A Translational Pathway for Recombinant Adeno-Associated Virus Human Gene Therapy: From Target Identification and Animal Modeling of the Disease to Non-Human Primate and Human Studies

Many steps go into developing a clinical viral gene therapy. The course starts with appropriate disease selection and moves through the many hurdles of in-vitro testing, animal model validation and proof-of-concept studies, all the way through pre-clinical large animal studies. In this thesis, I propose to outline the process of developing a translation pathway for a gene therapy using recombinant adeno-associated virus (rAAV). I will expand on this outline using data that I have generated during the course of my Ph.D. that ranges from animal model validation all the way through pre-clinical vector stability studies. Two disease models will be discussed throughout this thesis, Cockayne Syndrome (CS) and Alpha-1 Antitrypsin Deficiency (AATD). Cockayne Syndrome is a rare autosomal recessive genetic disorder involving mutations in either the CSA or CSB gene, leading to defects in DNA repair. Clinically this presents as progressive degeneration of the central nervous system, retina, cardiovascular system, and cochlea, which leads to mental retardation, post-natal growth defects, ocular abnormalities, and shortened life expectancy. Alpha-1 antitrypsin is a serine protease inhibitor largely produced in the liver that mainly functions to inhibit neutrophil elastase within the lung. AATD leads to an increased risk of emphysema, with shortened life expectancy, and also results in accumulations of mutant AAT polymers in the liver, sometimes leading to liver failure. Using these two disease models I will outline the upstream and downstream pre-clinical work as well as the transition to clinical trials of a rAAV based gene therapy

Rare-gas optics-free stable extreme-ultraviolet photon spectrometer for solar system studies

We have developed a prototype spectrometer for space applications that require long-term stable EUV photon flux measurements. In this recently developed spectrometer, the energy spectrum of the incoming photons is transformed directly into an electron energy spectrum by taking advantage of the photoelectric effect in one of several rare gases at low pressures. Using an electron energy spectrometer operating at a few electron volts, and followed by an electron multiplying detector, pulses due to individual electrons are counted. The overall efficiency of this process is essentially independent of gain drifts in the signal path, and the secular degradation of optical components that is often a problem in other techniques is avoided

Interpretation of Pioneer 10 heliospheric Ly ? glow data obtained beyond 30 AU

International audienceThe Pioneer 10 (P10) Ly ? dataset is the only dataset that is available for the study of the very local interstellar medium (VLISM) in the downstream direction relative to the incoming interstellar neutral hydrogen flow, at very large distances from the sun. Selected P10 data obtained in 1984 and 1986 at distances between 31.81 to 32.25 and 37.29 to 37.74 AU have been used to estimate the local interstellar neutral hydrogen and proton densities. State of the art, stationary neutral-plasma and radiative transfer models have been used in the interpretation of the data. No stationary VLISM heliospheric model was found that best fitted both the P10 1984 data and the 1986 data. The failure to find a single best fit model is most probably due to the fact that the heliospheric model used here did not incorporate time-dependence and interstellar magnetic field effects

CONCERNING THE PROBLEM OF COLLISIONAL HEATING OF THE INTERSTELLAR HELIUM FLOW BY SOLAR WIND PROTONS

Abstract-In the past heating by elastic collisions with the solar wind protons of the interstellar helium flow into the heliosphere has been a subject ofconsideration by several authors. The approaches used in this context are critically analysed in this paper and it is shown that the concept of a continuous momentum transfer is not likely to be applicable. It is argued in contrast that large angle deflections though being rare events give the predominant contributions to the momentum transfer to the neutral helium atoms. This may have consequences for a progressed modelling of the interstellar helium distribution in the heliosphere

Rare-gas optics-free stable extreme-ultraviolet photon spectrometer for solar system studies

We have developed a prototype spectrometer for space applications that require long-term stable EUV photon flux measurements. In this recently developed spectrometer, the energy spectrum of the incoming photons is transformed directly into an electron energy spectrum by taking advantage of the photoelectric effect in one of several rare gases at low pressures. Using an electron energy spectrometer operating at a few electron volts, and followed by an electron multiplying detector, pulses due to individual electrons are counted. The overall efficiency of this process is essentially independent of gain drifts in the signal path, and the secular degradation of optical components that is often a problem in other techniques is avoided

Muscle-Directed Delivery of an AAV1 Vector Leads to Capsid-Specific T Cell Exhaustion in Nonhuman Primates and Humans

With the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) approvals for Zolgensma, Luxturna, and Glybera, recombinant adeno-associated viruses (rAAVs) are considered efficient tools for gene transfer. However, studies in animals and humans demonstrate that intramuscular (IM) AAV delivery can trigger immune responses to AAV capsids and/or transgenes. IM delivery of rAAV1 in humans has also been described to induce tolerance to rAAV characterized by the presence of capsid-specific regulatory T cells (Tregs) in periphery. To understand mechanisms responsible for tolerance and parameters involved, we tested 3 muscle-directed administration routes in rhesus monkeys: IM delivery, venous limb perfusion, and the intra-arterial push and dwell method. These 3 methods were well tolerated and led to transgene expression. Interestingly, gene transfer in muscle led to Tregs and exhausted T cell infiltrates in situ at both day 21 and day 60 post-injection. In human samples, an in-depth analysis of the functionality of these cells demonstrates that capsid-specific exhausted T cells are detected after at least 5 years post-vector delivery and that the exhaustion can be reversed by blocking the checkpoint pathway. Overall, our study shows that persisting transgene expression after gene transfer in muscle is mediated by Tregs and exhausted T cells

