Reversible masking using low-molecular-weight neutral lipids to achieve optimal-targeted delivery.

Intravenous injection of therapeutics is required to effectively treat or cure metastatic cancer, certain cardiovascular diseases, and other acquired or inherited diseases. Using this route of delivery allows potential uptake in all disease targets that are accessed by the bloodstream. However, normal tissues and organs also have the potential for uptake of therapeutic agents. Therefore, investigators have used targeted delivery to attempt delivery solely to the target cells; however, use of ligands on the surface of delivery vehicles to target specific cell surface receptors is not sufficient to avoid nonspecific uptake. PEGylation has been used for decades to try to avoid nonspecific uptake but suffers from many problems known as "The PEGylation Dilemma." We have solved this dilemma by replacing PEGylation with reversible masking using low-molecular-weight neutral lipids in order to achieve optimal-targeted delivery solely to target cells. Our paper will focus on this topic.

Oncolytic viruses for induction of anti-tumor immunity.

Oncolytic virotherapy is an evolving but, as yet, unrealized treatment option for cancer. This approach harnesses the cancer-restricted replicative activity of engineered viruses to achieve tumor cell kill. Tumors that are resistant to chemotherapy or radiotherapy can be susceptible to viral oncolysis because of distinct cell kill mechanisms. There is now compelling evidence that collateral induction of anti-tumor immune responses contributes substantially to viral antitumor activities. In addition to the expected anti-viral immune clearance, the "danger" signal created by virus-infected cells can generate immune co-stimulation known to override immune suppression and reverse tolerance within the tumor microenvironment. Our recent findings indicate that immune activation augments the clinical outcomes of oncolytic virotherapy. Strikingly similar and robust clinical response rates ( > 25%) were observed among advanced cancer patients following intratumoral treatments with adenoviral (AdΔ24) and herpes simplex (JS1/34.5-/47) constructs armed with an integrated granulocyte-macrophage colony-stimulating factor (GMCSF) payload. Both agents produced regressions in injected as well as distant, uninjected lesions, demonstrating systemic effectiveness. We discuss the innate and adaptive immune activating events that may contribute to these clinical outcomes, and examine systemic delivery strategies to tilt the immunological balance from viral clearance to tumor elimination.

Optimization of Non-Viral Gene Therapeutics Using Bilamellar Invaginated Vesicles.

Bilamellar invaginated vesicles (BIVs) are unique liposomal nanoparticles (NPs) that are highly efficient vehicles for intravenous (iv) delivery of encapsulated therapeutics including plasmid DNA. Systemic administration of therapeutics is required to effectively treat or cure metastatic cancer, certain cardiovascular diseases, and other acquired or inherited diseases. In addition to having extended half-life and stability in circulation, BIVs are nontoxic, nonimmunogenic, biodegradable and can be repeatedly administered without losing potency. Furthermore, BIVs encapsulating therapeutic agents can be modified to specifically enter the disease cells using small molecules that mimic beta turns incorporated on the surface of BIV complexes while focusing biodistribution by bypassing uptake in non-target organs and tissues using reversible masking. These modifications do not alter the unique properties of the BIV delivery system that provide for its robust treatment of disease demonstrated in small and large animal models and in Phase I clinical trials. This review will cover the unique properties of BIVs, including its fusogenic entry into cells and its ability to penetrate tight barriers in vivo. Methods to further improve the overall delivery-expression system including further purification of plasmid DNA to eliminate colanic acid from all current commercially produced preparations, and enhanced or prolonged expression provided by plasmid design will also be discussed.

Liposomes for gene transfer in cancer therapy.

We developed improved liposomes that produce efficacy for the treatment of cancer, cardiovascular diseases, and HIV-1-related diseases in small and large animal models. Because our processes are reproducible, we have standard operating procedures (SOPs) for the cGMP manufacture of these reagents that have been approved by the Food and Drug Administration for use in phase I/II clinical trials.

Nonviral delivery for genomic therapy of cancer.

We have developed improved liposomal nanoparticles that efficiently condense nucleic acids, proteins, viruses, drugs, and mixtures of these agents on the interior of bilamellar invaginated structures (BIVs) produced by a novel extrusion procedure. The liposomal complexes have extended half-life in the circulation, serum stability, and broad biodistribution; are targetable to specific organs and cell types; can penetrate through tight barriers in several organs; are fusogenic with cell membranes and avoid endosomes; are optimized for nucleic acid:lipid ratio and colloidal suspension in vivo; can be size fractionated to produce totally homogeneous populations of complexes prior to injection; are nontoxic, nonimmunogenic, and can be repeatedly administered; and they are stable in liquid suspensions and freeze-dried formulations. We can add specific ligands either by ionic interactions or by covalent attachments to the surface of these nucleic acid-liposome complexes to accomplish targeted delivery to specific cell surface receptors. Furthermore, the charge on the surface of these complexes can be modified to avoid uptake by nontarget cells using our novel technology called "reversible masking." We have also achieved high-dose systemic delivery of these complexes without toxicity in vivo by further purification of plasmid DNA. At present, these complexes are injected intravenously into patients in clinical trials to treat lung cancer and will be used in upcoming trials to treat breast, pancreatic, and head and neck cancers. Notably, BIV complexes are being injected intravenously into patients with non-small-cell lung carcinoma who have failed to respond to chemotherapy. These patients are living longer and have demonstrated objective responses, including tumor regression.

