Gene therapy: the first two decades and the current state-of-the-art.

The concept of gene therapy was envisioned soon after the emergence of restriction endonucleases and subcloning of mammalian genes in phage and plasmids. Over the ensuing decades, vectors were developed, including nonviral methods, integrating virus vectors (gammaretrovirus and lentivirus), and non-integrating virus vectors (adenovirus, adeno-associated virus, and herpes simplex virus vectors). Preclinical data demonstrated potential efficacy in a broad range of animal models of human diseases, but clinical efficacy in humans remained elusive in most cases, even after decades of experience in over 1000 trials.

In vivo expression of human ATP:cob(I)alamin adenosyltransferase (ATR) using recombinant adeno-associated virus (rAAV) serotypes 2 and 8.

Background: Methylmalonic aciduria (MMA) is an autosomal recessive disease with symptoms that include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000-fold greater than normal. CblB MMA, a subset of the mutations leading to MMA, is caused by a deficiency in the enzyme cob(I)alamin adenosyltransferase (ATR). No animal model currently exists for this disease.

Adeno-associated viral vector-mediated interleukin-10 prolongs allograft survival in a rat kidney transplantation model.

Interleukin-10 (IL-10) is a pleiotropic cytokine that plays a pivotal role in the regulation of immune responses. Hence, we evaluated the effects of a recombinant adeno-associated viral vector 1 (rAAV1) encoding rat IL-10 (rAAV1-IL-10) in a rat model of kidney allograft rejection. Dark Agouti rat kidneys were transplanted into Wistar-Furth (WF) rats 8 weeks following a single intramuscular administration of either rAAV1-IL-10 or rAAV1-green fluorescence protein (GFP).

Preclinical characterization of a recombinant adeno-associated virus type 1-pseudotyped vector demonstrates dose-dependent injection site inflammation and dissemination of vector genomes to distant sites.

To translate the potential advantages of recombinant adeno-associated virus type 1 (rAAV1) vectors into a clinical application for muscle-directed gene therapy for alpha1 -antitrypsin (AAT) deficiency, we performed safety studies in 170 C57BL/6 mice and 26 New Zealand White rabbits. A mouse toxicology study included 8 cohorts of 10 mice each (5 per sex). Mice were killed either 21 or 90 days after intramuscular injection of doses ranging up to 1x10(13)vector genomes (VG), equivalent to 4 x 10(14)VG/kg.

Fluorescence polarization of tetracycline derivatives as a technique for mapping nonmelanoma skin cancers.

Nonmelanoma skin cancer is the most common form of human cancer, often resulting in high morbidity. Low visual contrast of these tumors makes their delineation a challenging problem. Employing a linearly polarized monochromatic light source and a wide-field CCD camera, we have developed a technique for fluorescence polarization imaging of the nonmelanoma cancers stained using antibiotics from the tetracycline family.

Expression of a truncated cystic fibrosis transmembrane conductance regulator with an AAV5-pseudotyped vector in primates.

Gene therapy using recombinant adeno-associated virus (rAAV2) vectors for cystic fibrosis has shown gene transfer and remarkable safety, yet indeterminate expression. A new construct has been characterized with a powerful exogenous promoter, the cytomegalovirus enhancer/chicken beta-actin promoter, driving a truncated CF transmembrane conductance regulator (CFTR), pseudotyped in an AAV5 viral coat. Our goal is to demonstrate that airway delivery of a pseudotyped rAAV5 vector results in gene transfer as well as expression in non-human primates.

Viral vector-mediated and cell-based therapies for treatment of cystic fibrosis.

Gene and cell-based therapies are considered to be potentially powerful new approaches for the management of cystic fibrosis (CF) lung disease. Despite tremendous efforts that have been made, especially in studies to understand the obstacles to gene delivery, major challenges to the application of these approaches remain to be solved. This article will review the advancements made and challenges remaining in the development of viral vector-mediated and cell-based approaches to treat patients with CF.

Improved staining method for determining the extent of thermal damage to cells.

Background And Objective: Enzyme histochemical stains of frozen sections have been used by investigators to assess thermal damage. The assessment of thermal damage to cells in lipid-rich tissues such as subcutaneous tissue and sebaceous glands can be difficult due to the quality of frozen sections of such tissues. The purpose of this study is to develop an improved method for this type of evaluation.

Phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 alphal-antitrypsin (AAT) vector in AAT-deficient adults.

A phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 (rAAV2) alpha1-antitrypsin (AAT) vector was performed in 12 AAT-deficient adults, 10 of whom were male. All subjects were either homozygous for the most common AAT mutation (a missense mutation designated PI*Z) or compound heterozygous for PI*Z and another mutation known to cause disease. There were four dose cohorts, ranging from 2.

alpha-1 antitrypsin inhibits caspase-3 activity, preventing lung endothelial cell apoptosis.

alpha-1 Antitrypsin (A1AT) is an abundant circulating serpin with a postulated function in the lung of potently inhibiting neutrophil-derived proteases. Emphysema attributable to A1AT deficiency led to the concept that a protease/anti-protease imbalance mediates cigarette smoke-induced emphysema. We hypothesized that A1AT has other pathobiological relevant functions in addition to elastase inhibition.

Repeated intrathecal injections of plasmid DNA encoding interleukin-10 produce prolonged reversal of neuropathic pain.

Neuropathic pain is a major clinical problem unresolved by available therapeutics. Spinal cord glia play a pivotal role in neuropathic pain, via the release of proinflammatory cytokines. Anti-inflammatory cytokines, like interleukin-10 (IL-10), suppress proinflammatory cytokines.

