Gene editing for latent herpes simplex virus infection reduces viral load and shedding in vivo.

Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a potentially curative approach against HSV infection, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Gene editing performed with two anti-HSV-1 meganucleases delivered by a combination of AAV9, AAV-Dj/8, and AAV-Rh10 can eliminate 90% or more of latent HSV DNA in mouse models of orofacial infection, and up to 97% of latent HSV DNA in mouse models of genital infection. Using a pharmacological approach to reactivate latent HSV-1, we demonstrate that ganglionic viral load reduction leads to a significant decrease of viral shedding in treated female mice. While therapy is well tolerated, in some instances, we observe hepatotoxicity at high doses and subtle histological evidence of neuronal injury without observable neurological signs or deficits. Simplification of the regimen through use of a single serotype (AAV9) delivering single meganuclease targeting a duplicated region of the HSV genome, dose reduction, and use of a neuron-specific promoter each results in improved tolerability while retaining efficacy. These results reinforce the curative potential of gene editing for HSV disease.

Kinetics of the Gas-Phase Reactions of syn- and anti-CH3CHOO Criegee Intermediate Conformers with SO2 as a Function of Temperature and Pressure.

Kinetics of reactions between SO2 and CH3CHOO Criegee intermediate conformers have been measured at temperatures between 242 and 353 K and pressures between 10 and 600 Torr using laser flash photolysis of CH3CHI2/O2/N2/SO2 gas mixtures coupled with time-resolved broadband UV absorption spectroscopy. The kinetics of syn-CH3CHOO + SO2 are pressure-dependent and exhibit a negative temperature dependence, with the observed pressure dependence reconciling apparent discrepancies between previous measurements performed at ∼298 K. Results indicate a rate coefficient of (4.80 ± 0.46) × 10-11 cm3 s-1 for the reaction of syn-CH3CHOO with SO2 at 298 K and 760 Torr. In contrast to the behavior of the syn-conformer, the kinetics of anti-CH3CHOO + SO2 display no significant dependence on temperature or pressure over the ranges investigated, with a mean rate coefficient of (1.18 ± 0.21) × 10-10 cm3 s-1 over all conditions studied in this work. Results indicate that the reaction of syn-CH3CHOO with SO2 competes with unimolecular decomposition and reaction with water vapor in areas with high SO2 concentration and low humidity, particularly at lower temperatures.

Effect of Viral Illness on Procalcitonin as a Predictor of Bacterial Infection in Febrile Infants.

OBJECTIVE: The impact of confirmed viral infections (CVI) on procalcitonin (PCT) levels in febrile infants aged 8-60 days with a bacterial illness (BI) is unknown. The objectives of the study were to (1) examine the association of CVI with PCT levels in patients with/without a concurrent BI, defined as bacteremia, meningitis, or urinary tract infection, and (2) assess PCT as a predictor of BI in infants with a concurrent CVI. METHODS: In this single-center, retrospective cohort study, we examined febrile infants aged 8-60 days presenting between January 1, 2018 and December 31, 2020. PCT levels were compared between groups, according to results of bacterial cultures and viral tests, using the Wilcoxon rank test. The prediction ability of PCT to detect BI with/without concurrent CVI was assessed by using area under the curve from logistic regression. RESULTS: Patients included: 404 BI-/CVI+, 73 BI+/CVI-, 48 BI+/CVI+, and 138 BI-/CVI-. Median PCT level in the BI+/CVI+ group was significantly lower when compared to BI+/CVI- (0.36 ng/mL vs 0.89 ng/mL), but significantly higher than the BI-/CVI- group (0.36 ng/mL vs 0.1 ng/mL). The presence of a CVI reduced the sensitivity of PCT in BI detection (68% vs 44%), with minimal impact specificity (93% vs 96%). CONCLUSIONS: In previously healthy febrile infants 8-60 days old, the presence of a CVI reduces the sensitivity of PCT BI detection without impacting its specificity. The impact of a CVI on PCT levels in febrile infants has implications for how this marker of infection should be considered when assessing risk of BI in infants.

Liver-Humanized NSG-PiZ Mice Support the Study of Chronic Hepatitis B Virus Infection and Antiviral Therapies.

