Identification of host essential factors for recombinant AAV transduction of the polarized human airway epithelium.

IMPORTANCE: The essential steps of successful gene delivery by recombinant adeno-associated viruses (rAAVs) include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene WDR63, whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.

Rickettsia africae infections in sub-Saharan Africa: A systematic literature review of epidemiological studies and summary of case reports.

Rickettsia africae is a tick-borne bacteria known to cause African tick bite fever (ATBF). While the disease was first described more than 100 years ago, knowledge of transmission risk factors and disease burden remain poorly described. To better understand the burden of R. africae, this article reviewed and summarized the published literature related to ATBF epidemiology and clinical management. Using a systematic approach, consistent with the PRISMA guidelines, we identified more than 100 eligible articles, including 65 epidemiological studies and 41 case reports. Most reports described R. africae in ticks and livestock, while human studies were less common. Human disease case reports were exclusively among returning travellers from non-endemic areas, which limits our disease knowledge among at-risk populations: people living in endemic regions. Substantial efforts to elucidate the ATBF risk factors and clinical manifestations among local populations are needed to develop effective preventative strategies and facilitate appropriate and timely diagnosis.

Configurable Organic Charge Carriers toward Stable Perovskite Photovoltaics.

Due to their solution processability and unique photoelectric characteristics, perovskite solar cells (PSCs) have shown considerable promise in the area of renewable energy. Although their power conversion efficiency (PCE) has risen from 3.8% to 25.7% in only a few years, their short lifetime and high material prices continue to be key roadblocks to commercial viability. Charge transporting materials (CTMs), such as hole/electron transporting materials, are critical components in PSCs because they not only govern hole or electron extraction and transporting from the perovskite layer to the electrodes but also protect the perovskite from direct contact with the ambient environment. CTMs are split into two types: inorganic CTMs (ICTMs) and organic CTMs (OCTMs). Because of their inexpensive prices, well-adjusted energy levels, and low temperature solution-processed features, OCTMs have been more frequently explored and employed than ICTMs. Various forms of OCTMs with more straightforward synthetic pathways and better performance have been thoroughly researched. Recent achievements in the development of OCTMs will be discussed and evaluated on a molecular level in this study, which will include a systematic categorization of OCTMs based on molecular functionalization techniques. In order to achieve highly efficient and stable PSCs, we will present insights on the structure-property relationship in the design of OCTMs as well as device stability. We hope that this analysis will serve as a comprehensive reference to molecular design guidelines for various types of OCTMs, spurring greater research toward designing highly efficient and OCTMs for stable PSCs.

Suppressing ion migration in metal halide perovskite via interstitial doping with a trace amount of multivalent cations.

Cations with suitable sizes to occupy an interstitial site of perovskite crystals have been widely used to inhibit ion migration and promote the performance and stability of perovskite optoelectronics. However, such interstitial doping inevitably leads to lattice microstrain that impairs the long-range ordering and stability of the crystals, causing a sacrificial trade-off. Here, we unravel the evident influence of the valence states of the interstitial cations on their efficacy to suppress the ion migration. Incorporation of a trivalent neodymium cation (Nd3+) effectively mitigates the ion migration in the perovskite lattice with a reduced dosage (0.08%) compared to a widely used monovalent cation dopant (Na+, 0.45%). The photovoltaic performances and operational stability of the prototypical perovskite solar cells are enhanced with a trace amount of Nd3+ doping while minimizing the sacrificial trade-off.

Single-nucleus RNA sequencing of mouse auditory cortex reveals critical period triggers and brakes.

Auditory experience drives neural circuit refinement during windows of heightened brain plasticity, but little is known about the genetic regulation of this developmental process. The primary auditory cortex (A1) of mice exhibits a critical period for thalamocortical connectivity between postnatal days P12 and P15, during which tone exposure alters the tonotopic topography of A1. We hypothesized that a coordinated, multicellular transcriptional program governs this window for patterning of the auditory cortex. To generate a robust multicellular map of gene expression, we performed droplet-based, single-nucleus RNA sequencing (snRNA-seq) of A1 across three developmental time points (P10, P15, and P20) spanning the tonotopic critical period. We also tone-reared mice (7 kHz pips) during the 3-d critical period and collected A1 at P15 and P20. We identified and profiled both neuronal (glutamatergic and GABAergic) and nonneuronal (oligodendrocytes, microglia, astrocytes, and endothelial) cell types. By comparing normal- and tone-reared mice, we found hundreds of genes across cell types showing altered expression as a result of sensory manipulation during the critical period. Functional voltage-sensitive dye imaging confirmed GABA circuit function determines critical period onset, while Nogo receptor signaling is required for its closure. We further uncovered previously unknown effects of developmental tone exposure on trajectories of gene expression in interneurons, as well as candidate genes that might execute tonotopic plasticity. Our single-nucleus transcriptomic resource of developing auditory cortex is thus a powerful discovery platform with which to identify mediators of tonotopic plasticity.

