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We recently reported a novel coupling strategy involving salicylhydroxamic acid and phenyl(di)boronic acid molecules to attach the CNGRC peptide to PEI/DNA for CD13 targeting in tumors. Here, we doubly coupled Simian Virus (SV) 40 peptide-(nuclear localization signal)) and oligonucleotide-based (DNA nuclear targeting signal) nuclear signals to the same vector using peptide nucleic acid chemistry. This vector, CNGRC/PEG/PEI/DNA-betagal/NLS/DNTS, was predominantly localized in the cell nucleus, yielding about 200-fold higher betagal gene expression in vitro, more than 20-fold increase in tumor-specific gene delivery, and a robust betagal gene expression as demonstrated in stained tumor sections. For gene therapy purposes, we further engineered a similar targeting polyplex, CNGRC/PEG/PEI/DNA-p53/NLS/DNTS, with EBV-based episomal vector for sustained p53 gene expression. A distribution of vector DNA and apoptosis in p53-containing tumors was observed, yielding a significant tumor regression and 95% animal survival after 60 days. This multicomponent vector also co-targeted tumor and tumor-associated endothelial cells but not normal cells, and had more efficient therapeutic index than each vector administered as a single modality. The use of an efficient coupling strategy without compromising the vector's integrity for DNA condensation and endosomal escape; nuclear import; tumor-specific and persistent p53 gene expression clearly provides a basis for developing a single combinatorial approach for non-viral gene therapy.

We have utilized a novel polyethylenimine (PEI)/DNA-betagal vector to investigate the specificity and efficiency of immuno-targeting prostate-specific membrane antigen (PSMA). Coupling of the PSMA-specific monoclonal antibody, J591, to the vector was facilitated via the high-affinity interaction between phenyl(di)boronic acid and salicylhydroxamic acid molecules. Highly efficient gene delivery by this prostate cancer (PCA)-targeted J591/polyethylene glycol (PEG)/PEI/DNA-betagal vector was demonstrated in PSMA-positive cells relative to controls, resulting in significant growth inhibition in vitro when the J591/PEG/PEI/DNA-p53 was used. Competition with free antibody resulted in about 90% reduction in both J591 internalization and betagal gene delivery, indicating specificity for PSMA-positive cells. More importantly, testing the efficiency of the J591/PEG/PEI/DNA-betagal targeting vector in an orthotopic PCA model in nude mice resulted in up to a 20-fold increase in gene delivery over the untargeted vector controls. The in vivo organ distribution profile also revealed betagal expression predominantly in the tumor, which was more than 1 log higher than the next highest level of expression in the lung. Furthermore, with the targeted vector containing the gene for yellow fluorescent protein or biotinylated J591, we further demonstrate in vivo that vector-mediated gene delivery is specific for both tumor cells and tumor-associated neovasculature in PSMA-positive tumors. These results suggest the potential for further optimization of this novel vector in the context of therapeutic gene delivery.

Successful systemic gene therapy has been hindered by vector-related limitations, including toxicity and inefficient gene delivery to tumor cells after i.v. administration. To circumvent these problems, we developed a novel formulation between the polycation polyethyleneimine and DNA that mediates high-level tumor cell transduction in vitro and efficient i.v. gene delivery in that greater reporter gene expression occurred in tumor than in lung. Strikingly, administration of just 6 micro g of the polyethyleneimine/DNA-p53 vector every 3 days for 3 weeks indicated restoration of normal cell cycle regulation and apoptotic mechanisms as demonstrated by efficient p53 expression, increased apoptosis, and a 70% reduction in tumor size in an orthotopic bladder cancer model. This novel vector formulation represents a new method to increase i.v. delivery of genes to tumors.

Hemophilia B is a bleeding disorder caused by mutations in the factor IX gene. The disorder is X-linked recessive with a prevalence of about 1 in 30,000 Caucasian males. Factor IX is naturally synthesized in the liver and secreted into blood. Here we report the construction of recombinant adenoviral vectors containing the canine factor IX cDNA that are capable of transducing hepatocytes in mice at high efficiencies in vivo without partial hepatectomy. The recombinant viral vector was used to treat hemophilia B dogs by direct vector infusion into the portal vasculature of deficient animals. Plasma factor IX concentrations in the treated hemophilia B dogs increased from 0 to 300% of the level present in normal dogs, resulting in complete amelioration of the disease as demonstrated by normal blood coagulation and hemostatic measurements. Although plasma factor IX concentration started to decline after a few days, therapeutic levels of factor IX persisted for 1-2 months in the treated animals. The results validate the principle of in vivo hepatic gene delivery to reconstitute the genetic deficiency in a large animal model and suggest that gene therapy is achievable when long-acting vectors are developed.