Hepatitis B virus (HBV) is a pathogen of major public health importance that is largely incurable once a chronic infection is established. Only humans and great apes are fully permissive to HBV infection, and this species restriction has impacted HBV research by limiting the utility of small animal models. To combat HBV species restrictions and enable more in vivo studies, liver-humanized mouse models have been developed that are permissive to HBV infection and replication. Unfortunately, these models can be difficult to establish and are expensive commercially, which has limited their academic use. As an alternative mouse model to study HBV, we evaluated liver-humanized NSG-PiZ mice and showed that they are fully permissive to HBV. HBV selectively replicates in human hepatocytes within chimeric livers, and HBV-positive (HBV+) mice secrete infectious virions and hepatitis B surface antigen (HBsAg) into blood while also harboring covalently closed circular DNA (cccDNA). HBV+ mice develop chronic infections lasting at least 169 days, which should enable the study of new curative therapies targeting chronic HBV, and respond to entecavir therapy. Furthermore, HBV+ human hepatocytes in NSG-PiZ mice can be transduced by AAV3b and AAV.LK03 vectors, which should enable the study of gene therapies that target HBV. In summary, our data demonstrate that liver-humanized NSG-PiZ mice can be used as a robust and cost-effective alternative to existing chronic hepatitis B (CHB) models and may enable more academic research labs to study HBV disease pathogenesis and antiviral therapy. IMPORTANCE Liver-humanized mouse models have become the gold standard for the in vivo study of hepatitis B virus (HBV), yet their complexity and cost have prohibited widespread use of existing models in research. Here, we show that the NSG-PiZ liver-humanized mouse model, which is relatively inexpensive and simple to establish, can support chronic HBV infection. Infected mice are fully permissive to hepatitis B, supporting both active replication and spread, and can be used to study novel antiviral therapies. This model is a viable and cost-effective alternative to other liver-humanized mouse models that are used to study HBV.

Adeno-associated virus vectors and neurotoxicity-lessons from preclinical and human studies.

Over 15 years after hepatotoxicity was first observed following administration of an adeno-associated virus (AAV) vector during a hemophilia B clinical trial, recent reports of treatment-associated neurotoxicity in animals and humans have brought the potential impact of AAV-associated toxicity back to prominence. In both pre-clinical studies and clinical trials, systemic AAV administration has been associated with neurotoxicity in peripheral nerve ganglia and spinal cord. Neurological signs have also been seen following direct AAV injection into the brain, both in non-human primates and in a clinical trial for late infantile Batten disease. Neurotoxic events appear variable across species, and preclinical animal studies do not fully predict clinical observations. Accumulating data suggest that AAV-associated neurotoxicity may be underdiagnosed and may differ between species in terms of frequency and/or severity. In this review, we discuss the different animal models that have been used to demonstrate AAV-associated neurotoxicity, its potential causes and consequences, and potential approaches to blunt AAV-associated neurotoxicity.

A multiplexed barcode approach to simultaneously evaluate gene delivery by adeno-associated virus capsid variants in nonhuman primates.

BACKGROUND AND AIMS: Adeno-associated virus (AAV) vectors are widely used to deliver therapeutic transgenes to distinct tissues, including the liver. Vectors based on naturally occurring AAV serotypes as well as vectors using engineered capsids have shown variations in tissue tropism and level of transduction between different mouse models. Moreover, results obtained in rodents frequently lack translatability into large animal studies. In light of the increasing interest in AAV vectors for human gene therapy, an increasing number of studies are being performed in nonhuman primates. To keep animal numbers to a minimum and thus optimize the process of AAV capsid selection, we developed a multiplex barcoding approach to simultaneously evaluate the in vivo vector performance for a set of serotypes and capsid-engineered AAV vectors across multiple organs. APPROACH AND RESULTS: Vector biodistribution and transgene expression were assessed by quantitative PCR, quantitative reverse transcription PCR, vector DNA amplicon Illumina sequencing and vRNAseq in male and female rhesus macaques simultaneously dosed with a mixture of barcoded naturally occurring or engineered AAV vectors encoding the same transgene. As expected, our findings show animal-to-animal variation in both the biodistribution and tissue transduction pattern, which was partly influenced by each animal's distinctive serological status. CONCLUSIONS: This method offers a robust approach to AAV vector optimization that can be used to identify and validate AAV vectors for gene delivery to potentially any anatomical site or cell type.

Kinetics of the gas phase reaction of the Criegee intermediate CH2OO with SO2 as a function of temperature.

