Lyophilized DNA Formulations for Enhanced Expression of Plasmid DNA

Abstract

The present invention provides for a method of treating or preventing ischemic or liver disease in a subject by administering a composition reconstituted from a lyophilized hepatocyte growth factor (HGF) DNA formulation, where the DNA formulation comprises an HGF plasmid DNA, salt and a carbohydrate. The invention further provides for a method of making such a lyophilized DNA formulation that preserves or enhances gene expression both in vitro and in vivo, thus maintaining or stimulating the biological activity of the expressed protein. The invention also provides for the DNA formulation, or the lyophilized DNA formulation according to the methods disclosed.

Description

[0001]This application claims priority to U.S. Provisional Appl. No. 61 / 043,605, filed on Apr. 9, 2008, the entire contents of which are hereby incorporated by reference in their entirety.REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB[0002]The content of the electronically submitted sequence listing (Name: Sequence_Listing_Ascii.txt, Size: 68,654 bytes; and Date of Creation: Apr. 8, 2009) filed herewith the application is incorporated by reference in its entirety.BACKGROUND OF THE INVENTION[0003]Lyophilization is often a preferred formulation for therapeutic materials because the long-term stability of many materials increases in the lyophilized state. However, for plasmid DNA, lyophilized formulations are not the formulations of choice. In most clinical trials using naked (non-complexed plasmid) DNA as a delivery vector, the preferred formulation has been a liquid formulation.[0004]While lyophilized plasmid DNA may be a preferred form of storage, lyophilized f...

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Benefits of technology

[0018]The steps for lyophilization may include subjecting a DNA formulation of the invention to the process of being frozen at subzero temperatures (e.g, −10° C. to −500° C.), and then subjected to one or more drying cycles which comprises gradually heating the DNA formulation to a temperature of about 20° C. to less than or equal to about 30° C., wherein the lyophilization occurs over a period of about 50 to about 100 hours. In a further aspect of the invention, the method for lyophilization comprises: (a) forming an aqueous DNA formulation comprising a plasmid DNA, a salt and a carbohydrate, where the plasmid DNA comprises an HGF gene, or variant thereof; (b) cooling the DNA formulation solution to a temperature of about −10° C. to about −50° C., until frozen; (c) drying the DNA formulation by heating to a temperature of about 20° C. to about 30° C.; and (d) recovering a lyophilized DNA formulation composition having a water content of from about 0.1 weight percent to about 5 weight percent based on the total weight of the recovered DNA formulation.

[0019]In certain embodiments, the DNA formulation is lyophilized under conditions comprising (a) about 30 hours to about 50 hours at a temperature greater than or equal to about −50° C. and less than about 0° C., and (b) about 20 hours to about 50 hours at ...

Problems solved by technology

However, for plasmid DNA, lyophilized formulations are not the formulations of choice.

While lyophilized plasmid DNA may be a preferred form of storage, lyophilized formulations for plasmid DNA have been considered to cause a reduction in gene expression efficiency.

Thus, lyophilization may increase the stability of DNA under long-term storage, but may also cause some damage upon the initial lyophilization process, potentially through changes in the DNA secondary structure or the concentration of reactive elements such as contaminating metals.

In Poxon et al, Pharmaceutical Development and Technology 5:115-122 (2000), the authors demonstrated that lyophilization of a plasmid DNA (pRL-CMV) resulted in a statistically significant loss of transfection efficiency.

While Poxon et al used carbohydrates to ameliorate the in vitro decreased transfection activity of a non-therapeutic plasmid, pRL-CMV expressing Renilla luciferase, stored in EDTA buffer, Poxon et al did not address the use of lyophilized naked DNA formulations in vivo for disease treatment or prevention.

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