Gene therapy vectors based on adeno-associated virus (AAV) are currently in clinical trials for numerous disease targets, such as muscular dystrophy, hemophilia, Parkinson's disease, Leber's congenital amaurosis and macular degeneration. Despite its considerable promise and emerging clinical success, several challenges impede the broader implementation of AAV gene therapy, including the prevalence of neutralizing antibodies in the human population, low transduction of a number of therapeutically relevant cell and tissue types, an inability to overcome physical and cellular barriers in vivo and a relatively limited carrying capacity. These challenges arise as the demands we place on AAV vectors are often different from or even at odds with the properties nature bestowed on their parent viruses. Viral-directed evolution-the iterative generation of large, diverse libraries of viral mutants and selection for variants with specific properties of interest-offers an approach to address these problems. Here we outline progress in creating novel classes of AAV variant libraries and highlight the successful isolation of variants with novel and advantageous in vitro and in vivo gene delivery properties.

Posttranslational modification of tyrosine residues in proteins, to produce 3-nitrotyrosine (3-NT), is associated with over 50 disease states including transplant rejection, lung infection, central nervous system and ocular inflammation shock, cancer, and neurological disorders (for example, Alzheimer's disease, Parkinson's disease, and stroke). The levels of 3-NT increase in aging tissue, and levels of 3-NT in proteins are a predictor of disease risk. Here we report the evolution and characterization of an aminoacyl-tRNA synthetase/tRNA pair for the cotranslational, site-specific incorporation of 3-NT into proteins at genetically encoded sites. To demonstrate the utility of our approach for studying the effect on protein function of nitration on sites defined in vivo, we prepared manganese superoxide dismutase (MnSOD) that is homogeneously nitrated at a site known to be modified in disease-related inflammatory responses, and we measured the effect of this defined modification on protein function.

The physiological components that contribute to cystic fibrosis (CF) lung disease are steadily being elucidated. Gene therapy could potentially correct these defects. CFTR-null pigs provide a relevant model to test gene therapy vectors. Using an in vivo selection strategy that amplifies successful capsids by replicating their genomes with helper adenovirus coinfection, we selected an adeno-associated virus (AAV) with tropism for pig airway epithelia. The evolved capsid, termed AAV2H22, is based on AAV2 with 5 point mutations that result in a 240-fold increased infection efficiency. In contrast to AAV2, AAV2H22 binds specifically to pig airway epithelia and is less reliant on heparan sulfate for transduction. We administer AAV2H22-CFTR expressing the CF transmembrane conductance regulator (CFTR) cDNA to the airways of CF pigs. The transduced airways expressed CFTR on ciliated and nonciliated cells, induced anion transport, and improved the airway surface liquid pH and bacterial killing. Most gene therapy studies to date focus solely on Cl− transport as the primary metric of phenotypic correction. Here, we describe a gene therapy experiment where we not only correct defective anion transport, but also restore bacterial killing in CFTR-null pig airways.

Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. Enhanced vectors are required to extend these landmark successes to other indications, however, and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties, and to gain insights into AAV’s evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes, and in general ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not utilize sialic acids, galactose, or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19–31 fold higher gene expression in muscle compared to AAV1, a clinically utilized serotype for muscle delivery, highlighting their promise for gene therapy.

BackgroundThree billion people use solid cooking fuels, and 4 million people die from household air pollution annually. Shifting households to clean fuels, like liquefied petroleum gas (LPG), may protect health only if stoves are consistently used. Few studies have used an implementation science framework to systematically assess “de-implementation” of traditional stoves, and none have done so with pregnant women who are more likely to adopt new behaviors. We evaluated an introduced LPG stove coupled with a phased behavioral intervention to encourage exclusive gas stove use among pregnant women in rural Guatemala.MethodsWe enrolled 50 women at < 20 weeks gestation in this prospective cohort study. All women received a free 3-burner LPG stove and ten tank refills. We conducted formative research using COM-B Model and Theoretical Domains Framework (TDF). This included thematic analysis of focus group findings and classes delivered to 25 pregnant women (Phase 1). In Phase 2, we complemented classes with a home-based tailored behavioral intervention with a different group of 25 pregnant women. We mapped 35 TDF constructs onto survey questions. To evaluate stove use, we placed temperature sensors on wood and gas stoves and estimated fraction of stove use three times during pregnancy and twice during the first month after infant birth.ResultsClass attendance rates were above 92%. We discussed feasible ways to reduce HAP exposure, proper stove use, maintenance and safety. We addressed food preferences, ease of cooking and time savings through cooking demonstrations. In Phase 2, the COM-B framework revealed that other household members needed to be involved if the gas stove was to be consistently used. Social identity and empowerment were key in decisions about stove repairs and LPG tank refills. The seven intervention functions included training, education, persuasion, incentivization, modelling, enablement and environmental restructuring. Wood stove use dropped upon introduction of the gas stove from 6.4 h to 1.9 h.ConclusionsThis is the first study using the COM-B Model to develop a behavioral intervention that promotes household-level sustained use of LPG stoves. This study lays the groundwork for a future LPG stove intervention trial coupled with a behavioral change intervention.Trial registrationNCT02812914, registered 3 June 2016, retrospectively registered.

