Disruption of Microtubules Post-Virus Entry Enhances Adeno-Associated Virus Vector Transduction

Perinuclear retention of viral particles is a poorly understood phenomenon observed during many virus infections. In this study, we investigated whether perinuclear accumulation acts as a barrier to limit recombinant adeno-associated virus (rAAV) transduction. After nocodazole treatment to disrupt microtubules at microtubule-organization center (MT-MTOC) after virus entry, we observed higher rAAV transduction. To elucidate the role of MT-MTOC in rAAV infection and study its underlying mechanisms, we demonstrated that rAAV's perinuclear localization was retained by MT-MTOC with fluorescent analysis, and enhanced rAAV transduction from MT-MTOC disruption was dependent on the rAAV capsid's nuclear import signals. Interestingly, after knocking down RhoA or inhibiting its downstream effectors (ROCK and Actin), MT-MTOC disruption failed to increase rAAV transduction or nuclear entry. These data suggest that enhancement of rAAV transduction is the result of increased trafficking to the nucleus via the RhoA-ROCK-Actin pathway. Ten-fold higher rAAV transduction was also observed by disrupting MT-MTOC in brain, liver, and tumor in vivo. In summary, this study indicates that virus perinuclear accumulation at MT-MTOC is a barrier-limiting parameter for effective rAAV transduction and defines a novel defense mechanism by which host cells restrain viral invasion

Quantitative 3D Tracing of Gene-delivery Viral Vectors in Human Cells and Animal Tissues

Trafficking through a variety of cellular structures and organelles is essential for the interaction between gene-delivery vectors (i.e., adeno-associated virus (AAV) and liposomes) and host cells/tissues. Here, we present a method of computer-assisted quantitative 3D biodistribution microscopy that samples the whole population of fluorescently-labeled vectors and document their trafficking routes. Using AAV as a working model, we first experimentally defined numerical parameters for the singularity of Cy5-labeled particles by combining confocal microscopy and atomic force microscopy (AFM). We then developed a robust approach that integrates single-particle fluorescence imaging with 3D deconvolution and isosurface rendering to quantitate viral distribution and trafficking in human cells as well as animal tissues at the single-particle level. Using this quantitative method, we uncovered an as yet uncharacterized rate-limiting step during viral cell entry, while delineating nuclear accumulation of virions during the first 8 hours postinfection. Further, our studies revealed for the first time that following intramuscular injection, AAV spread progressively across muscle tissues through endomysium between myofibers instead of traversing through target cells. Such 3D resolution and quantitative dissection of vector–host interactions at the subcellular level should significantly improve our ability to resolve trafficking mechanisms of gene-delivery particles and facilitate the development of enhanced viral vectors

K137R Mutation on Adeno-Associated Viral Capsids Had Minimal Effect on Enhancing Gene Delivery In Vivo

The adeno-associated viral (AAV) vector has emerged as an attractive vector for gene therapy applications. Development of AAV vectors with enhanced gene transduction efficiency is important to ease the burden of AAV production and minimize potential immune responses. Rational mutations on AAV capsids have gained attention as a simple method of enhancing AAV transduction efficiency. A single-amino acid mutation, K137R, on AAV1 and AAV8 was recently reported to increase liver transgene expression by 5–10-fold. To determine whether the same mutation on other AAV serotypes would result in similar gene enhancement effects, K137R mutants were generated on AAV7, AAV8, and AAV9, and their effects were evaluated in vivo. Two reporter genes were utilized: the nuclear LacZ gene driven by the cytomegalovirus promoter and the luciferase gene driven by the CB promoter. Surprisingly, we found no difference in luciferase gene expression in the liver or other tissues using either the wild-type AAV8 capsid or AAV8-K137R. LacZ gene expression in the liver by AAV8-K137R was about onefold higher than that of wild-type AAV8. However, no difference was found in other tissues, such as skeletal muscle and cardiac muscle. In addition, no difference was found in transgene expression with either AAV7-K137R or AAV9-K137R mutants. Our results indicated that the K137R mutation on AAV7, AAV8, and AAV9 had minimal to no effect on transduction efficiency in vivo