Hepatic Changes Associated with Chronic Alcohol Exposure in an Alpha-1 Antitrypsin PiZ Mouse Model

The PiZ mutation in the alpha-1 antitrypsin (AAT) gene causes the PiZ mutant protein to be sequestered in the endoplasmic reticulum of hepatocytes, causing significant liver pathology in ~10% of PiZZ homozygous AAT disease patients. Current transgenic mouse models of the disease include the liver-specific over-expression of mutant PiZ protein. However, these animal models do not efficiently recapitulate the liver damage found in PiZZ homozygous patients. Since only a small percentage of patients develop liver disease and it is not reproducible in animal models of AATD, it suggests that there are other factors that participate in disease pathogenesis. Here, we propose that in the presence of alcohol, liver injury will be initiated and that the intensity of the disease will be exacerbated by the presence of accumulated PiZ mutant protein. To test this hypothesis, we have administered alcohol via the Lieber-DeCarli diet regimen to PiZ transgenic and control C57Bl/6 mice for 12 weeks. We found no difference in alcohol and non-alcohol fed mice in terms of elevations in liver enzymes (AST and ALT). We did find a difference in the degree of steatosis and inflammation in the livers of alcohol fed PiZ mice over those of control alcohol fed mice. These findings are consistent with a chronic low-level hepatic insult seen in chronic alcohol consumption. The difference between PiZ and control mice will allow us to test gene therapies that prevent the accumulation of PiZ aggregates within hepatocytes to determine if they will prevent the exacerbation of alcoholic liver disease

Kismeth: Analyzer of plant methylation states through bisulfite sequencing

<p>Abstract</p> <p>Background</p> <p>There is great interest in probing the temporal and spatial patterns of cytosine methylation states in genomes of a variety of organisms. It is hoped that this will shed light on the biological roles of DNA methylation in the epigenetic control of gene expression. Bisulfite sequencing refers to the treatment of isolated DNA with sodium bisulfite to convert unmethylated cytosine to uracil, with PCR converting the uracil to thymidine followed by sequencing of the resultant DNA to detect DNA methylation. For the study of DNA methylation, plants provide an excellent model system, since they can tolerate major changes in their DNA methylation patterns and have long been studied for the effects of DNA methylation on transposons and epimutations. However, in contrast to the situation in animals, there aren't many tools that analyze bisulfite data in plants, which can exhibit methylation of cytosines in a variety of sequence contexts (CG, CHG, and CHH).</p> <p>Results</p> <p>Kismeth <url>http://katahdin.mssm.edu/kismeth</url> is a web-based tool for bisulfite sequencing analysis. Kismeth was designed to be used with plants, since it considers potential cytosine methylation in any sequence context (CG, CHG, and CHH). It provides a tool for the design of bisulfite primers as well as several tools for the analysis of the bisulfite sequencing results. Kismeth is not limited to data from plants, as it can be used with data from any species.</p> <p>Conclusion</p> <p>Kismeth simplifies bisulfite sequencing analysis. It is the only publicly available tool for the design of bisulfite primers for plants, and one of the few tools for the analysis of methylation patterns in plants. It facilitates analysis at both global and local scales, demonstrated in the examples cited in the text, allowing dissection of the genetic pathways involved in DNA methylation. Kismeth can also be used to study methylation states in different tissues and disease cells compared to a reference sequence.</p

The TWINS exospheric neutral H-density distribution under solar minimum conditions

Terrestrial exospheric atomic hydrogen (H) resonantly scatters solar Lyman-α (121.567 nm) radiation, observed as the glow of the H-geocorona. The Two Wide-angle Imaging Neutral-atom Spectrometers (TWINS) satellites are equiped with two Lyman-α line-of-sight Detectors (LADs) each. Since during the past solar minimum conditions the relevant solar control parameters practically did not vary, we are using LAD data between June and September 2008 to create a time averaged hydrogen geocorona model representative for these solar minimum conditions. In this averaged model we assume that the H-geocorona is longitudinally symmetric with respect to the earth-sun line. We find a 3-dimensional H-density distribution in the range from 3 to 8 earth radii which with some caution can also be extrapolated to larger distances. For lower geocentric distances than 3 earth radii a best fitting r-dependent Chamberlain (1963)-like model is adapted. Main findings are larger than conventionally expected H-densities at heights above 5 <I>R</I>_E and a pronounced day-to-night side H-density asymmetry. The H-geocorona presented here should serve as a reference H-atmosphere for the earth during solar minimum conditions