The kinetics of the gas phase reaction of the Criegee intermediate CH2OO with SO2 have been studied as a function of temperature in the range 223-344 K at 85 Torr using flash photolysis of CH2I2/O2/SO2/N2 mixtures at 248 nm coupled to time-resolved broadband UV absorption spectroscopy. Measurements were performed under pseudo-first-order conditions with respect to SO2, revealing a negative temperature dependence. Analysis of experimental results using the Master Equation Solver for Multi-Energy well Reactions (MESMER) indicates that the observed temperature dependence, combined with the reported lack of a pressure dependence in the range 1.5-760 Torr, can be described by a reaction mechanism consisting of the formation of a pre-reaction complex leading to a cyclic secondary ozonide which subsequently decomposes to produce HCHO + SO3. The temperature dependence can be characterised by kCH2OO+SO2 = (3.72 ± 0.13) × 10-11 (T/298)(-2.05±0.38) cm3 molecule-1 s-1. The observed negative temperature dependence for the title reaction in conjunction with the decrease in water dimer (the main competitor for the Criegee intermediate) concentration at lower temperatures means that Criegee intermediate chemistry can play an enhanced role in SO2 oxidation in the atmosphere at lower temperatures.

Leveraging Genetic Reports and Electronic Health Records for the Prediction of Primary Cancers: Algorithm Development and Validation Study.

BACKGROUND: Precision oncology has the potential to leverage clinical and genomic data in advancing disease prevention, diagnosis, and treatment. A key research area focuses on the early detection of primary cancers and potential prediction of cancers of unknown primary in order to facilitate optimal treatment decisions. OBJECTIVE: This study presents a methodology to harmonize phenotypic and genetic data features to classify primary cancer types and predict cancers of unknown primaries. METHODS: We extracted genetic data elements from oncology genetic reports of 1011 patients with cancer and their corresponding phenotypical data from Mayo Clinic's electronic health records. We modeled both genetic and electronic health record data with HL7 Fast Healthcare Interoperability Resources. The semantic web Resource Description Framework was employed to generate the network-based data representation (ie, patient-phenotypic-genetic network). Based on the Resource Description Framework data graph, Node2vec graph-embedding algorithm was applied to generate features. Multiple machine learning and deep learning backbone models were compared for cancer prediction performance. RESULTS: With 6 machine learning tasks designed in the experiment, we demonstrated the proposed method achieved favorable results in classifying primary cancer types (area under the receiver operating characteristic curve [AUROC] 96.56% for all 9 cancer predictions on average based on the cross-validation) and predicting unknown primaries (AUROC 80.77% for all 8 cancer predictions on average for real-patient validation). To demonstrate the interpretability, 17 phenotypic and genetic features that contributed the most to the prediction of each cancer were identified and validated based on a literature review. CONCLUSIONS: Accurate prediction of cancer types can be achieved with existing electronic health record data with satisfactory precision. The integration of genetic reports improves prediction, illustrating the translational values of incorporating genetic tests early at the diagnosis stage for patients with cancer.

Optimization of AAV vectors to target persistent viral reservoirs.

Gene delivery of antiviral therapeutics to anatomical sites where viruses accumulate and persist is a promising approach for the next generation of antiviral therapies. Recombinant adeno-associated viruses (AAV) are one of the leading vectors for gene therapy applications that deliver gene-editing enzymes, antibodies, and RNA interference molecules to eliminate viral reservoirs that fuel persistent infections. As long-lived viral DNA within specific cellular reservoirs is responsible for persistent hepatitis B virus, Herpes simplex virus, and human immunodeficiency virus infections, the discovery of AAV vectors with strong tropism for hepatocytes, sensory neurons and T cells, respectively, is of particular interest. Identification of natural isolates from various tissues in humans and non-human primates has generated an extensive catalog of AAV vectors with diverse tropisms and transduction efficiencies, which has been further expanded through molecular genetic approaches. The AAV capsid protein, which forms the virions' outer shell, is the primary determinant of tissue tropism, transduction efficiency, and immunogenicity. Thus, over the past few decades, extensive efforts to optimize AAV vectors for gene therapy applications have focused on capsid engineering with approaches such as directed evolution and rational design. These approaches are being used to identify variants with improved transduction efficiencies, alternate tropisms, reduced sequestration in non-target organs, and reduced immunogenicity, and have produced AAV capsids that are currently under evaluation in pre-clinical and clinical trials. This review will summarize the most recent strategies to identify AAV vectors with enhanced tropism and transduction in cell types that harbor viral reservoirs.

CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice.

Chronic hepatitis B virus (HBV) infection is a major public health problem. New treatment approaches are needed because current treatments do not target covalently closed circular DNA (cccDNA), the template for HBV replication, and rarely clear the virus. We harnessed adeno-associated virus (AAV) vectors and CRISPR-Staphylococcus aureus (Sa)Cas9 to edit the HBV genome in liver-humanized FRG mice chronically infected with HBV and receiving entecavir. Gene editing was detected in livers of five of eight HBV-specific AAV-SaCas9-treated mice, but not control mice, and mice with detectable HBV gene editing showed higher levels of SaCas9 delivery to HBV+ human hepatocytes than those without gene editing. HBV-specific AAV-SaCas9 therapy significantly improved survival of human hepatocytes, showed a trend toward decreasing total liver HBV DNA and cccDNA, and was well tolerated. This work provides evidence for the feasibility and safety of in vivo gene editing for chronic HBV infections, and it suggests that with further optimization, this approach may offer a plausible way to treat or even cure chronic HBV infections.

Gene Transfer in Adeno-Associated Virus Seropositive Rhesus Macaques Following Rapamycin Treatment and Subcutaneous Delivery of AAV6, but Not Retargeted AAV6 Vectors.

Adeno-associated virus (AAV) vectors such as AAV6, which shows tropism for primary human CD4+ T cells in vitro, are being explored for delivery of anti-HIV therapeutic modalities in vivo. However, pre-existing immunity and sequestration in nontarget organs can significantly hinder their performance. To overcome these challenges, we investigated whether immunosuppression would allow gene delivery by AAV6 or targeted AAV6 derivatives in seropositive rhesus macaques. Animals were immune suppressed with rapamycin before intravenous (IV) or subcutaneous (SC) delivery of AAV, and we monitored vector biodistribution, gene transfer, and safety. Macaques received phosphate-buffered saline, AAV6 alone, or an equal dose of AAV6 and an AAV6-55.2 vector retargeted to CD4 through a direct ankyrin repeat protein (DARPin). AAV6 and AAV6-55.2 vector genomes were found in peripheral blood mononuclear cells and most organs up to 28 days postadministration, with the highest levels seen in liver, spleen, lymph nodes (LNs), and muscle, suggesting that retargeting did not prevent vector sequestration. Despite vector genome detection, gene expression from AAV6-55.2 was not detected in any tissue. SC injection of AAV6 facilitated efficient gene expression in muscle adjacent to the injection site, plus low-level gene expression in spleen, LNs, and liver, whereas gene expression following IV injection of AAV6 was predominantly seen in the spleen. AAV vectors were well tolerated, although elevated liver enzymes were detected in three of four AAV-treated animals 14 days after rapamycin withdrawal. One SC-injected animal had muscle inflammation proximal to the injection site, plus detectable T cell responses against transgene and AAV6 capsid at study finish. Overall, our data suggest that rapamycin treatment may offer a possible strategy to express anti-HIV therapeutics such as broadly neutralizing antibodies from muscle. This study provides important safety and efficacy data that will aid study design for future anti-HIV gene therapies.

Modeling cancer clinical trials using HL7 FHIR to support downstream applications: A case study with colorectal cancer data.

BACKGROUND AND OBJECTIVE: Identification and Standardization of data elements used in clinical trials may control and reduce the cost and errors during the operational process, and enable seamless data exchange between the electronic data capture (EDC) systems and Electronic Health Record (EHR) systems. This study presents a methodology to comprehensively capture the clinical trial data element needs. MATERIALS AND METHODS: Case report forms (CRF) for clinical trial data collection were used to approximate the clinical information need, whereby these information needs were then mapped to a semantically equivalent field within an existing FHIR cancer profile. For items without a semantically equivalent field, we considered these items to be information needs that cannot be represented in current standards and proposed extensions to support these needs. RESULTS: We successfully identified 62 discrete items from a preliminary survey of 43 base questions in four CRFs used in colorectal cancer clinical trials, in which 28 items are modeled with FHIR extensions and their associated responses for colorectal cancer. We achieved promising results in the data population of the CRFs with average Precision 98.5 %, Recall 96.2 %, and F-measure 96.8 % for all base questions. We also demonstrated the auto-filled answers in CRFs can be used to discover patient subgroups using a topic modeling approach. CONCLUSION: CRFs can be considered as a proxy for representing information needs for their respective cancer types. Mining the information needs can serve as a valuable resource for expanding existing standards to ensure they can comprehensively represent relevant clinical data without loss of granularity.