Clinical characteristics of Plasmodium falciparum infection among symptomatic patients presenting to a major urban military hospital in Cameroon.

BACKGROUND: Urban malaria has received insufficient attention in the literature. The prevalence and clinical characteristics of Plasmodium falciparum infection amongst patients presenting with suspected malaria were investigated at a major urban hospital in Douala, Cameroon with a particular focus on anaemia. METHODS: A cross-sectional, 18-week demographic and clinical survey was conducted of patients presenting to the Emergency Department of Douala Military Hospital with suspected malaria, largely defined by the presence or recent history of fever. Venous samples were tested for P. falciparum using rapid diagnostic tests and PCR, and anaemia was defined by haemoglobin level according to WHO definitions. Likelihood ratios (LR), odds ratios (OR), and population attributable risk percent (PARP) were calculated. RESULTS: Participants were ages 8 months to 86 years, 51% were women (257/503), and all districts of Douala were represented. Overall, 38.0% (n = 189/497) were anaemic, including 5.2% (n = 26/497) with severe anaemia. Anaemia prevalence was significantly higher (OR: 2.20, 95% CI 1.41-3.45) among children < 15 years (53.1%, n = 52/98) compared to adults (34%, n = 133/392). Plasmodium falciparum was detected in 37.2% by nested PCR. Among all participants, several factors were associated with clinically significant LR for P. falciparum infection, including age 10-14 years (positive LR: 3.73), living in the island district of Douala VI (positive LR: 3.41), travel to any of three northern regions (positive LR: 5.11), and high fever > 40 °C at presentation (positive LR: 4.83). Among all participants, 8.7% of anaemia was associated with P. falciparum infection, while the PARP was 33.2% among those < 15 years of age and 81.0% among 10-14-year-olds. CONCLUSIONS: The prevalence of P. falciparum infection in the urban hospital was high. Mirroring trends in many rural African settings, older children had the highest positivity rate for P. falciparum infection. Anaemia was also common in all age groups, and for those 10-14 years of age, 80% of the risk for anaemia was associated with P. falciparum infection. Malaria rates in major urban population centres can be high, and more research into the multifactorial causes of anaemia across the age spectrum are needed.

Professionalism: COVID-19 made me do it!

PURPOSE OF REVIEW: COVID-19 pandemic has created profound ethical challenges, not only for clinical decision-making but also for defining physician professional conduct. RECENT FINDINGS: Multiple ethical questions arose as the COVID-19 pandemic ravaged globally, including physician obligations in a pandemic, allotment of personal protective equipment, care of unvaccinated patients, discern between evidence-based and unreliable information, addressing end-of-life wishes, implications of involving medical students in a public health crisis, and finally physician burnout aggravated by a pandemic. SUMMARY: There is a need to redefine existing medical professionalism standards so that future healthcare professionals are well prepared to deal with similar public health crisis.

The Effect of Activated Protein C on Attenuation of Ischemia-Reperfusion Injury in a Rat Muscle Flap Model.

