Methods and compositions for preserving adenoviral vectors

Details Methods and compositions for preserving adenoviral vectors Methods and compositions for preserving adenoviral vectors

1998-12-10

2001-05-01

Travers, Russell (Department: 1614)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C514S777000, C424S093200

Reexamination Certificate

active

06225289

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention relates to methods and compositions useful in preserving adenoviral vectors, particularly adenoviral gene transfer vectors.
BACKGROUND OF THE INVENTION
Modified viruses have proven convenient vector systems for investigative and therapeutic gene transfer applications. Adenoviral vector systems present several advantages for such uses because they are generally associated with benign pathologies in humans, and the 36 kb of the adenoviral genome has been extensively studied. Adenoviral vectors can be produced in high titers (e.g., about 10
13
particle/ml), and such vectors can transfer genetic material to non-replicating, as well as replicating, cells (in contrast with, for example, retroviral vectors which only transfer genetic material to replicating cells). The adenoviral genome can be manipulated to carry a large amount of exogenous DNA (up to about 8 kb), and the adenoviral capsid can potentiate the transfer of even longer sequences (Curiel et al.,
Hum. Gene Ther.,
3, 147-154 (1992)). Additionally, adenoviruses generally do not integrate into the host cell chromosome, but rather are maintained as a linear episome, thus minimizing the likelihood that a recombinant adenovirus will interfere with normal cell function. Aside from being a superior vehicle for transferring genetic material to a wide variety of cell types, adenoviral vectors represent a safe choice for gene transfer, a particular concern for therapeutic applications.
A variety of recombinant adenoviral vectors have been described. Most of the vectors in use today derive from either the adenovirus serotype 2 (Ad2) or serotype 5 (Ad5), members of subgroup C. An exogenous gene of interest typically is inserted into the early region 1 (E1) of the adenovirus. Disruption of the E1 region decreases the amount of viral proteins produced by both the early regions (DNA binding protein) and late regions (penton, hexon, and fiber proteins), preventing viral propagation. These replication deficient adenoviral vectors require growth in either a complementary cell line or in the presence of an intact helper virus, which provides, in trans, the essential E1 functions (Berker et al.,
J. Virol.,
61, 1213-1220 (1987); Davidson et al.,
J. Virol.,
61, 1226-1239 (1987); Mansour et al.,
Mol. Cell Biol.,
6, 2684-2694 (1986)). More recently, adenoviral vectors deficient in both E1 and the early region 4 (E4) have been used to substantially abolish expression of viral proteins. In order to insert the larger genes (up to 8 kb) into the adenoviral genome, adenoviral vectors additionally deficient in the nonessential early region 3 (E3) are used. Multiply deficient adenoviral vectors are described in published PCT patent application WO 95/34671.
The use of adenoviral vectors in investigative and therapeutic applications necessitates that the vectors be transported and stored for a period of time. During this period of storage, the adenoviral vectors desirably are maintained without significant loss of infectivity and/or viability. Adenoviral vectors can be stored frozen at very low temperatures, e.g., −80° C., without significant loss of activity; however, the need for low temperature freezers, which are not widely available, limits the practicality of this approach. Lyophilization, or freeze-drying, is another option for storage of adenoviral vectors. This method has disadvantages as it is expensive, and, upon reconstitution, the adenoviral vector composition is often left for extended periods of time at room temperature (i.e., 20-25° C.). Adenoviral vectors rapidly lose viability when stored at room temperature. Similar problems arise when adenoviral vectors are dried at room temperature.
In view of the above, there exists a need for further methods of, and compositions useful in, the storage or preservation of adenoviral vectors. In particular, there is a need for methods and compositions for storage of adenoviral vectors in a liquid state, rather than a dried or frozen state. The present invention provides such methods and compositions. 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 a method for stabilizing a viral gene transfer vector comprising (a) preparing a pharmaceutical composition comprising an adenoviral vector, a stabilizing agent and a pharmaceutically acceptable liquid carrier, and (b) maintaining the composition in liquid form at a temperature above 0° C. for at least 7 days. The present invention also provides a pharmaceutical composition comprising an adenoviral gene transfer vector, a pharmaceutically acceptable liquid carrier, and a stabilizing agent selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof, wherein the composition can be maintained in liquid form at a temperature above 0° C. for at least 7 days such that the activity of said composition decreases 20% or less after 7 days.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for satisfactorily preserving (i.e., storing) an adenoviral gene transfer vector in a pharmaceutical composition at a temperature above 0° C. for at least 7 days. This invention further provides the pharmaceutical composition for such a purpose, which pharmaceutical composition comprises an adenoviral gene transfer vector, a pharmaceutically acceptable liquid carrier, and a stabilizing agent. The present inventive method and composition desirably preserves an adenoviral gene transfer vector with an acceptable decrease in activity during storage at relatively high temperatures for extended periods of time.
The term “activity,” as used in describing the present invention, is a measure of the viability of a composition of an adenoviral gene transfer vector. Activity is a measure of the amount of gene product produced by cells (e.g., 293 cells or preferably A549 cells) infected by a sample comprising the adenoviral vector containing the gene (i.e., the adenoviral gene transfer vector).
The adenoviral gene transfer vector composition is maintained at a temperature such that the composition remains in liquid form (i.e., temperatures above freezing, thereby preventing viral inactivation). Typically, the adenoviral gene transfer vector composition is maintained at a temperature above 0° C., preferably at 4° C. or higher (e.g., 4-10° C.). In some embodiments, it is desirable to maintain the adenoviral gene transfer vector composition at a temperature of 10° C. or higher (e.g., 10-20° C.), 20° C. or higher (e.g., 20-25° C.), or even 30° C. or higher (e.g., 30-40° C.), such as may be encountered under non-environmentally controlled ambient conditions (which can result in the adenoviral gene transfer vector composition being exposed to a variety of non-freezing temperatures of, for example, 4-37° C.).
The adenoviral gene transfer vector composition is maintained for various periods of time. The adenoviral gene transfer vector desirably is maintained at the aforementioned temperature(s) for at least 1 day (e.g., 7 days (1 week) or more), though typically the time period will be longer, such as at least 3, 4, 5, or 6 weeks, or even longer, such as at least 10, 11 or 12 weeks. During that time period, the adenoviral gene transfer vector optimally loses no, or substantially no, activity, although some loss of activity is acceptable, especially with relatively higher storage temperatures and/or relatively longer storage times.
The present inventive method and composition desirably preserve an adenoviral gene transfer vector at a temperature above 0° C., preferably at a temperature of 4° C., such that the activity of the adenoviral gene transfer vector composition decreases about 20% or less, preferably about 10% or less, and more preferably about 5% or less, after any of the aforementioned time periods, especially after 7 days. Most preferably, the present inventi

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