A multifunctional PEI-based cationic polyplex for enhanced systemic p53-mediated gene therapy.

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.

Successful in vivo tumor targeting of prostate-specific membrane antigen with a highly efficient J591/PEI/DNA molecular conjugate.

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.

Insulin-like growth factor binding protein-6 activates programmed cell death in non-small cell lung cancer cells.

Insulin-like growth factor binding proteins (IGFBPs) are secreted into the extra-cellular matrix and inhibit cell growth through IGF-dependent and -independent mechanisms. In this study, we investigated the role of IGFBP-6, a relatively unexplored member of the IGFBP family, in the proliferation of non-small cell lung cancer (NSCLC) cells. Infection of NSCLC cell lines in vitro with an adenovirus expressing human IGFBP-6 under the control of a CMV promoter (Ad5CMV-BP6) reduced NSCLC cell number through activation of programmed cell death, as shown by cell staining with Hoechst 33342 or DNA end-labeling with bromodeoxyuridine triphosphate. The growth regulatory effect of IGFBP-6 was investigated in vivo by intratumoral injection of Ad5CMV-BP6 in NSCLC xenografts established in nu/nu mice. A single injection of Ad5CMV-BP6 reduced the size of NSCLC xenografts by 45%. These findings indicate that IGFBP-6 is a potent inducer of programmed cell death in cancer cells and support investigations into IGFBP-6 as a potential target in cancer therapeutics.

Down-regulation of bcl-2 is associated with p16INK4-mediated apoptosis in non-small cell lung cancer cells.

We introduced a functional p16 cDNA into non-small cell lung cancer (NSCLC) cell lines expressing different combinations of normal and mutated p16, p53, and Rb genes via a recombinant adenovirus to determine the effect of exogenous p16 expression on cell growth. Analysis of p16-deficient cells infected with Adv/p16 identified growth arrest of the cells in the G0 - G1 phase early on. Apoptosis was identified to occur by the 5th day after infection which corresponded with increased p16 expression, reduced Rb expression, and increased Rb hypophosphorylation, but only occurred in cells expressing functional p53. Further analysis indicated that the expression of the anti-apoptotic protein bcl-2 was greatly reduced in the NSCLC cell lines H460 and A549 (both -p16, +p53, +Rb), again only by the 5th day after Adv/p16 infection, but no affect on Bax expression was observed. H1299 cells (-p16, -p53, +Rb) infected with Adv/p16 only exhibited apoptosis by an additional infection with Adv/p53 which also corresponded with a down-regulation of bcl-2. In addition, the infection of A549 cells with Adv/p16 followed by a subsequent infection with Adv/Rb lead to a significant decrease in apoptosis which correlated with an increase in bcl-2 expression. These studies suggest that p16 is capable of mediating apoptosis in NSCLC cell lines expressing wild-type p53, through a direct down-regulation of Rb and an indirect down-regulation of the anti-apoptotic protein bcl-2.

The contribution of poly-L-lysine, epidermal growth factor and streptavidin to EGF/PLL/DNA polyplex formation.

High-level targeted gene delivery has been demonstrated by molecular conjugates in vitro; however, in vivo delivery has been limited. The complexity of the resulting protein/DNA polyplex and a lack of understanding of its formation are persistent limitations. In this report, we show the effect of the DNA-binding agent poly-L-lysine (PLL), the ligand epidermal growth factor (EGF), and the coupling protein streptavidin on particle size, charge and gene delivery. Smaller (< 80 nm) and more stable polyplexes were obtained with PLL1116 than with shorter versions of PLL, especially in 0.15 M NaCl. Stability was increased by adding streptavidin to the polyplex; however, EGF increased particle size (> 1000 nm) and decreased gene delivery when > 300 EGF molecules per polyplex were used, indicating that a critical number of EGF molecules was needed for efficient gene delivery. The correct combination of these components resulted in the most efficient gene delivery in vitro and now provide for testing a more stable protein/DNA polyplex to aid in enhancing gene delivery in vivo.

Gene delivery into malignant cells in vivo by a conjugated adenovirus/DNA complex.