Ischemia-reperfusion injury is often the final and irreversible factor causing flap failure in microsurgery. The salvage of a microsurgical flap with an ischemia-reperfusion injury contributes to the success of microsurgical flap transfers. Activated protein C (APC), a serine protease with anticoagulant and anti-inflammatory activities, has been shown to improve ischemic flap survival. To date, APC has yet to be applied to models of free flap with ischemia-reperfusion injury. In this study, we aimed to investigate the effect of APC on gracilis flap ischemia-reperfusion injury induced by gracilis vessels clamping and reopening. Sixty male Sprague-Dawley rats were randomly divided into 2 groups. After 4 hours of clamping for ischemia, flaps were reperfused and recombinant human APC (25 µg/kg) or saline was injected in the flaps through pedicles. At 0, 1, 4, 18, and 24 hours after injection (n = 6 for each time point), the tissue samples were harvested. The muscle viability at 24 hours in saline group was 54.8% (15.1%), whereas the APC-treated group was 90.0% (4.3%) (P < 0.05). The induced nitric oxide synthase (iNOS) mRNA expression increased with the time after reperfusion, which were 0.93 (0.25) to 2.09 (0.22) in saline group, and 0.197 (0.15) to 0.711 (0.15) in the APC-treated group. iNOS mRNA expression in the APC-treated group was significantly higher than the saline group at 1, 18, and 24 hours (P < 0.05). Numerous inflammatory cells were observed infiltrating and invading the muscle fibers in the saline group more than the APC-treated group. Increased number of polymorphonuclear cells was also noted in the saline group compared with the APC-treated group (P < 0.05). In conclusion, APC treatment can significantly attenuate ischemia-reperfusion injury and increase the survival of the free flap through down-regulating iNOS mRNA expression and reducing the inflammatory cells. Further research is still needed to be done on various mechanisms in which APC is protective to prevent tissue damage.

Pneumocystis Pneumonia in Previously Undiagnosed Advanced HIV/AIDS and Importance of HIV Screening.

The diagnosis of human immunodeficiency virus (HIV) in its late stages, also known as acquired immunodeficiency syndrome (AIDS), leads to increased morbidity and mortality. This is mostly due to the time given for opportunistic infections to arise, which present with their own complications. In this case report, we present an otherwise healthy 38-year-old male, who presented with general systemic symptoms and was later found to have HIV and AIDS, ultimately resulting in his death during this hospital admission. This case report will discuss HIV-associated opportunistic infections, focusing more on Pneumocystis jirovecii pneumonia (PCP), and the following complications that arose during the course of this patient's hospitalization. The overarching goal of this case report is to highlight the importance of routine HIV screening in the United States in efforts to decrease mortality from AIDS-related opportunistic infections. In addition to mandating in-hospital screening, educating the public on lifestyle behaviors that can put one at risk for developing HIV is crucial to decreasing the prevalence of cases that go undiagnosed, as well as the disease itself.

Influence of an extreme event-the COVID-19 pandemic-On establishment of and data collection by a citizen science project.

The recent rising incidence of extreme natural events may significantly influence the implementation of citizen science projects, including the success of outreach strategies and the quality and scope of data collection. The MassMammals Watch and subsidiary MassBears citizen science projects, initiated during the height of the pandemic, recruit volunteers to submit sightings of black bears and other mammals. In this study, we evaluated the methods we employed for engaging and retaining community volunteers during a period of intense social restrictions, and we assessed whether such conditions were associated with spatial biases in our collected data. Newspaper features were more likely to recruit volunteers who engaged with the project multiple times, but social media and internet presence were important for reaching a larger audience. Bear sighting submissions peaked in number and were more likely to be in forested areas during 2020, the height of the pandemic, compared to later years, a pattern which we suggest stems from an increased desire to participate in outdoor activities in light of social distancing measures during that year. Such shifts in patterns of data collection are likely to continue, particularly in response to increasing extreme weather events associated with climate change. Here, we both make recommendations on optimal outreach strategies for others initiating citizen science programs and illustrate the importance of assessing potential biases in data collection imposed by extreme circumstances.

The risk and benefit profiles of US eligible lung cancer screening attendees vs. non-attendees.

