Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
1999-02-16
2001-04-03
Clark, Deborah J. R. (Department: 1633)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S320100, C435S325000, C435S455000, C435S456000, C424S093200
Reexamination Certificate
active
06210946
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of vector biology and gene therapy. More specifically, the present invention relates to the production of recombinant adenoviral vectors with replacement of fibers for cell-specific targeting with concomitant elimination of endogenous tropism.
2. Description of the Related Art
Adenovirus interacts with eukaryotic cells by virtue of specific receptor recognition of domains in the knob portion of the fiber protein (21-23) which protrude from each of the twelve vertices of the icosahedral capsid. Recombinant adenovirus vectors are used in a number of gene therapy applications (21, 35, 38). This fact has derived principally from the high levels of gene transfer achievable with this vector approach both in vitro and in vivo.
Recombinant adenovirus vectors are distinguished from other systems by their unique ability to accomplish in situ gene delivery to differentiated target cells in a variety of organ contexts (5, 6, 9, 10, 12, 20, 25, 27, 29, 31). This has allowed the utilization of recombinant adenoviral vectors as an approach to treat inherited genetic diseases, such as cystic fibrosis, whereby the delivered vector may be contained within the target organ (4-13). In addition, the ability of the adenoviral vector to accomplish in situ tumor transduction has allowed the development of a variety of anti-cancer gene therapy approaches for loco-regional disease (14-18).
Adenoviral vectors can accomplish in vivo gene delivery to a variety of organs after intravenous injection. In these instances, gene transfer frequencies have been sufficiently high to correct inherited metabolic abnormalities in various murine models. Thus, adenoviral vectors fulfill two requirements of an intravenously administered vector for gene therapy: systemic stability and the ability to accomplish long-term gene expression following high efficiency transduction of muscle cells.
Adenoviruses suffer, however, from the disadvantage that the widespread distribution of the adenovirus cellular receptor precludes the targeting of specific cell types. This lack of tropism of adenoviral vectors would result in a decrease in the efficiency of transduction, as the number of virus particles available for delivery to the target cells would be decreased by sequestration by nontarget cells. Furthermore, this would allow ectopic expression of the delivered gene, with unknown and possibly deleterious consequences. Therefore, a means must be developed to redirect the tropism of the adenovirus vector specifically to target cells and permit gene delivery only to affected organs.
To this end, several groups have reported genetic modifications to the knob domain of adenovirus fiber protein and incorporation of such chimeric fibers into virion. For instance, Stevenson et al. (35) and others (24) reported successful generation of Ad5 virions containing fibers consisting of the tail and shaft domains of Ad5 fiber and the knob domain of Ad3, respectively. In addition, Michael et al. (30) demonstrated the incorporation of the gastrin-releasing peptide into the carboxy terminus of recombinant Ad5 fiber. This finding was extended by Legrand et al. (30a) who achieved rescue of recombinant adenovirus vectors containing such fibers. Wickham et al. (41) described the generation of recombinant virus containing fibers with carboxy-terminal polylysine sequences. These studies have established key feasibility issues with respect to this genetic approach but have also demonstrated a number of limiting factors, including the size of the ligand, which may disrupt the correct folding of the fiber protein.
The prior art remains deficient in the lack of effective means to produce recombinant adenoviral vectors with combination of novel targeting and ablation of native tropism. The present invention fulfills this longstanding need and desire in the art.
SUMMARY OF THE INVENTION
The present invention describes the next generation of recombinant, cell-specific adenoviral vectors. More particularly, the instant specification discloses that there are two aspects to consider in the modification of adenoviral tropism: (1) ablation of endogenous tropism; and (2) introduction of novel tropism. To expand the utility of recombinant adenoviruses for gene therapy applications, methods to alter native vector tropism to achieve cell-specific transduction are necessary. To achieve such targeting, one can incorporate ligands into the adenoviral fiber protein, which mediates primary binding of adenovirus to its cell surface receptor. As described herein, the present invention discloses the development of a targeted adenovirus created by replacement of the adenovirus fiber protein. The present invention discloses recombinant adenovirus vectors comprising fiber replacement or substitution proteins composed of the fiber tail domain, a portion of the fibritin gene from the bacteriophage T4 and a ligand domain. The recombinant adenoviral vector may also encode a therapeutic gene i.e. the herpes simplex virus thymidine kinase gene, in the presence of ganclicovir, the adenovirus is able to mediate the specific killing of cells which express the targeted receptor. The present invention thus represents the demonstration of the retargeting of a recombinant adenoviral vector via a non-adenoviral cellular receptor.
In one embodiment of the present invention, there is provided a recombinant adenovirus vector lacking endogenous viral tropism but having novel tropism, wherein the adenovirus vector is modified to produce a replacement adenoviral fiber protein so as to modify viral tropism, wherein the replaced fiber gene comprises the amino-terminal portion of the adenoviral fiber gene including the tail domain, the carboxy-terminal portion of the T4 bacteriophage fibritin gene and a ligand, wherein the replaced adenovirus fiber retains its ability to trimerize and retains its native biosynthesis profile, wherein the ligand is selected from the group consisting of physiological ligands, anti-receptor antibodies and cell-specific peptides, wherein the adenoviral vector further contains a therapeutic gene, wherein said therapeutic gene is the herpes simplex virus-thymidine kinase gene.
In another embodiment of the present invention, there is provided a method of killing tumor cells in an individual in need of such treatment, comprising the steps of pretreating said individual with an effective amount of the recombinant adenoviral vector and administering ganciclovir to said individual.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
REFERENCES:
patent: 5770442 (1998-06-01), Wickham
patent: 5846782 (1998-12-01), Wickham et al.
patent: 5877011 (1999-03-01), Armentano et al.
patent: 5885808 (1999-03-01), Spooner
patent: 6057155 (2000-05-01), Wickham et al.
Eck et al. Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Edition, Chapter 5, Gene-Based Therapy, 1996.*
Verma et al. Gene Therapy-Promises, Problems, and Prospects. Nature, vol. 389, pp. 239-242, Sep. 18, 1997.*
Gall et al. Adenovirus Type 5 and 7 Capsid Chimera: Fiber Replacement Alters Receptor Tropism without Affecting Primary Immune Neutralization Epitopes. Journal of Virology, vol. 70, No. 4, pp. 2116-2123, Apr. 1996.
Curiel David T.
Krasnykh Victor N.
Adler Benjamin Aaron
Clark Deborah J. R.
Sorbello Eleanor
UAB Research Foundation
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