Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-10-30
2003-07-29
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C435S006120, C435S069100, C435S235100, C435S325000, C435S091400, C435S091410, C435S091420, C435S456000, C536S023100, C536S024100, C514S04400A, C424S093200, C424S093210
Reexamination Certificate
active
06599737
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to adenoviral gene transfer vectors, as well as methods of making and using the same.
BACKGROUND OF THE INVENTION
The 49 serotypes of human adenovirus are divided into six serogroups, A-F. All human adenoviruses have a capsid that contains 12 fiber proteins and other capsid proteins, such as penton base protein and hexon protein. The 12 fiber proteins extend from the surface of the capsid protein and bind with a native receptor that is expressed on the surface of cells that adenoviruses efficiently infect. This initial binding step is usually followed by a second virus-host cell interaction in which the penton base protein binds to an integrin. The binding of penton base protein to the cell is necessary for integrin mediated endocytosis of the virus. However, it is the binding characteristics of the fiber protein that are normally dominant (e.g., in non-recombinant adenoviruses) in selecting which cell types are infected by an adenovirus.
Of the 49 different serotypes of human adenoviruses, the subgroup F viruses (Ad40 and Ad41) are unique. They are the only serotypes that contain two distinct fiber genes (one short and one long) in the major late transcription unit. Both of these fiber genes are expressed and form homotrimers in equimolar ratios on the surface of group F adenoviruses. However, group F viruses are extremely fastidious (i.e., have complex requirements for viral propagation), and, in general, do not grow well in cell types normally used to grow adenoviruses, such as A549 cells, HeLa cells, and HEK-293 cells. For these and other reasons, it has not been desirable to use group F adenoviruses to make adenoviral vectors that comprise and direct the expression (in target cells) of heterologous genes (i.e., as gene transfer vectors).
In contrast to the group F adenoviruses, Ad2 (group C), Ad5 (group C), and some other adenoviruses have been well studied and are among the serotypes that are commonly adapted to gene transfer methods. Such adenoviral gene transfer vectors have been successfully employed as vehicles to transfer therapeutic, immunogenic or prophylactic, and experimental genes to mammals in vivo, as vehicles to transfer genes to cells and tissues in vitro (including for subsequent ex vivo therapies or studies), as models of regulated eukaryotic transcription, and for other purposes. Adenoviral vectors are among the preferred contemporary gene transfer vectors employed, because adenoviral vectors have relatively low toxicity to host cells, efficiently infect a broad range of host cells, do not typically integrate into the host cell genome, and have a substantial number of other advantages.
There are, however, a substantial number of cell types that adenoviral vectors do not efficiently infect. Moreover, for some applications, there has been a desire in the art to limit the host cell range of adenoviral vectors. Accordingly, there has been a significant effort to make chimeric adenoviral vectors having modified coat proteins which change and control the efficiency with which adenoviral vectors infect host cells in vivo and in vitro (see, e.g., U.S. Pat. No. 4,593,002 (Dulbecco), U.S. Pat. No. 5,521,291 (Curiel et al.), U.S. Pat. No. 5,543,328 (McClelland et al.), U.S. Pat. No. 5,547,932 (Curiel et al.), U.S. Pat. No. 5,559,099 (Wickham et al.), U.S. Pat. No. 5,695,991 (Lindholm et al.), U.S. Pat. No. 5,712,136 (Wickham et al.), and International Patent Application WO 94/10323 (Spooner et al.)). These modified coat proteins bind or selectively bind to a protein on the surface of a cell, which mediates the uptake of the receptor. However, many of these chimeric coat proteins substantially reduce the infection efficiency into preferred production cell lines (e.g., HEK-293 cells). The result of this decreased infection efficiency (in preferred production cell lines) can include lower yields and titers, the need to produce novel cell lines to support propagation of the novel vectors, and other deleterious effects.
In view of the foregoing, there exists a need for an adenoviral gene transfer vector or a method for producing an adenoviral vector that has a novel target cell range and/or allows the desired control of vector tropism, and which also allows easy and efficient vector production. The present invention provides such a vector and method. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The present invention provides an adenoviral gene transfer vector comprising a passenger gene and two types of fiber genes as well as the genome of the present inventive gene transfer vector. Optionally, one type of the fiber proteins of the adenoviral gene transfer vector has affinity with a natural adenoviral receptor, while the other type of fiber does has affinity for a novel cell surface binding site, but does not have affinity for a natural adenoviral receptor. Advantageously, the ratio of the types of fiber proteins on the adenoviral gene transfer vector can be manipulated to impart selectivity of binding to cells in vivo for research or other uses. The present invention also provides a chimeric adenoviral fiber gene that encodes a fiber protein that has a Factor Xa cleavage site.
DETAILED DESCRIPTION OF THE INVENTION
While applicants do not wish to be bound to any particular theory, it is believed that the adenoviral fiber protein is dominant in determining which types of cells will be efficiently infected when contacted with an adenovirus. Accordingly, recombinant fiber proteins have been made which have affinity for cellular receptors other than the native adenoviral fiber receptor (hereinafter referred to as CAR, which stands for Coxsackievirus-Adenovirus Receptor; see Bergelson et al.,
Science
, 275, 1320-1323 (1997), and Hong et al.,
EMBOJ.
, 16, 2294-2306 (1997)). In many embodiments of these chimeric adenoviral fiber proteins, the binding moiety of the native fiber protein is destroyed, deleted, or sterically obscured. Therefore, adenoviruses comprising the chimeric adenoviral fiber protein must be propagated in specialized cells expressing or overexpressing a receptor for the chimeric fiber protein. Several embodiments of the present invention provide a novel adenovirus comprising, and directing the expression of (in a target cell), a heterologous gene of interest (i.e., a gene transfer vector) and a convenient method of obviating this and other problems.
One embodiment of the present inventive method comprises making and using the present inventive adenovirus to facilitate propagation of an adenoviral vector that has a chimeric fiber protein that does not bind to CAR. The present inventive adenovirus is an adenovirus having two types of fiber proteins, at least one of which does not efficiently bind to CAR. The second type of fiber gene can be a wildtype fiber protein or is a chimeric fiber protein that retains the ability to bind to CAR. Thus, the inventive adenovirus can have one type of fiber protein with the ability to bind to CAR (or another receptor present on the surface of a preferred production cell line for propagating a particular virus) and a second type of fiber protein that binds to a receptor present on the surface of a target cell of interest.
Effective adenoviral infection does not require more than one or a few fiber proteins that have the capacity to bind to a cell surface receptor for the fiber protein. Therefore, the second type of fiber gene efficiently mediates the uptake of the virus by production cells, despite the fact that not all twelve fiber proteins—and perhaps only one fiber protein per capsid—have affinity for the production cell; especially when the production cell is maintained in vitro, where it is possible to obtain high viral particle to cell ratios. The present invention is especially useful where it is difficult to make or maintain the production cell (e.g., cells expressing an essential gene function of both
Cornell Research Foundation Inc.
Guzo David
Marvich Maria
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