Gene editing and elimination of latent herpes simplex virus in vivo.

We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection.

Kinetics of the Gas Phase Reactions of the Criegee Intermediate CH2OO with O3 and IO.

The kinetics of the gas phase reactions of the Criegee intermediate CH2OO with O3 and IO have been studied at 296 K and 300 Torr through simultaneous measurements of CH2OO, the CH2OO precursor (CH2I2), O3, and IO using flash photolysis of CH2I2/O2/O3/N2 mixtures at 248 nm coupled to time-resolved broadband UV absorption spectroscopy. Experiments were performed under pseudo-first-order conditions with respect to O3, with the rate coefficients for reactions of CH2OO with O3 and IO obtained by fitting to the observed decays of CH2OO using a model constrained to the measured concentrations of IO. Fits were performed globally, with the ratio between the initial concentration of O3 and the average concentration of IO varying in the range 30-700, and gave kCH2OO+O3 = (3.6 ± 0.8) × 10-13 cm3 molecule-1 s-1 and kCH2OO+IO = (7.6 ± 1.4) × 10-11 cm3 molecule-1 s-1 (where the errors are at the 2σ level). The magnitude of kCH2OO+O3 has a significant effect on the steady state concentration of CH2OO in chamber studies. Atmospheric implications of the results are discussed.

Developing an FHIR-Based Computational Pipeline for Automatic Population of Case Report Forms for Colorectal Cancer Clinical Trials Using Electronic Health Records.

PURPOSE: The Fast Healthcare Interoperability Resources (FHIR) is emerging as a next-generation standards framework developed by HL7 for exchanging electronic health care data. The modeling capability of FHIR in standardizing cancer data has been gaining increasing attention by the cancer research informatics community. However, few studies have been conducted to examine the capability of FHIR in electronic data capture (EDC) applications for effective cancer clinical trials. The objective of this study was to design, develop, and evaluate an FHIR-based method that enables the automation of the case report forms (CRFs) population for cancer clinical trials using real-world electronic health records (EHRs). MATERIALS AND METHODS: We developed an FHIR-based computational pipeline of EDC with a case study for modeling colorectal cancer trials. We first leveraged an existing FHIR-based cancer profile to represent EHR data of patients with colorectal cancer, and then we used the FHIR Questionnaire and QuestionnaireResponse resources to represent the CRFs and their data population. To test the accuracy of and overall quality of the computational pipeline, we used synoptic reports of 287 Mayo Clinic patients with colorectal cancer from 2013 to 2019 with standard measures of precision, recall, and F1 score. RESULTS: Using the computational pipeline, a total of 1,037 synoptic reports were successfully converted as the instances of the FHIR-based cancer profile. The average accuracy for converting all data elements (excluding tumor perforation) of the cancer profile was 0.99, using 200 randomly selected records. The average F1 score for populating nine questions of the CRFs in a real-world colorectal cancer trial was 0.95, using 100 randomly selected records. CONCLUSION: We demonstrated that it is feasible to populate CRFs with EHR data in an automated manner with satisfactory performance. The outcome of the study provides helpful insight into future directions in implementing FHIR-based EDC applications for modern cancer clinical trials.

Comparative fate of CrAssphage with culturable and molecular fecal pollution indicators during activated sludge wastewater treatment.

Wastewater treatment plants are typically monitored using fecal indicator bacteria to ensure adequate microbial water quality of the treated effluent. Fecal indicator bacteria exhibit poor correlation with virus fate in the environment, including during wastewater treatment. Viral-based microbial source tracking methods have the potential to overcome this limitation. The recently discovered human gut bacteriophage crAssphage is a promising viral human fecal indicator. In this current study, primary influent, primary effluent, secondary effluent, and final effluent of a conventional activated sludge wastewater treatment plant were analyzed for a suite of fecal indicators to evaluate the suitability of crAssphage as a wastewater process indicator for virus removal. CrAssphage was the most abundant fecal indicator measured through the wastewater treatment process. Culturable and molecular bacterial fecal pollution indicators showed higher removal than viral fecal pollution indicators, including crAssphage, confirming the necessity of a viral-specific fecal monitoring target. CrAssphage was strongly correlated with adenovirus and polyomavirus molecular indicators through the wastewater treatment process. Literature comparison demonstrated site-specific removal of molecular fecal indicators during wastewater treatment highlighting the need for local performance validation. The high abundance of crAssphage and correlation with pathogenic viruses suggests the potential suitability of crAssphage as a viral fecal pollution process indicator during wastewater treatment.