PDX-1 acts as a potential molecular target for treatment of human pancreatic cancer.

OBJECTIVES: The purpose of this study was to investigate whether pancreatic and duodenal homeobox factor 1 (PDX-1) could serve as a potential molecular target for the treatment of pancreatic cancer. METHODS: Cell proliferation, invasion capacity, and protein levels of cell cycle mediators were determined in human pancreatic cancer cells transfected with mouse PDX-1 (mPDX-1) alone or with mPDX-1 short hairpin RNA (shRNA) and/or human PDX-1 shRNA (huPDX-1 shRNA). Tumor cell growth and apoptosis were also evaluated in vivo in PANC-1 tumor-bearing severe combined immunodeficient mice receiving multiple treatments of intravenous liposomal huPDX-1 shRNA. RESULTS: mPDX-1 overexpression resulted in the significant increase of cell proliferation and invasion in MIA PaCa2, but not PANC-1 cells. This effect was blocked by knocking down mPDX-1 expression with mPDX-1 shRNA. Silencing of huPDX-1 expression in PANC-1 cells inhibited cell proliferation in vitro and suppressed tumor growth in vivo which was associated with increased tumor cell apoptosis. PDX-1 overexpression resulted in dysregulation of the cell cycle with up-regulation of cyclin D, cyclin E, and Cdk2 and down-regulation of p27. CONCLUSIONS: PDX-1 regulates cell proliferation and invasion in human pancreatic cancer cells. Down-regulation of PDX-1 expression inhibits pancreatic cancer cell growth in vitro and in vivo, implying its use as a potential therapeutic target for the treatment of pancreatic cancer.

Oral immunization of rhesus macaques with adenoviral HIV vaccines using enteric-coated capsules.

Targeted delivery of vaccine candidates to the gastrointestinal (GI) tract holds potential for mucosal immunization, particularly against mucosal pathogens like the human immunodeficiency virus (HIV). Among the different strategies for achieving targeted release in the GI tract, namely the small intestine, pH sensitive enteric coating polymers have been shown to protect solid oral dosage forms from the harsh digestive environment of the stomach and dissolve relatively rapidly in the small intestine by taking advantage of the luminal pH gradient. We developed an enteric polymethacrylate formulation for coating hydroxy-propyl-methyl-cellulose (HPMC) capsules containing lyophilized Adenoviral type 5 (Ad5) vectors expressing HIV-1 gag and a string of six highly-conserved HIV-1 envelope peptides representing broadly cross-reactive CD4(+) and CD8(+) T cell epitopes. Oral immunization of rhesus macaques with these capsules primed antigen-specific mucosal and systemic immune responses and subsequent intranasal delivery of the envelope peptide cocktail using a mutant cholera toxin adjuvant boosted cellular immune responses including, antigen-specific intracellular IFN-gamma-producing CD4(+) and CD8(+) effector memory T cells in the intestine. These results suggest that the combination of oral adenoviral vector priming followed by intranasal protein/peptide boosting may be an effective mucosal HIV vaccination strategy for targeting viral antigens to the GI tract and priming systemic and mucosal immunity.

Widespread, exceptionally high levels of histone H3 lysine 4 trimethylation largely mediate "privileged" gene expression.

We examined the molecular determinants for sustained high-level expression of "privileged" genes, defined as the 0.03% most highly expressed genes within any specific cell. We identified histone modifications by chromatin immunoprecipitation analyses on Keratin 8, the most highly expressed gene in the human breast cancer cell line, MCF-7, based on serial analysis of gene expression. Quantitative comparisons to the "normal" counterpart cell line, MCF-10A, expressing 350-fold lower levels of Keratin 8 and other breast cancer cell lines expressing higher levels were performed using real-time PCR. Extraordinarily high levels of trimethyl histone H3 lysine 4 (H3K4) were found primarily in the first intron of the Keratin 8 gene stretching from 400 to 2000 bp downstream from the promoter in all breast cancer cells lines but not in MCF-10A cells. The highest levels of histone H3K4 trimethylation in MCF-7 cells ranged from 70% to 80% over input within 1200 bp of this region. Knockdown of mixed-lineage leukemia (MLL), the specific methyltransferase for histone H3K4, with MLL-specific siRNA decreased histone H3K4 trimethylation on the Keratin 8 gene and decreased Keratin 8 mRNA levels. Histone H3K4 trimethylation mediates approximately 86% of the elevated, sustained expression of the Keratin 8 gene in MCF-7 cells.

Optimization of gene expression in nonactivated circulating lymphocytes.

