Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-09-22
2003-04-29
Nguyen, Dave T. (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C435S455000, C435S456000, C536S023100, C536S023400
Reexamination Certificate
active
06555368
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to adenoviral gene therapy vectors. More specifically, the present invention relates to adenoviral gene therapy vectors in which the adenoviral tropism has been genetically modified.
2. Description of the Related Art
Adenoviral vectors (Ad) have proven to be of enormous utility for a variety of gene therapy applications. This usefulness is derived largely from the unparalleled delivery efficiency of these vectors for in vitro and in vivo applications. Despite this property, however, the full benefit of these vectors is undermined currently by the lack of cell-specific gene delivery capability. Specifically, the promiscuous tropism of the adenovirus hinders gene delivery in a targeted, cell-specific manner. Thus, for the many gene therapy applications where such cell-specific transduction is required, current adenoviral vectors have limited utility.
To address the issue of efficient, cell-specific gene delivery, a variety of strategies have been developed to alter adenoviral tropism. These approaches have included direct chemical modifications of the adenoviral capsid proteins, bi-specific complexes (e.g., a capsid protein and a targeting moiety), and genetic capsid modifications (e.g., genetic replacement/insertion). Whereas the former two strategies have established the feasibility of adenoviral re-targeting, practical production issues as well as regulatory approval considerations have placed the utmost importance on the approach in which modifications to the adenoviral tropism are introduced genetically.
To this end, methods that alter adenoviral tropism via modifications of the adenoviral major capsid proteins, fiber, penton and hexon, have expanded tropism such that it is independent of the native adenoviral receptor (CAR). These methods additionally may ablate the native tropism of the adenovirus. Experimentally, tropism expansion has been achieved via the incorporation of peptide ligands with specificity for target cellular markers. This has largely been via the incorporation of the peptide, RGD-4C, at fiber and hexon locales. RGD-4C recognizes integrins of the &agr;v&bgr;3 and &agr;v&bgr;5 class. In addition, other small peptide markers have been employed to the same end. These studies have established that genetic modification(s) to the capsid can indeed alter adenoviral vector tropism to achieve a limited and/or specific range of gene delivery.
Of note, the locales employed in the context of modifying the major capsid proteins for targeting purposes have allowed only the incorporation of small peptides. To date, these have consisted of peptides identified via phage display methods, or short physiologic peptide ligands. Both of these types of targeting motifs, however, are suboptimal with respect to accomplishing the goal of cell-specific delivery. With respect to the former, only a n extremely limited repertoire of useful peptides have been identified heretofore via phage display techniques. In addition, these peptides have tended to be of low affinity. Furthermore, the fidelity of such targeting peptides, when in the context of the adenoviral vector, is not always preserved. With respect to the latter, available physiologic peptides do not allow targeting to the range of cells required for practical gene therapy approaches.
In this regard, single chain antibodies (scFvs) represent motifs with highly diverse specificities that can be exploited for adenoviral targeting. In addition, single chain antibodies possess high affinities for cognate targets. On this basis, the ability to incorporate single chain antibodies into the adenoviral capsid, and for the single chain antibody specificity/affinity to be preserved following display of the chimeric/recombinant capsid protein would dramatically enhance the utility of genetic capsid modification methods for adenoviral retargeting. The inability to configure single chain antibodies at fiber, hexon, and penton locales has indicated the need to examine the ability of single chain antibodies to be incorporated into alternate capsid proteins.
Thus, the prior art is deficient in alternate adenoviral capsid proteins that allows for the genetic introduction of a useful targeting moiety. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The present invention describes incorporation of targeting peptides such as single chain antibodies into the “minor” capsid proteins, pIIIa and pIX of adenovirus. pIIIa and pIX are present on the adenoviral capsid as monomers and the proteins have extended amino-terminus ectodomains. Thus, both locale and structural considerations indicate that pIIIa and pIX are the ideal capsid proteins for incorporating single chain antibodies and other targeting peptides and achieving genetic modification and retargeting of the adenovirus.
One object of the present invention is to provide a genetically modified adenovirus vector with cell-specific targeting capability and methods of making this genetically modified adenovirus vector.
In one embodiment of the present invention, there is provided a recombinant adenovirus, wherein the adenovirus comprises a modified gene encoding a modified adenoviral capsid protein.
In another embodiment of the present invention, a method of providing gene therapy to an individual in need of such treatment is described, comprising the steps of: administering to the individual an effective amount of a recombinant adenovirus, wherein the adenovirus comprises a modified gene encoding a modified adenoviral capsid protein.
In yet another embodiment of the present invention, there is provided a method of increasing the ability of an adenovirus to transduce a specific cell type, comprising the step of: modifying a gene encoding an adenoviral capsid protein.
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. These embodiments are given for the purpose of disclosure.
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Adler Benjamin Aaron
Nguyen Dave T.
Nguyen Quang
UAB Research Foundation
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