The lysosomal-autophagic pathway is activated by starvation and plays an important role in both cellular clearance and lipid catabolism. However, the transcriptional regulation of this pathway in response to metabolic cues is currently uncharacterized. Here we show that the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is induced by starvation through an autoregulatory feedback loop and exerts a global transcriptional control on lipid catabolism via PGC1α and PPARα. Thus, during starvation a transcriptional mechanism links the autophagic pathway to cellular energy metabolism. The conservation of this mechanism in Caenorhabditis elegans suggests a fundamental role for TFEB in the evolution of the adaptive response to food deprivation. Viral delivery of TFEB to the liver prevented weight gain and metabolic syndrome in both diet-induced and genetic mouse models of obesity, suggesting a novel therapeutic strategy for disorders of lipid metabolism.

Helper-dependent adenoviral vectors (HDAds) are devoid of all viral coding sequences and have demonstrated tremendous potential for gene therapy by providing increased cloning capacity (up to 37 kb) and long-term, high-level transgene expression in vivo with negligible toxicity. Currently, the most widely used method of producing HDAds is the Cre/loxP system developed by Graham and co-workers. However, two major obstacles currently hinder progress of this promising technology: (1) the difficulty of large-scale vector production and (2) helper virus (HV) contamination. We have developed an improved producer cell line, HV, and protocols that have successfully addressed these problems. With this system, >1 x 10(13) viral particles (vp) can be easily produced from 3 liters of cells within 2 weeks of vector rescue, with specific yields of >10,000 vp/cell and with exceedingly low HV contamination of 0.4-0.1% without relying on density-based vector purification and 0.02-0.01% following CsCl purification. This new system represents a major improvement over the original method in terms of simplicity, speed, vector yield, and purity, and it will significantly improve our ability to assess this promising gene therapy technology, especially in large animal models and, ultimately, for clinical applications.

Adenoviruses are robust gene delivery vectors in vivo, but are limited by their propensity to provoke strong innate and adaptive responses. Previous work has demonstrated that polyethylene glycol (PEG) modification of adenovirus can protect the vectors from preexisting and adaptive immune responses by reducing protein-protein interactions. To test whether PEGylation can reduce innate immune responses to adenovirus by reducing their interactions with immune cells, first-generation (FG-Ad) and helper-dependent (HD-Ad) Ad5 vectors were PEGylated with SPA-PEG and tested in vitro and in vivo. We demonstrate that increasing PEGylation ablated in vitro transduction, but surprisingly had no negative effect on the level or distribution of in vivo gene delivery. This poor in vitro transduction could be rescued in part by physically forcing the PEGylated vectors onto cells, suggesting that physiological forces in vivo may enable transduction via heparin sulfate proteoglycan and integrin interactions. While transduction remained the same as for unmodified vectors, the PEGylated vectors reduced innate IL-6 responses by 70 and 50% in vivo for FG-Ad and HD-Ad. These reduced innate responses paralleled similar reductions in vector uptake by macrophages in vitro and Kupffer cells in vivo. These data suggest that PEGylation of Ad vectors can reduce innate immune responses without reducing transduction in vivo. These data also suggest that nonspecific vector uptake by macrophages and Kupffer cells may be critically involved in the initial activation of innate immune responses.

Systemic intravascular delivery of adenoviral (Ad) vectors for liver-directed gene therapy has been widely employed because of its simplicity, noninvasiveness, and potential for high transduction. For first-generation Ad vectors (FGAd), this results in high but transient levels of transgene expression and long-term hepatotoxicity due to viral gene expression from the vector backbone. Furthermore, high doses also result in an acute innate inflammatory response with potentially lethal consequences. Unlike FGAd, helper-dependent Ad vectors (HDAd) contain no viral genes and can provide sustained, high-level transgene expression with negligible long-term toxicity. However, whether the absence of viral gene expression leads to any decrease of acute toxicity in nonhuman primates has yet to be determined. To address this, we injected one baboon with 5.6 x 10(12) HDAd viral particles (VP)/kg and a second with 1.1 x 10(13) VP/kg. Approximately 50% hepatocyte transduction, accompanied by mild and transient acute toxicity, was observed in the animal receiving the lower dose. In the animal receiving the higher dose, 100% hepatocyte transduction, accompanied by lethal acute toxicity, was observed. These results indicate that systemic delivery of HDAd, like FGAd, results in acute toxicity in baboons consistent with activation of the innate inflammatory response, the severity of which is dose dependent, and confirm the hypothesis that Ad-mediated acute toxicity is independent of viral gene expression.