Safety in nonhuman primates of ocular AAV2-RPE65, a candidate treatment for blindness in Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is a molecularly heterogeneous disease group that leads to blindness. LCA caused by RPE65 mutations has been studied in animal models and vision has been restored by subretinal delivery of AAV-RPE65 vector. Human ocular gene transfer trials are being considered.

The role of photosensitizer molecular charge and structure on the efficacy of photodynamic therapy against Leishmania parasites.

Photodynamic therapy (PDT) is emerging as a potential therapeutic modality in the clinical management of cutaneous leishmaniasis (CL). In order to establish a rationale for effective PDT of CL, we investigated the impact of the molecular charge and structure of photosensitizers on the parasitic phototoxic response. Two photosensitizers from the benzophenoxazine family that bear an overall cationic charge and two anionic porphyrinoid molecules were evaluated.

IL-10 regulation of lupus in the NZM2410 murine model.

Multiple studies have reported high levels of IL-10 in SLE patients and in murine models of lupus. IL-10 is a regulatory cytokine mainly produced by B cells, which use this cytokine to support their proliferation, and by myeloid cells, which use IL-10 to reduce proinflammatory responses. IL-10 is also produced by a subset of CD4+ T regulatory cells.

Comparative oncogenomics identifies NEDD9 as a melanoma metastasis gene.

Genomes of human cancer cells are characterized by numerous chromosomal aberrations of uncertain pathogenetic significance. Here, in an inducible mouse model of melanoma, we characterized metastatic variants with an acquired focal chromosomal amplification that corresponds to a much larger amplification in human metastatic melanomas. Further analyses identified Nedd9, an adaptor protein related to p130CAS, as the only gene within the minimal common region that exhibited amplification-associated overexpression.

Cystic fibrosis transmembrane conductance regulator deficiency exacerbates islet cell dysfunction after beta-cell injury.

The cause of cystic fibrosis-related diabetes (CFRD) remains unknown, but cystic fibrosis transmembrane conductance regulator (CFTR) mutations contribute directly to multiple aspects of the cystic fibrosis phenotype. We hypothesized that susceptibility to islet dysfunction in cystic fibrosis is determined by the lack of functional CFTR. To address this, glycemia was assessed in CFTR null (CFTR(-/-)), C57BL/6J, and FVB/NJ mice after streptozotocin (STZ)-induced beta-cell injury.

Safety of recombinant adeno-associated virus type 2-RPE65 vector delivered by ocular subretinal injection.

AAV2 delivery of the RPE65 gene to the retina of blind RPE65-deficient animals restores vision. This strategy is being considered for human trials in RPE65-associated Leber congenital amaurosis (LCA), but toxicity and dose efficacy have not been defined. We studied ocular delivery of AAV-2/2.

In vivo complementation of complex I by the yeast Ndi1 enzyme. Possible application for treatment of Parkinson disease.

Recent studies suggest that dysfunction of the NADH-quinone oxidoreductase (complex I) is associated with a number of human diseases, including neurodegenerative disorders such as Parkinson disease. We have shown previously that the single subunit rotenone-insensitive NADH-quinone oxidoreductase (Ndi1) of Saccharomyces cerevisiae mitochondria can restore NADH oxidation in complex I-deficient mammalian cells. The Ndi1 enzyme is insensitive to complex I inhibitors such as rotenone and 1-methyl-4-phenylpyridinium ion, known as a metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Therapeutic level of functional human alpha 1 antitrypsin (hAAT) secreted from murine muscle transduced by adeno-associated virus (rAAV1) vector.

Alpha 1 antitrypsin (AAT) is a serine proteinase inhibitor (serpin). One well-known function of this protein is to inactivate neutrophil elastase and other neutrophil-derived proteinases, and prevent the destruction of pulmonary extracellular matrix. Deficiency of AAT can cause emphysema due to degradation of interstitial elastin by elastase.

A novel antiapoptotic role for alpha1-antitrypsin in the prevention of pulmonary emphysema.

Rationale: There is growing evidence that alveolar cell apoptosis plays an important role in emphysema pathogenesis, a chronic inflammatory lung disease characterized by alveolar destruction. The association of alpha1-antitrypsin deficiency with the development of emphysema has supported the concept that protease/antiprotease imbalance mediates cigarette smoke-induced emphysema.

Objectives: We propose that, in addition to its antielastolytic effects, alpha1-antitrypsin may have broader biological effects in the lung, preventing emphysema through inhibition of alveolar cells apoptosis.

Enhanced IgE allergic response to Aspergillus fumigatus in CFTR-/- mice.

To gain insight into aberrant cytokine regulation in cystic fibrosis (CF), we compared the phenotypic manifestations of allergen challenge in gut-corrected CFTR-deficient mice with background-matched C57Bl6 (B6) mice. Aspergillus fumigatus (Af) antigen was used to mimic allergic bronchopulmonary aspergillosis, a peculiar hyper-IgE syndrome with a high prevalence in CF patients. CFTR-/-, C57BL/6 and FVB/NJ mice were sensitized with Af antigen by serial intraperitoneal injections.

Systemic correction of a fatty acid oxidation defect by intramuscular injection of a recombinant adeno-associated virus vector.

Mitochondrial beta-oxidation of fatty acids is required to meet physiologic energy requirements during illness and periods of fasting or physiologic stress, and is most active in liver and striated muscle. Acyl-CoA dehydrogenases of varying chain-length specificities represent the first step in the mitochondria for each round of beta-oxidation, each of which removes two-carbon units as acetyl-CoA for entry into the tricarboxylic acid cycle. We have used recombinant adeno-associated virus (rAAV) vectors expressing short-chain acyl-CoA dehydrogenase (SCAD) to correct the accumulation of fatty acyl-CoA intermediates in deficient cell lines.