BACKGROUND: The United States Preventive Services Task Force (USPSTF) recommend lung-cancer screening for individuals aged 50-80 with ≥20 pack-years and ≤15 quit-years, but uptake is low. The risk and benefit profiles of screening attendees are unknown; consequently, the impact and lost opportunity of ongoing lung-cancer screening in the US remains unclear. METHODS: We estimated lung-cancer death risk (using the Lung Cancer Death Risk Assessment Tool) and life gained from screening (using the LYFS-CT model) for individuals 50-79 who ever-smoked in the US-representative 2022 Behavioral Risk Factor Surveillance System. We compared lung-cancer death risk and life-gained among USPSTF-eligible individuals by screening status (self-reported screened vs not screened in past year), and estimated the number of lung-cancer deaths averted and life-years gained under current screening levels and if everyone eligible was screened. RESULTS: USPSTF-eligibility was 33.7% (95%CI:33.1-34.4%), of whom 17.9% (95%CI : 17.0-18.8%) self-reported screening. Screening uptake increased with increasing lung-cancer death risk quintile (Q1 = 5.2% (95%CI : 3.0%-8.8%); Q5 = 21.8% (95%CI : 20.3%-23.3%)) and life-gain from screening quintile (Q1 = 6.2% (95%CI : 3.8%-9.9%); Q5 = 20.8% (95%CI : 19.5%-22.2%)). Screened individuals had higher lung-cancer death risk (Risk Ratio [RR]=1.35, 95%CI : 1.26-1.46) and life-years gained (RR = 1.19, 95%CI : 1.12-1.25) than unscreened individuals. Currently screening averts 19,306 lung-cancer deaths and gains 237,564 life-years; screening everyone eligible would additionally avert 56,956 lung-cancer deaths and gain 751,850 life-years. Two-thirds of USPSTF-lung-eligible women were up-to-date with breast-cancer screening, but only 17.3% attended lung screening in the past year. CONCLUSIONS: Eligible screening attendees had higher lung-cancer death risk and benefit from screening. Higher rates of screening could substantially increase the number of lung-cancer deaths prevented.

Survival by sex and HIV status in patients with anal cancer in the USA between 2001 and 2019: a retrospective cohort study.

BACKGROUND: The risk of anal cancer is increased among people with HIV, particularly men who have sex with men. Estimating survival by HIV status and sex and identifying groups at high risk is crucial for documenting prognostic differences between populations. We aimed to compare all-cause and anal cancer-specific survival in patients with anal cancer with and without HIV, stratified by sex, and to identify predictors of survival, stratified by HIV status. METHODS: In this retrospective cohort study, we used data from the HIV/AIDS Cancer Match Study of 13 population-based HIV and cancer registries throughout the USA. We included individuals aged 20-79 years diagnosed with invasive anal cancer between 2001 and 2019. To estimate associations between HIV status and both all-cause and anal cancer-specific mortality overall, we used Cox proportional hazards models, adjusting for year of and age at diagnosis, sex, race and ethnicity, histology, cancer stage, region, and treatment. We also calculated sex-specific adjusted hazard ratios (HRs). By HIV status, we identified characteristics associated with mortality. Models among people with HIV were further adjusted for AIDS status and HIV transmission risk group. FINDINGS: Between Jan 1, 2001, and Dec 31, 2019, 1161 (43·6%) of 2662 patients with anal cancer and HIV and 7722 (35·4%) of 21 824 patients without HIV died. HIV was associated with a 1·35 times (95% CI 1·24-1·47) increase in all-cause mortality among male patients and a 2·47 times (2·10-2·90) increase among female patients. Among patients with HIV, all-cause mortality was increased among non-Hispanic Black individuals (adjusted HR 1·19, 95% CI 1·04-1·38), people with AIDS (1·36, 1·10-1·68), people who inject drugs (PWID; 1·49, 1·17-1·90), patients with adenocarcinoma (2·74, 1·82-4·13), and those with no or unknown surgery treatment (1·34, 1·18-1·53). HIV was associated with anal cancer-specific mortality among female patients only (1·52, 1·18-1·97). Among patients with HIV, anal cancer-specific mortality was increased among patients with adenocarcinoma (3·29, 1·89-5·72), those with no or unknown treatment (1·59, 1·17-2·17), and PWID (1·60, 1·05-2·44). INTERPRETATION: HIV was associated with all-cause mortality among patients with anal cancer, especially women. Anal cancer-specific mortality was elevated among female patients with HIV. As screening for anal cancer becomes more widespread, examining the effects of screening on survival by HIV status and sex is crucial. FUNDING: US National Cancer Institute Intramural Research Program.

Dense, Continuous Membrane Labeling and Expansion Microscopy Visualization of Ultrastructure in Tissues.

Lipid membranes are key to the nanoscale compartmentalization of biological systems, but fluorescent visualization of them in intact tissues, with nanoscale precision, is challenging to do with high labeling density. Here, we report ultrastructural membrane expansion microscopy (umExM), which combines a novel membrane label and optimized expansion microscopy protocol, to support dense labeling of membranes in tissues for nanoscale visualization. We validated the high signal-to-background ratio, and uniformity and continuity, of umExM membrane labeling in brain slices, which supported the imaging of membranes and proteins at a resolution of ~60 nm on a confocal microscope. We demonstrated the utility of umExM for the segmentation and tracing of neuronal processes, such as axons, in mouse brain tissue. Combining umExM with optical fluctuation imaging, or iterating the expansion process, yielded ~35 nm resolution imaging, pointing towards the potential for electron microscopy resolution visualization of brain membranes on ordinary light microscopes.