Phase 1 Gene Therapy for Duchenne Muscular Dystrophy Using a Translational Optimized AAV Vector

Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer

Production prediction for fracture-vug carbonate reservoirs using electric imaging logging data

Considering the fluid flow non-darcy characteristics in fracture-vug carbonate reservoirs, a new multi-scale conduit flow model production prediction method for fracture-vug carbonate reservoirs was presented using image segmentation technique of electric imaging logging data. Firstly, based on Hagen-Poiseuille's law of incompressible fluid flow and the different cross-sectional areas in single fractures and vugs in carbonate reservoirs, a multi-scale conduit flow model for fracture-vug carbonate reservoir was established, and a multi-scale conduit radial fluid flow equation was deduced. Then, conduit flow production index was introduced. The conduit flow production index was calculated using fracture-vug area extracted from the result of electrical image segmentation. Finally, production prediction of fracture-vug carbonate reservoir was realized by using electric imaging logging data. The method has been applied to Ordovician fracture-vug carbonate reservoirs in the Tabei area, and the predicted results are in good agreement with the oil testing data. Key words: Tarim Basin, Ordovician, carbonate, fracture-vug carbonate reservoir, electric imaging logging, conduit flow model, production index, production predictio

Sex differences in the relationship between post-vaccination adverse reactions, decision regret, and WTP for the booster dose of COVID-19 vaccine in Taizhou, China

This study investigated sex differences in the relationship between post-vaccination adverse reactions, decision regret, and willingness to pay (WTP) for the booster dose of COVID-19 vaccines. This research carried out an online cross-sectional investigation among healthcare workers (HCWs) in Taizhou, China. In total, 1,054 respondents (165 males and 889 females) have received two-dose COVID-19 vaccination. We performed descriptive analysis, chi-square test, and mediation analysis on the exported data. In this study, 67 (40.6%) males and 429 (48.3%) females had WTP for the booster dose. Our study presented that decision regret mediated the effect of adverse reactions after vaccination on WTP for the booster dose in both male and female groups. In males, decision regret played a completely mediating role, while in females, it acted as a partial mediator. Sex differences in the relationship between post-vaccination side effects, decision regret, and WTP for the third dose were demonstrated in a sample of healthcare workers

COVID-19 Vaccination in China: Adverse Effects and Its Impact on Health Care Working Decisions on Booster Dose

Although many research studies have concentrated on people’s willingness to take the COVID-19 vaccine, little attention has been paid to the underlying mechanism of consent. An understanding of potential factors and mechanisms that affect the willingness to receive a vaccination can contribute information critical for containing the pandemic. This study explored the effects of post-vaccination adverse reactions on the willingness to take the booster dose and the role of decision regret. A self-administered online survey was carried out in Taizhou, China. Questionnaires were completed by 1085 healthcare workers (HCWs), 1054 (97.1%) of whom had completed two doses of the COVID-19 vaccine. Mediation analysis methodology was applied in this study. Our study showed that post-vaccination adverse reactions in HCWs could decrease their willingness to take the booster dose. Of note, HCWs who experienced adverse reactions after vaccination would be more likely to regret their previous vaccination decisions, which, in turn, further reduced their willingness to receive a booster shot. Decision regret mediated the relationship between adverse post-vaccination reactions and a willingness to take the booster dose. The findings implied inextricable relationships among post-vaccination adverse reactions, decision regret, and willingness to take the booster dose. It is suggested that notice of these post-vaccination adverse reactions should be further incorporated into vaccine communication campaigns and policy interventions advocating booster doses to improve vaccine uptake intent and increase the willingness to receive booster doses of a COVID-19 vaccine