid) formed by the multimeric assembly of multiple copies of one or more virus-encoded proteins. The capsid houses the viral nucleic acid (RNA or DNA) and may or may not be enveloped in a lipid bilayer which is studded with virus-encoded oligomeric spike glycoproteins, visible on electron micrographs as spikes projecting from the surface of the virus.
The initial event in the virus life cycle is binding to the surface of the eukaryotic target cell. Binding is mediated by the direct interaction of specialised proteins or glycoproteins on the surface of the virus (antireceptors) with receptors on the surface of the target cell, or indirectly via soluble ligands which bind the virus to receptors on the surface of the target cell. In some instances, the interaction between a virus and a target cell receptor may transmit a metabolic signal to the interior of the target cell. Binding is followed by penetration of the target cell membrane and entry of the viral nucleic acid into the cytosol (reviewed in Marsh and Helenius 1989 Adv Virus Res 36 p107-151). Some nonenveloped viruses undergo conformational changes which result in their direct translocation across the target cell membrane, whereas others, such as adenovirus, are first endocytosed and then cause disruption of the wall of the acidified endosomal vesicle. Enveloped viruses fuse with the target cell plasma membrane whereupon the virus capsid (or core particle), housing the viral nucleic acid is released into the cytoplasm of the target cell. This envelope fusion event is catalysed by oligomeric viral membrane spike glycoproteins which are anchored in the viral envelope and may, or may not be dependent on the prior endocytosis of bound virus and its exposure to an acidic environment within the endosomal vesicle. The mechanisms by which viral spike glycoproteins catalyse membrane fusion may involve their proteolytic cleavage, the dissociation of noncovalently linked subunits or other conformational alterations which expose buried hydrophobic moieties capable of penetrating the lipid membrane of the target cell. Thus, virus-mediated delivery of nucleic acid is a complex, multistage process.
After delivery of the viral nucleic acid into the target cell, further steps in the viral life cycle which lead to viral gene expression, genome replication and the production of progeny viruses are often critically dependent on variable host cell factors. For example, division of the infected target cell is required for efficient integration of a reverse-transcribed retroviral genome into the host cell chromosome and subsequent retroviral gene expression (Harel et al, 1981 Virology 110 p202-207).
The spike glycoproteins of one virus can be incorporated into the viral particles of another strain. Thus with dual viral infection of a single cell by two enveloped mammalian viruses, the host range of either virus may be predictably extended due to promiscuous incorporation of spike glycoproteins encoded by both viruses. This has been shown for closely or distantly related retroviruses (Levy, 1977 Virology 77 p811-825; Weiss and Wong, 1977 Virology 76 p826-834; Besmer and Baltimore, 1977 J Virol 21 p965-973; Canivet et al, 1990 Virology 178 p543-541; Lusso et al, 1990 Science 247 p848-852; Spector et al, 1990 J Virol 64 p2298-2308) and for unrelated viruses from different families (Schnitzer et al, 1977 J Virol 23 p449-454; Metsikko and Garoff, 1989 J Virol 63 p5111-5118; Schubert et al, 1992 J Virol 66 p1579-1589). The spike glycoproteins of certain closely or distantly related retroviruses have also proved to be entirely interchangable, allowing production of infectious hybrid virions with envelope spike glycoproteins of one retrovirus and core particles of another retrovirus (Mann et al, 1983 Cell 33 p153-159; Wilson et al, 1989 J Virol 63 p2374-2378). Similar results have been demonstrated with insect/plant viruses (Briddon et al, 1990 Virology 177 p85-94). Furthermore virus host range was predictably altered by exchanging the N-terminal receptor binding domains of envelope spike glycoproteins from related retroviruses with distinct cellular tropisms (Battini et al, J Virol 1992:66 p1468-1475). When viral spike glycoproteins from closely related virus strans were coexpressed in the same cell, mixed oligomers were formed with high efficiency (Boulay et al, 1988 3 Cell Biol 106 p629-639; Doms et al, 1990 J Virol 64 p3537-3540). Thus, there is considerable scope for altering the host ranges of recombinant viruses by exchanging, mixing and recombining the viral spike glycoproteins of naturally occurring viruses.
However, a relatively small minority of the universe of eukaryotic cell surface structures are actually used as receptors by naturally occurring viruses and it is often difficult to identify a virus with a host range which coincides with the requirements of a particular gene therapy application. For example, to genetically modify a population of normally quiescent haemopoietic stem cells in vivo, a most desirable gene transfer vehicle would be a recombinant retrovirus whose spike glycoproteins do not bind to nontarget cells, but which bind to haemopoietic stem cells and mediate membrane fusion and which also signal the target cell to divide as the nucleic acid is delivered. There are no known naturally occurring viral spike glycoproteins which meet these requirements, and there is therefore a need for new technologies to facilitate the generation of novel spike glycoproteins which can enhance the specificity and efficiency of virus-mediated gene delivery and expression.
Preformed viral particles can be attached to cells which lack virus receptors by way of a (multivalent) molecular bridge. This is clearly demonstrated by the phenomenon of antibody-dependent enhancement of viral infectivity. Thus, antibody-complexed foot-and-mouth disease virus (a nonenveloped picornavirus) has been shown to infect normally insusceptible cells via the Fc receptor (Mason et al, 1993 Virology 192 p568-577). The phenomenon of antibody-dependent enhancement of viral infectivity, mediated through binding of antibody-complexed viruses to cellular Fc receptors and complement receptors has been demonstrated for several enveloped and nonenveloped viruses (Porterfield, 1986 Adv Virus Res 31 p335-355). Moreover, bivalent antibodies that bind dengue virus to cell surface components other than the Fc receptor were recently shown to enhance infection (Mady et al, 1991 J Immunol 147 p3139-3144). Also, Baird et al (1990 Nature 348 p344-346) showed that herpes simplex virio
Hawkins Robert E.
Russell Stephen J.
Winter Gregory P.
Cambridge Drug Discovery Holding, LTD
Palmer & Dodge LLP
Schwartzman Robert A.
Williams Kathleen M.
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