Pharmaceutical compositions and methods of use thereof comprising polyanionic polymers and amphiphilic block copolymers to improve gene expression

#The invention relates to the field of gene delivery, such as gene therapy and genetic vaccination.

#The unique features of smooth, skeletal, and cardiac muscles, have presented numerous challenges for the development and administration of effective polynucleotide compositions for intramuscular administration. Direct injection of purified plasmids (“naked DNA”) in isotonic saline into muscle was found to result in DNA uptake and gene expression in smooth, skeletal, and cardiac muscles of various species. Rolland A., Critical Reviews in Therapeutic Drug Carrier Systems, Begell House, 143 (1998). It is believed that the unique cytoarchitectural features of muscle tissue are responsible for the uptake of polynucleotides because skeletal and cardiac muscle cells appear to be better suited to take-up and express injected foreign DNA vectors relative to other types of tissues. Dowty & Wolff, Gene Therapeutics: Methods and Applications of Direct Gene Transfer, Birkhäuser, Boston, p. 182 (1994). The relatively low expression levels attained by this method, however, have limited its applications. See Aihara and Miyazaki, Nature Biotechnology, 16:867 (1998). Additionally, traditional gene delivery systems such as polycations, cationic liposomes, and lipids that are commonly proposed to boost gene expression in other tissues usually result in inhibition of gene expression in skeletal and cardiac muscles. Dowty & Wolff, Gene Therapeutics: Methods and Applications of Direct Gene Transfer, Birkhäuser, Boston, p. 82 (1994).

#Anionic polymers such as dextran sulfate and salmon DNA can decrease gene expression in the muscle. Rolland A., Critical Reviews in Therapeutic Drug Carrier Systems, Begell House, 1998, p. 143. Various noncondensive interactive polymers have been used with a limited success to modify gene expression in striated muscle. Nonionic polymers such as poly(vinyl pyrrolidone) poly(vinyl alcohol) interact with plasmids through hydrogen bonding. Rolland A., Critical Reviews in Therapeutic Drug Carrier Systems, Begell House, 1998, p. 143. These polymers may facilitate the uptake of polynucleotides in muscle cells and cause up to 10-fold enhancement of gene expression. However, to achieve a significant increase in gene expression, high concentrations of polymers (about 5% and more) need to be administered. Mumper et al., Pharmacol. Res., 13, 701-709 (1996); March et al., Human Gene Therapy, 6(1), 41-53 (1995). This high concentration of poly(vinyl pyrrolidone) poly(vinyl alcohol) needed to improve gene expression can be associated with toxicity, inflammation, and other adverse effects in muscle tissues. Block copolymers have been used to improve gene expression in muscle or to modify the physiology of the muscle for subsequent therapeutic applications. See U.S. Pat. Nos. 5,552,309; 5,470,568; 5,605,687; and 5,824,322. For example, block copolymers can be used in a gel-like form (more than 1% of block copolymers) to formulate virus particles used to perform gene transfer in the vasculature. In the same range of block copolymers concentration (1-10%), it is possible with block copolymer to modify the permeability of damaged muscle tissue (radiation and electrical injury, and frost bite). In addition DNA molecules can be incorporated into cells following membrane permeabilization with block copolymers. For these applications, block copolymers were used at concentrations giving gel-like structures and viscous delivery systems. These systems are unlikely to enable diffusion of the DNA injected into the muscle, however, thus limiting infusion of the DNA into the myofibers.

#FIG. 1A is a graph of luciferase activity for CpG ODN phosphodiester.

#FIG. 1B is a graph of luciferase activity for non-CpG ODN phosphorothio.

#FIG. 2 is a graph of poloxamers with polyacrylic acid.

#Definitions

#As used herein, the terms below have the following meaning:

#Backbone: Used in graft copolymer nomenclature to describe the chain onto which the graft is formed.