• Mobilized hematopoietic stem cells transduced with IV injected HD-Ad5/35 11 vectors home to the BM persist long term.• Our approach allows for the stable genetic modification of primitive, long-term persisting HSPCs.Current protocols for hematopoietic stem/progenitor cell (HSPC) gene therapy, involving the transplantation of ex vivo genetically modified HSPCs are complex and not without risk for the patient. We developed a new approach for in vivo HSPC transduction that does not require myeloablation and transplantation. It involves subcutaneous injections of granulocyte-colony-stimulating factor/AMD3100 to mobilize HSPCs from the bone marrow (BM) into the peripheral blood stream and the IV injection of an integrating, helper-dependent adenovirus (HD-Ad5/35 11 ) vector system. These vectors target CD46, a receptor that is uniformly expressed on HSPCs. We demonstrated in human CD46 transgenic mice and immunodeficient mice with engrafted human CD34 1 cells that HSPCs transduced in the periphery home back to the BM where they stably express the transgene. In hCD46 transgenic mice, we showed that our in vivo HSPC transduction approach allows for the stable transduction of primitive HSPCs. Twenty weeks after in vivo transduction, green fluorescent protein (GFP) marking in BM HSPCs (Lin 2 Sca1 1 Kit 2 cells) in most of the mice was in the range of 5% to 10%. The percentage of GFP-expressing primitive HSPCs capable of forming multilineage progenitor colonies (colony-forming units [CFUs]) increased from 4% of all CFUs at week 4 to 16% at week 12, indicating transduction and expansion of long-term surviving HSPCs. Our approach was well tolerated, did not result in significant transduction of nonhematopoietic tissues, and was not associated with genotoxicty. The ability to stably genetically modify HSPCs without the need of myeloablative conditioning is relevant for a broader clinical application of gene therapy. (Blood. 2016;128(18):2206-2217

The physics of confined water has stimulated extensive research in recent years, in particular, regarding the role of hydrogen bonding as a significant factor in the observed dynamics. In this work, two-dimensional infrared spectroscopy was employed to investigate the response of the OH moiety of water in phospholipid membrane samples. The results show strong evidence for three distinct hydrogen bonding motifs (H2O with zero, one, or both OH moieties hydrogen bonded), whose relative proportions at the membrane interface are estimated.

Gene transfer of CFTR cDNA to airway epithelia is a promising approach to treat cystic fibrosis (CF). Most gene transfer vectors use strong viral promoters even though the endogenous CFTR promoter is very weak. To learn whether expressing CFTR at a low level in a fraction of cells would correct Cl(-) transport, we mixed freshly isolated wild-type and CF airway epithelial cells in varying proportions and generated differentiated epithelia. Epithelia with approximately 20% wild-type cells generated approximately 70% the transepithelial Cl(-) current of epithelia containing 100% wild-type cells. These data were nearly identical to those previously obtained with CFTR expressed under control of a strong promoter in a CF epithelial cell line. We also tested high level CFTR expression using the very strong cytomegalovirus (CMV) promoter as well as the cytokeratin-18 (K18) promoter. In differentiated airway epithelia, the CMV promoter generated 50-fold more transgene expression than the K18 promoter, but the K18 promoter generated more transepithelial Cl(-) current at high vector doses. Using functional studies, we found that with marked overexpression, some CFTR channels were present in the basolateral membrane where they shunted Cl(-) flow, thereby reducing net transepithelial Cl(-) transport. These results suggest that very little CFTR is required in a fraction of CF epithelial cells to complement Cl(-) transport because transepithelial Cl(-) flow is limited at the basolateral membrane. Thus they suggest a broad leeway in promoter strength for correcting the CF gene transfer, although at very high expression levels CFTR may be mislocalized to the basolateral membrane.

Alpha-1-anti-trypsin deficiency is the most common genetic cause of liver disease in children and liver transplantation is currently the only available treatment. Enhancement of liver autophagy increases degradation of mutant, hepatotoxic alpha-1-anti-trypsin (ATZ). We investigated the therapeutic potential of liver-directed gene transfer of transcription factor EB (TFEB), a master gene that regulates lysosomal function and autophagy, in PiZ transgenic mice, recapitulating the human hepatic disease. Hepatocyte TFEB gene transfer resulted in dramatic reduction of hepatic ATZ, liver apoptosis and fibrosis, which are key features of alpha-1-anti-trypsin deficiency. Correction of the liver phenotype resulted from increased ATZ polymer degradation mediated by enhancement of autophagy flux and reduced ATZ monomer by decreased hepatic NFκB activation and IL-6 that drives ATZ gene expression. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease of alpha-1-anti-trypsin deficiency. This study may pave the way towards applications of TFEB gene transfer for treatment of a wide spectrum of human disorders due to intracellular accumulation of toxic proteins.