ASPYRE-Lung: validation of a simple, fast, robust and novel method for multi-variant genomic analysis of actionable NSCLC variants in FFPE tissue.

Introduction: Genomic variant testing of tumors is a critical gateway for patients to access the full potential of personalized oncology therapeutics. Current methods such as next-generation sequencing are costly and challenging to interpret, while PCR assays are limited in the number of variants they can cover. We developed ASPYRE® (Allele-Specific PYrophosphorolysis REaction) technology to address the urgent need for rapid, accessible and affordable diagnostics informing actionable genomic target variants of a given cancer. The targeted ASPYRE-Lung panel for non-small cell carcinoma covers 114 variants in 11 genes (ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET & NTRK1/2/3) to robustly inform clinical management. The assay detects single nucleotide variants, insertions, deletions, and gene fusions from tissue-derived DNA and RNA simultaneously. Methods: We tested the limit of detection, specificity, analytical accuracy and analytical precision of ASPYRE-Lung using FFPE lung tissue samples from patients with non-small cell lung carcinoma, variant-negative FFPE tissue from healthy donors, and FFPE-based contrived samples with controllable variant allele fractions. Results: The sensitivity of ASPYRE-Lung was determined to be ≤ 3% variant allele fraction for single nucleotide variants and insertions or deletions, 100 copies for fusions, and 200 copies for MET exon 14 skipping. The specificity was 100% with no false positive results. The analytical accuracy test yielded no discordant calls between ASPYRE-Lung and expected results for clinical samples (via orthogonal testing) or contrived samples, and results were replicable across operators, reagent lots, runs, and real-time PCR instruments with a high degree of precision. Conclusions: The technology is simple and fast, requiring only four reagent transfer steps using standard laboratory equipment (PCR and qPCR instruments) with analysis via a cloud-based algorithm. The ASPYRE-Lung assay has the potential to be transformative in facilitating access to rapid, actionable molecular profiling of tissue for patients with non-small cell carcinoma.

Gads regulates the expansion phase of CD8+ T cell-mediated immunity.

The Gads adaptor protein is critical for TCR-mediated Ca(2+) mobilization. We investigated the effect of Gads deficiency on the proliferation of CD8(+) T cells following peptide stimulation and in the context of infection with an intracellular pathogen. We stimulated CD8(+) T cells from Gads(+/+) OT-I and Gads(-/-) OT-I mice with cognate Ag (SIINFEKL) or altered peptide ligand. In vitro experiments revealed that Gads was required for optimal proliferation of CD8(+) T cells. This defect was most evident at the early time points of proliferation and when low doses of Ag were used as stimuli. Cell cycle analysis demonstrated that Gads(-/-) CD8(+) T cells had impaired TCR-mediated exit from the G(0) phase of the cell cycle. Furthermore, Gads(-/-) CD8(+) T cells had delayed expression of c-myc and CD69 upon the stimulation with SIINFEKL. We then investigated how Gads deficiency would impact CD8(+) T cell-mediated immunity in the context of infection with an intracellular pathogen. At early time points, Gads(+/+) and Gads(-/-) CD8(+) T cells proliferated to a similar extent, despite the fact that expression of CD69 and CD25 was reduced in the absence of Gads. After 5 d postinfection, Gads was required to sustain the expansion phase of the immune response; the peak response of Gads(-/-) cells was significantly lower than for Gads(+/+) cells. However, Gads was not required for the differentiation of naive CD8(+) T cells into memory cells. We conclude that the primary function of Gads is to regulate the sensitivity of the TCR to Ag ligation.

Recipient vessels in the free flap reconstruction around the knee.

Microsurgical free tissue transfer is playing a critical role in reconstruction of the soft tissue around the knee to salvage the limb, especially when the defects exist with a wide zone of injury or with a poor soft tissue condition, where local flaps are unavailable. For a successful free flap transfer, proper selection of a recipient vessel is essential and challenging. The survival and other outcomes of the transferred flaps were closely related to which recipient vessel was used and the location of anastomosis. In this article, we review most of the clinical reports about using free flaps to reconstruct the soft tissue around the knee, excluding the cases of postamputation, and discuss about the recipient vessels that can be used.