Current viral delivery systems suffer from disadvantages that may limit the rate at which therapeutic gene expressing constructs can be tested both in vitro and in vivo. In this study, our focus was to develop a simple gene delivery system for the rapid and reproducible testing of therapeutic genes in cancer cells both in vitro and in vivo. We report here that a delivery system based on using a conjugated adenovirus in complex from with a DNA plasmid can be used for not only delivering genes in vitro but also for efficient and reproducible delivery in vivo. Replication defective adenoviral particles were chemically modified by covalent attachment of poly-L-lysine (PLL) to the viral capsid, allowing for direct interaction with DNA. The adenovirus/PLL conjugate (Adv/PLL) was used to deliver the plasmid pCMV/beta-gal to several different cancer cell lines (i.e., lung, cervical) in vitro and resulted in transduction efficiencies as high as 52% as determined by histochemical staining. On direct intralesional injection of the Adv/PLL/DNA complex into subcutaneous tumors, transduction efficiencies greater than 35% could also be achieved. As a result, this system provides a simple method for delivering and testing therapeutic genes in cells both in vitro and in vivo, prior to the further development of gene therapy vectors for both malignant and benign disease.

Delivery of the p53 tumor suppressor gene into lung cancer cells by an adenovirus/DNA complex.

An adenovirus/DNA complex was constructed by chemically linking poly-L-lysine to the capsid of the replication-defective adenovirus dl312, allowing for coupling with plasmid DNA by an ionic interaction. We have previously demonstrated that this adenovirus/DNA complex can efficiently transduce malignant cells with a plasmid expressing the beta-galactosidase gene both in vitro and in vivo. In this report, we show that this system can deliver a therapeutic gene that encodes for the tumor suppressor protein p53 to lung cancer cells, both in vitro and in vivo, leading to significant biological effects. Transfection of the p53-negative human lung cancer cell line H1299 with the adenovirus/DNA complex carrying a plasmid expressing the p53 gene resulted in high levels of p53 protein and induction of apoptosis. Injection of the complex carrying the p53 gene to subcutaneous tumor sites 5 days after tumor cell implantation resulted in a significant inhibition of tumorigenicity as measured by the number and size of tumors that developed 21 days after treatment. Three and six injections of the complex carrying the p53 gene into H1299 subcutaneous tumor nodules led to significant dose-related tumor growth suppression 18 days after the first injection compared with control-treated tumors. This adenovirus/DNA complex, therefore, is capable of efficiently delivering the p53 gene into malignant cells in vitro and in vivo and now provides a general gene delivery vector that is simple to construct and capable of testing therapeutic genes in malignant cells.

In vivo hepatic gene therapy: complete albeit transient correction of factor IX deficiency in hemophilia B dogs.

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.

The role of microvascular damage in photodynamic therapy: the effect of treatment on vessel constriction, permeability, and leukocyte adhesion.

Intravital microscopy of the rat cremaster muscle was used to evaluate changes in vessel constriction, vessel permeability, and leukocyte adhesion during and after photodynamic therapy (PDT). Animals were given Photofrin doses of 0-25 mg/kg i.v. 24 h before treatment. Cremaster muscles were exposed to 135 J/cm2 light at 630 nm. Animals given 5 mg/kg Photofrin showed no vessel constriction or increase in vessel permeability to albumin. Doses of 10 and 25 mg/kg Photofrin caused a dose-related constriction of arterioles which was observed within the first minutes of illumination at the higher drug dose. After the initial constriction, arteriole response to PDT was biphasic in nature, with some vessels relaxing to nearly control levels while others remained fully constricted. Constriction of venules occurred only at the highest porphyrin dose studied (25 mg/kg) and was delayed in comparison to arteriole constriction. Photofrin doses which produced arteriole constriction also caused an increase in venule permeability to albumin, which occurred shortly after the start of light treatment and was progressive with time. Leakage began at specific sites along the venule wall but became uniform along the entire length of the venule by 1 h after treatment. Changes in the adherence of polymorphonuclear leukocytes to venule endothelium were also observed with PDT. Photofrin doses of 25 mg/kg and 45 J/cm2 light were sufficient to cause polymorphonuclear leukocytes to become adherent to the vessel wall. A second group of animals was given indomethacin trihydrate to examine the involvement of cyclooxygenase products such as thromboxane in vessel response to PDT. Animals given 5 mg/kg indomethacin intraarterially 1 h before light treatment showed no constriction of arterioles or venules at all Photofrin and light doses studied. No increases in venule permeability to albumin were seen in this group of animals. This suggests that cyclooxygenase products including thromboxane are important in causing vessel constriction and changes in permeability during PDT. The initiating event which causes the release of these vasoactive agents remains unknown.