Background Household air pollution is a major contributor to death and disability worldwide. Over 95% of rural Guatemalan households use woodstoves for cooking or heating. Woodsmoke contains carcinogenic or fetotoxic polycyclic aromatic hydrocarbons (PAHs) and volatile organic compounds (VOCs). Increased PAHs and VOCs have been shown to increase levels of oxidative stress. Objective We examined PAH and VOC exposures among recently pregnant rural Guatemalan women exposed to woodsmoke and compared exposures to levels seen occupationally or among smokers. Methods Urine was collected from 23 women who were 3 months post-partum 3 times over 72-hours: morning (fasting), after lunch, and following dinner or use of wood-fired traditional sauna baths (samples=68). Creatinine-adjusted urinary concentrations of metabolites of 4 PAHs and 8 VOCs were analyzed by liquid chromatography—mass spectrometry. Creatinine-adjusted urinary biomarkers of oxidative stress, 8-isoprostane and 8-OHdG, were analyzed using enzyme-linked immunosorbent assays (ELISA). Long-term (pregnancy through 3 months prenatal) exposure to particulate matter and airborne PAHs were measured. Results Women using wood-fueled chimney stoves are exposed to high levels of particulate matter (median 48-hour PM2.5 105.7 μg/m3; inter-quartile range (IQR): 77.6–130.4). Urinary PAH and VOC metabolites were significantly associated with woodsmoke exposures: 2-naphthol (median (IQR) in ng/mg creatinine: 295.9 (74.4–430.9) after sauna versus 23.9 (17.1–49.5) fasting; and acrolein: 571.7 (429.3–1040.7) after sauna versus 268.0 (178.3–398.6) fasting. Urinary PAH (total PAH: ρ = 0.89, p < 0.001) and VOC metabolites of benzene (ρ=0.80, p < 0.001) and acrylonitrile (ρ=0.59, p < 0.05) were strongly correlated with long-term exposure to particulate matter. However urinary biomarkers of oxidative stress were not correlated with particulate matter (ρ = 0.01 to 0.05, p > 0.85) or PAH and VOC biomarkers (ρ =−0.20 to 0.38, p > 0.07). Urinary metabolite concentrations were significantly greater than those of heavy smokers (mean cigarettes/day = 18) across all PAHs. In 15 (65%) women, maximum 1-hydroxypyrene concentrations exceeded the occupational exposure limit of coke-oven workers. Conclusions The high concentrations of urinary PAH and VOC metabolites among recently pregnant women is alarming given the detrimental fetal and neonatal effects of prenatal PAH exposure. As most women used chimney woodstoves, cleaner fuels are critically needed to reduce smoke exposure.

BackgroundPreterm birth is the leading cause of death among children <5 years of age. Accurate determination of prematurity is necessary to provide appropriate neonatal care and guide preventive measures. To estimate the most accurate method to identify infants at risk for adverse outcomes, we assessed the validity of two widely available methods—last menstrual period (LMP) and the New Ballard (NB) neonatal assessment—against ultrasound in determining gestational age and preterm birth in highland Guatemala.MethodsPregnant women (n = 188) were recruited with a gestational age <20 weeks and followed until delivery. Ultrasound was performed by trained physicians and LMP was collected during recruitment. NB was performed on infants within 96 hours of birth by trained study nurses. LMP and NB accuracy at determining gestational age and identifying prematurity was assessed by comparing them to ultrasound.ResultsBy ultrasound, infant mean gestational age at birth was 38.3 weeks (SD = 1.6) with 16% born at less than 37 gestation. LMP was more accurate than NB (mean difference of +0.13 weeks for LMP and +0.61 weeks for NB). However, LMP and NB estimates had low agreement with ultrasound-determined gestational age (Lin’s concordance<0.48 for both methods) and preterm birth (κ<0.29 for both methods). By LMP, 18% were judged premature compared with 6% by NB. LMP underestimated gestational age among women presenting later to prenatal care (0.18 weeks for each additional week). Gestational age for preterm infants was overestimated by nearly one week using LMP and nearly two weeks using NB. New Ballard neuromuscular measurements were more predictive of preterm birth than those measuring physical criteria.ConclusionIn an indigenous population in highland Guatemala, LMP overestimated prematurity by 2% and NB underestimated prematurity by 10% compared with ultrasound estimates. New, simple and accurate methods are needed to identify preterm birth in resource-limited settings worldwide.

The use of surface‐based methods for the delivery of therapeutics has recently generated increasing interest. These platforms have tremendous potential to minimize detrimental side effects associated with systemic delivery by localizing the therapeutic vehicle, and thus provide higher local doses for improved efficacy. Cationic lipids are one of the most commonly used synthetic carriers for the delivery of genetic cargo, such as DNA and RNA. However, reports on the use of lipid‐based films for gene delivery are scarce. Here we investigate the use of a lipid‐based film for the in vitro delivery of plasmid DNA. Solid DNA‐lipid films show very low levels of transfection, while identical complexes prepared for bolus delivery provide high levels of transfection when used directly. We investigate the mechanism, whereby the activity of these solid‐state films is lost and suggest methods for circumventing these challenges and restoring the efficacy of these films as gene delivery platforms. © 2013 American Institute of Chemical Engineers AIChE J, 59: 3203–3213, 2013