Vascularized fibular grafts in patients with avascular necrosis of femoral head: a systematic review and meta-analysis.

BACKGROUND: Vascularized fibular grafting (VFG) has been initiated to treat avascular necrosis of the femoral head (ANFH) since the late 1970s. There are a number of review articles updating the use of VFG to treat the ANFH. None of them applied statistical analysis for combining results from different studies to obtain a quantitative estimate of the overall effect and potential harm of VFG in comparison to other treatment. METHODS: Several electronic databases were searched to find studies using VFG to treat ANFH. The outcomes sought included Harris Score, failure rate (conversion to total hip arthroplasty (THA) and/or femoral head collapse), and complications rate. Included studies were assessed for methodological bias and estimates of effect were calculated. Potential reasons for heterogeneity were explored. RESULTS: The clinical results of 69.0 % of VFG-treated patients and 25.0 % of non-VFG-treated patients were good to excellent (OR 0.13; p < 0.01). The conversion rate to THA of VFG-treated and that of other methods treated hips was 16.5 % and 42.6 % (OR 0.19; p < 0.001). Collapse rate of VFG-treated and that of non-VFG-treated hips was 16.7 % and 63.6 % (OR 0.09; p < 0.05). The complication rate of VFG-treated and that of other methods treated patients was 23.8 % and 8.9 % (OR 3.44; p = 0.09). For Steinberg stage I, II ANFH, failure rate of VFG-treated and that of non-VFG-treated hips was 9.8 % and 40.2 % (OR 0.17; p < 0.001). For Steinberg stage II, III ANFH, failure rate of VFG-treated and that of non-VFG-treated hips was 16.5 % and 42.8 %, respectively (OR 0.17; p < 0.001). CONCLUSIONS: VFG is a justified method that can prevent the ANFH from progressing to collapse, and that can retard or avoid hip replacement, especially in the hips of Steinberg stage I, II, and III.

Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors.

Parvovirus B19 (B19V) infects human erythroid progenitor cells (EPCs) and causes several hematological disorders and fetal hydrops. Amino acid (aa) 5-68 of minor capsid protein VP1 (VP1u5-68aa) is the minimal receptor binding domain for B19V to enter EPCs. Here, we carried out a genome-wide CRISPR-Cas9 guide RNA screen and identified tyrosine protein kinase receptor UFO (AXL) as a proteinaceous receptor for B19V infection of EPCs. AXL gene silencing in ex vivo expanded EPCs remarkably decreased B19V internalization and replication. Additions of the recombinant AXL extracellular domain or a polyclonal antibody against it upon infection efficiently inhibited B19V infection of ex vivo expanded EPCs. Moreover, B19V VP1u interacted with the recombinant AXL extracellular domain in vitro at a relatively high affinity (KD = 103 nM). Collectively, we provide evidence that AXL is a co-receptor for B19V infection of EPCs.

Identification of Host Restriction Factors Critical for Recombinant AAV Transduction of Polarized Human Airway Epithelium.

Recombinant (r)AAV2.5T was selected from the directed evolution of an AAV capsid library in human airway epithelium (HAE). The capsid gene of rAAV2.5T is a chimera of the N-terminal unique coding sequence of AAV2 VP1 unique (VP1u) and the VP2- and VP3-coding sequence of AAV5 with a single amino acid mutation of A581T. We conducted two rounds of genome wide CRISPR gRNA library screening for host factors limiting rAAV2.5T transduction in HeLa S3 cells. The screen identified several genes that are critical for rAAV2.5T transduction in HeLa S3 cells, including previously reported genes KIAA0319L , TM9SF2 , VPS51 , and VPS54 , as well as a novel gene WDR63 . We verified the role of KIAA0319L and WDR63 in rAAV2.5T transduction of polarized HAE by utilizing CRISPR gene knockouts. Although KIAA0319L, a proteinaceous receptor for multiple AAV serotypes, played an essential role in rAAV2.5T transduction of polarized HAE either from apical or basolateral side, our findings demonstrated that the internalization of rAAV2.5T was independent of KIAA0319L. Importantly, we confirmed WDR63 is an important player in rAAV2.5T transduction of HAE, while not being involved in vector internalization and nuclear entry. Furthermore, we identified that the basal stem cells of HAE can be significantly transduced by rAAV2.5T. Significance: The essential steps of a successful gene delivery by rAAV include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene WDR63 , whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.