John R. Weinstein

Directed evolution of novel adeno-associated viruses for therapeutic gene delivery

Gene therapy vectors based on adeno-associated virus (AAV) are currently in clinical trials for numerous disease targets, such as muscular dystrophy, hemophilia, Parkinsons disease, Lebers 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.

CFTR gene transfer with AAV improves early cystic fibrosis pig phenotypes

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.

AAV ANCESTRAL RECONSTRUCTION LIBRARY ENABLES SELECTION OF BROADLY INFECTIOUS VIRAL VARIANTS

Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. However, enhanced vectors are required to extend these landmark successes to other indications 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 use sialic acids, galactose or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19- to 31-fold higher gene expression in the muscle compared with AAV1, a clinically used serotype for muscle delivery, highlighting their promise for gene therapy.

Exposure to polycyclic aromatic hydrocarbons and volatile organic compounds among recently pregnant rural Guatemalan women cooking and heating with solid fuels

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 three times over 72h: morning (fasting), after lunch, and following dinner or use of wood-fired traditional sauna baths (samples=68). Creatinine-adjusted urinary concentrations of metabolites of four PAHs and eight 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 48h PM2.5 105.7μg/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.

Cracking the code: getting men tested in rural Africa.

The emphasis on women in the African AIDS epidemic has left men behind [1]. Although HIV disproportionately affects women in this context [2], they are more likely than men to come into contact with the healthcare system because of child bearing and rearing, and thus more likely to receive early HIV diagnosis and treatment, improving their treatment outcomes. In Malawi, the Option Bþ treatment policy has made dramatic gains for pregnant and breastfeeding women and their infants. Men do not have the same contact with and encouragement from the healthcare system; they get into care later and are disproportionately represented among AIDS deaths [3]. The ambitious UNAIDS 90–90–90 targets will not be met without concerted efforts to engage men [4].

Determining gestational age and preterm birth in rural Guatemala: A comparison of methods

Background Preterm 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. Methods Pregnant 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. Results By 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. Conclusion In 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.

Adoption of Liquefied Petroleum Gas Stoves in Guatemala: A Mixed-Methods Study

Household air pollution is the sixth leading risk factor for premature mortality in Guatemala. Households in Guatemala are gradually adopting liquefied petroleum gas (LPG) stoves, but a strong tradition of woodstove use persists. We conducted a mixed-methods study of LPG stove use in peri-urban Guatemala. We used knowledge, attitudes and practices surveys with 187 LPG stove owners who also used woodstoves to identify perceptions of stove and cooking practices. Barriers to sustained use of LPG stoves were evaluated through focus groups, participant observations with stove users, and key informant interviews with community leaders. Seven themes emerged that explain household decisions to use LPG stoves: (1) The “new technology” should be framed in terms of what the “old technology” lacks, (2) income is not a predictor of gas stove acquisition but may predict sustained use, (3) men are key decision-makers but messages about LPG do not target them, (4) when stoves are viewed as “prize possessions” they may not be used, (5) collective fear about gas stoves is not based on personal experience, but on “stories we hear,” (6) sustained LPG use is hampered by two major factors, seasonally available wood and LPG retailers who are perceived as dishonest, and (7) wood fuel collection is a time to enjoy the company of friends and family and is not “drudgery.” National policies should promote the use of clean cookstove technologies in peri-urban and rapidly urbanizing areas in Guatemala where LPG stoves are in use, but used intermittently, instead of the current plan to install 100,000 “improved” woodstoves by 2032. This could be done by improving dependable cylinder distribution services, targeting gas safety and promoting positive health messages that appeal to men, as well as women.

A directed evolution approach to select for novel Adeno-associated virus capsids on an HIV-1 producer T cell line

A directed evolution approach was used to select for Adeno-associated virus (AAV) capsids that would exhibit more tropism toward an HIV-1 producer T cell line with the long-term goal of developing improved gene transfer vectors. A library of AAV variants was used to infect H9 T cells previously infected or uninfected by HIV-1 followed by AAV amplification with wild-type adenovirus. Six rounds of biological selection were performed, including negative selection and diversification after round three. The H9 T cells were successfully infected with all three wild-type viruses (AAV, adenovirus, and HIV-1). Four AAV cap mutants best representing the small number of variants emerging after six rounds of selection were chosen for further study. These mutant capsids were used to package an AAV vector and subsequently used to infect H9 cells that were previously infected or uninfected by HIV-1. A quantitative polymerase chain reaction assay was performed to measure cell-associated AAV genomes. Two of the four cap mutants showed a significant increase in the amount of cell-associated genomes as compared to wild-type AAV2. This study shows that directed evolution can be performed successfully to select for mutants with improved tropism for a T cell line in the presence of HIV-1.

260. AAV Ancestral Reconstruction Library Enables Selection of Broadly Infectious Viral Variants

Adeno-associated virus (AAV) vectors have demonstrated clinical efficacy in treating several diseases. Improved vectors may extend these landmark successes to other indications, and protein engineering approaches are enhancing the gene delivery properties of AAV vectors to address such unmet medical needs. To access new viral sequences with potentially enhanced infectious properties, and to gain insights into AAVs evolutionary history, we computationally designed and experimentally characterized an ancestral reconstruction of the AAV capsid. To this end, we first generated a phylogenetic tree of AAV sequences through Bayesian Markov chain Monte Carlo simulations, then selected a node for reconstruction based on its relative confidence value and proximity to AAV serotypes of clinical interest. We next used HandAlign, a Markov chain Monte Carlo alignment sampler, to predict the ancestral sequence of the most likely alignment at the selected node. This generated a reconstruction where most amino acids were predicted with high confidence, while 32 of the positions were predicted with low confidence. In order to address uncertainty in these positions, we synthesized the inferred ancestral capsid as a large combinatorial library incorporating degenerate residues at the 32 variable amino acid sites. We then characterized the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to six rounds of selection on a panel of representative cell lines. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes, and in general the selected ancestral libraries were broadly infectious across the cell line panel, indicating that they favor promiscuity over specificity. Unlike many current serotypes, these ancestral AAVs do not utilize sialic acids, galactose, or heparan sulfate proteoglycans for cellular entry. Additionally, ancestral libraries were found to be as susceptible to neutralizing antibodies as extant serotypes, suggesting that such ancestral variant candidates exhibit immunogenic properties similar to modern descendants. Interestingly, ancestral AAVs retained infectivity at temperatures that completely ablated infectivity of modern serotypes; this higher thermostability may have supported increased evolvability through mutational tolerance. Finally, selected variants mediated 19-31 fold higher gene expression in muscle compared to AAV1, a serotype clinically utilized for muscle delivery, highlighting their potential promise for gene therapy.