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.

1 . A DNA formulation comprising a plasmid DNA, a salt and a carbohydrate, wherein said plasmid DNA comprises an HGF gene, or variant thereof. 
     
     
         2 . The DNA formulation of  claim 1 , wherein said carbohydrate is a mono-, oligo-, or polysaccharide selected from the group consisting of sucrose, glucose, lactose, trehalose, arabinose, pentose, ribose, xylose, galactose, hexose, idose, mannose, talose, heptose, fructose, gluconic acid, sorbitol, mannitol, methyl α-glucopyranoside, maltose, lactone, sorbose, glucaric acid, erythrose, threose, allose, altrose, gulose, erythrulose, ribulose, xylulose, psicose, tagatose, glucuronic acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, neuraminic acid, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans, levan, fucoidan, carrageenan, galactocarolose, pectins, pectic acids, amylose, pullulan, glycogen, amylopectin, cellulose, dextran, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xantham gum, starch, and admixtures thereof. 
     
     
         3 . The DNA formulation of  claim 1 , wherein said carbohydrate is in an amount selected from the group consisting of between about 0.05% to about 30%, between about 0.1% to about 15%, between about 0.2% to about 10%, between about 0.5% and 5%, between about 0.75% and 3%, between about 0.8% and 2%, and between about 0.8% and 1.5%. 
     
     
         4 . The DNA formulation of  claim 3 , wherein said carbohydrate is in an amount of about 0.1% to about 10%. 
     
     
         5 . (canceled) 
     
     
         6 . The DNA formulation of  claim 2 , wherein said carbohydrate is selected from the group consisting of sucrose, mannitol, and admixture thereof. 
     
     
         7 . The DNA formulation of  claim 1 , wherein said salt is selected from the group consisting of NaCl, KCl, and admixture thereof. 
     
     
         8 . The DNA formulation of  claim 1 , wherein said salt is in an amount selected from the group consisting of between about 0.001% and about 10%, between about 0.1% and 5%, between about 0.5% and 2%, between about 0.8% and 1.5%, and between about 0.8% and 1.2%. 
     
     
         9 . (canceled) 
     
     
         10 . The DNA formulation of  claim 1 , wherein said HGF gene or variant thereof is selected from the group consisting of flHGF, dHGF, NK1, NK2, NK4, and admixtures thereof. 
     
     
         11 . The DNA formulation of  claim 1 , wherein said plasmid DNA comprises a hybrid HGF gene. 
     
     
         12 . The DNA formulation of  claim 11 , wherein said hybrid HGF gene is selected from the group consisting of HGF-X2, HGF-X3, HGF-X6, HGF-X7, and HGF-X8. 
     
     
         13 . The DNA formulation of  claim 12 , wherein said plasmid DNA is selected from the group consisting of: pCK-HGF-X2, pCK-HGF-X3, pCK-HGF-X6, pCK-HGF-X7, pCK-HGF-X8, pCP-HGF-X2, pCP-HGF-X3, pCP-HGF-X6, pCP-HGF-X7 and pCP-HGF-X8. 
     
     
         14 . The DNA formulation of  claim 1 , wherein said plasmid DNA is at a concentration of about 1 ng/mL to about 30 mg/mL. 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . The DNA formulation of  claim 1 , wherein said DNA formulation is lyophilized. 
     
     
         18 . The lyophilized DNA formulation of  claim 17 , wherein the lyophilization of said DNA formulation comprises (a) loading a container with said DNA formulation into a lyophilizer; (b) cooling said DNA formulation to a subzero temperature; and (c) drying said DNA formulation. 
     
     
         19 - 21 . (canceled) 
     
     
         22 . A method of treating or preventing ischemic or liver disease in a subject, comprising administering a composition reconstituted from a lyophilized DNA formulation,
 wherein said lyophilized DNA formulation comprises a plasmid DNA, a salt and a carbohydrate; and   wherein said plasmid DNA comprises an HGF gene, or variant thereof.   
     
     
         23 - 37 . (canceled) 
     
     
         38 . The method of  claim 22 , wherein said lyophilized DNA is reconstituted in a pharmaceutically acceptable solution. 
     
     
         39 . The method of  claim 38 , wherein said pharmaceutically acceptable solution is selected from the group consisting of water, PBS, TE, Tris buffer, normal saline, and admixtures thereof. 
     
     
         40 . The method of  claim 22 , wherein said reconstituted composition is administered by direct injection. 
     
     
         41 . A method of making a lyophilized DNA formulation comprising:
 (a) preparing a DNA formulation comprising a plasmid DNA, a salt and a carbohydrate, wherein said plasmid DNA comprises an HGF gene, or variant thereof; and   (b) lyophilizing said DNA formulation, thereby making said lyophilized DNA formulation.   
     
     
         42 . The method of  claim 41 , wherein the lyophilization of said DNA formulation further comprises (a) loading a container with said DNA formulation into a lyophilizer; (b) cooling said DNA formulation to a subzero temperature; and (c) drying said DNA formulation. 
     
     
         43 . (canceled) 
     
     
         44 . (canceled)