Circulating lymphocytes are important target cells for the treatment of HIV-related and autoimmune diseases and for stimulating anti-tumor immunity. To date, gene transfection of these nonactivated cells after intravenous delivery of viral or nonviral vectors remains low although these circulating cells are highly accessible. Optimized lentiviral vectors currently can transduce less than 10% of nonactivated circulating lymphocytes. Here we report transfection of up to 15% of these nonactivated cells using liposomes directed to human CCR5 displayed on the surface of helper T cells and macrophages in transgenic mice. Attachment of modified MIP-1 beta to the surface of DNA-liposome complexes increased gene delivery and expression in nonactivated circulating lymphocytes approximately sixfold. In vitro data using these complexes to transfect PM1 cells that have elevated levels of CCR5 supported our data obtained in vivo. Therefore, ligands that bind to cell surface receptors on circulating lymphocytes can be used with optimized systemic liposomes to increase transfection and gene expression in these cells without activation.

Enhanced gene expression in breast cancer cells in vitro and tumors in vivo.

Gene therapy clinical trials for cancer frequently produce inconsistent results. Some of this variability could result from differences in transcriptional regulation that limit expression of therapeutic genes in specific cancers. Systemic liposomal delivery of a nonviral plasmid DNA showed efficacy in animal models for several cancers. However, we observed large differences in the levels of gene expression from a CMV promoter-enhancer between lung and breast cancers. To optimize gene expression in breast cancer cells in vitro and in vivo, we created a new promoter-enhancer chimera to regulate gene expression. Serial analyses of gene expression data from a panel of breast carcinomas and normal breast cells predicted that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter is highly active in breast cancers. Furthermore, GAPDH is up-regulated by hypoxia, which is common in tumors. We added the GAPDH promoter, including the hypoxia enhancer sequences, to our in vivo gene expression plasmid. The novel CMV-GAPDH promoter-enhancer showed up to 70-fold increased gene expression in breast tumors compared to the optimized CMV promoter-enhancer alone. No significant increase in gene expression was observed in other tissues. These data demonstrate tissue-specific effects on gene expression after nonviral delivery and suggest that gene delivery systems may require plasmid modifications for the treatment of different tumor types. Furthermore, expression profiling can facilitate the design of optimal expression plasmids for use in specific cancers.

Cationic liposome-mediated gene delivery in vivo.

Several improvements have been made in liposomal delivery, thus making this technology potentially useful for treatment of certain diseases in the clinic. Success in non-viral delivery is complicated and requires optimization of several components. These components include nucleic acid purification, plasmid design, formulation of the delivery vehicle, administration route and schedule, dosing, detection of gene expression, and others. With further improvements, broad use of non-viral delivery systems to treat human disorders should be possible.

Identification of heat shock protein 60 as a molecular mediator of alpha 3 beta 1 integrin activation.

The alpha 3 beta 1 integrin is involved in the adhesion of metastatic breast cancer cells to the lymph nodes and to osteoblasts in the bone. Regulation of the affinity or avidity of integrins for their ligands may result from conformational changes induced by changes in the microenvironment of the integrin. Two surface proteins, 55 and 32 kDa, coimmunoprecipitated with the alpha 3 beta 1 integrin from breast carcinoma cells. The 55-kDa protein preferentially associated with the active form of the alpha 3 beta 1 integrin. The protein was identified as HSP60 using two-dimensional electrophoresis and mass spectrometry and confirmed by reimmunoprecipitation of the integrin immune complex with an anti-HSP60 antibody. In cell spreading assays on a thrombospondin-1 substrate, addition of exogenous-recombinant HSP60 was sufficient to specifically activate alpha 3 beta 1 integrin but not to activate function of alpha 2 beta 1, alpha v beta 3, alpha 4 beta 1, or alpha 5 beta 1 integrins. Furthermore, mizoribine, an HSP60-binding drug, blocked activation of the alpha 3 beta 1 integrin induced by insulin-like growth factor 1 (IGF1) or exogenous recombinant HSP60 and inhibited the association of HSP60 with the integrin. Additionally, inhibiting the surface expression of endogenous HSP60 by nonactin inhibited activation of the alpha 3 beta 1 integrin by IGF1. These data demonstrate that HSP60 binding is sufficient to activate alpha 3 beta 1 integrin function and suggest that association of endogenous HSP60 with alpha 3 beta 1 integrin is necessary for IGF1-induced activation.

Bilamellar cationic liposomes protect adenovectors from preexisting humoral immune responses.

Adenoviral vectors have been widely used for gene therapy, but they are limited both by the presence of a humoral immune response that dramatically decreases the level of transduction after reinjection and by their requirement for target cells to express appropriate receptors such as Coxsackie adenovirus receptor (CAR). To overcome both limits, we encapsulated adenovectors using bilamellar DOTAP:chol liposomes. Electron micrography (EM) showed that these liposomes efficiently encapsulated the vectors, allowing CAR-independent adenovector transduction of otherwise resistant cells. DOTAP:chol-encapsulated adenovectors encoding LacZ or alpha(1)-antitrypsin inhibitor (AAT) were also functionally resistant ex vivo and in vivo to the neutralizing effects of human anti-adenoviral antibodies, unlike other liposomal systems. Hence, bilamellar DOTAP:chol liposomes may be useful for applications using adenovectors in which the target cells lack adenoviral receptors or in which the recipient already has or develops a neutralizing antibody response that would otherwise inactivate readministered vector.