Viral diversity is an obligate consideration in CRISPR/Cas9 designs for targeting the HIV reservoir.

BACKGROUND: RNA-guided CRISPR/Cas9 systems can be designed to mutate or excise the integrated HIV genome from latently infected cells and have therefore been proposed as a curative approach for HIV. However, most studies to date have focused on molecular clones with ideal target site recognition and do not account for target site variability observed within and between patients. For clinical success and broad applicability, guide RNA (gRNA) selection must account for circulating strain diversity and incorporate the within-host diversity of HIV. RESULTS: We identified a set of gRNAs targeting HIV LTR, gag, and pol using publicly available sequences for these genes and ranked gRNAs according to global conservation across HIV-1 group M and within subtypes A-C. By considering paired and triplet combinations of gRNAs, we found triplet sets of target sites such that at least one of the gRNAs in the set was present in over 98% of all globally available sequences. We then selected 59 gRNAs from our list of highly conserved LTR target sites and evaluated in vitro activity using a loss-of-function LTR-GFP fusion reporter. We achieved efficient GFP knockdown with multiple gRNAs and found clustering of highly active gRNA target sites near the middle of the LTR. Using published deep-sequence data from HIV-infected patients, we found that globally conserved sites also had greater within-host target conservation. Lastly, we developed a mathematical model based on varying distributions of within-host HIV sequence diversity and enzyme efficacy. We used the model to estimate the number of doses required to deplete the latent reservoir and achieve functional cure thresholds. Our modeling results highlight the importance of within-host target site conservation. While increased doses may overcome low target cleavage efficiency, inadequate targeting of rare strains is predicted to lead to rebound upon cART cessation even with many doses. CONCLUSIONS: Target site selection must account for global and within host viral genetic diversity. Globally conserved target sites are good starting points for design, but multiplexing is essential for depleting quasispecies and preventing viral load rebound upon therapy cessation.

In Vivo Hematopoietic Stem Cell Transduction.

Current protocols for hematopoietic stem cell (HSC) gene therapy, involving the transplantation of ex vivo lentivirus vector-transduced HSCs into myeloablated recipients, are complex and not without risk for the patient. In vivo HSC gene therapy can be achieved by the direct modification of HSCs in the bone marrow after intraosseous injection of gene delivery vectors. A recently developed approach involves the mobilization of HSCs from the bone marrow into peripheral the blood circulation, intravenous vector injection, and re-engraftment of genetically modified HSCs in the bone marrow. We provide examples for in vivo HSC gene therapy and discuss advantages and disadvantages.

In vivo dynamics of AAV-mediated gene delivery to sensory neurons of the trigeminal ganglia.

The ability to genetically manipulate trigeminal ganglion (TG) neurons would be useful in the study of the craniofacial nervous system and latent alphaherpesvirus infections. We investigated adeno-associated virus (AAV) vectors for gene delivery to the TG after intradermal whiskerpad delivery in mice. We demonstrated that AAV vectors of serotypes 1, 7, 8, and 9 trafficked from the whiskerpad into TG neurons and expressed transgenes within cell bodies and axons of sensory neurons in all three branches of the TG. Gene expression was highest with AAV1, and steadily increased over time up to day 28. Both constitutive and neuronal-specific promoters were able to drive transgene expression in TG neurons. Levels of vector genomes in the TG increased with input dose, and multiple transgenes could be co-delivered to TG neurons by separate AAV vectors. In conclusion, AAV1 vectors are suitable for gene delivery to TG sensory neurons following intradermal whiskerpad injection.

Characteristics of Providers Performing Urogynecologic Procedures on Medicare Patients 2012-2014.

OBJECTIVE: To analyze the characteristics of providers performing stress urinary incontinence (SUI) and pelvic organ prolapse (POP) procedures in the United States. METHODS: The Centers for Medicare Services public database, released for years 2012 through 2014, was queried for SUI-related and POP-related Healthcare Common Procedure Coding System. Providers were categorized as Female Pelvic Medicine and Reconstructive Surgery (FPMRS) providers and non-FPMRS providers, using a list of FPMRS board-certified providers compiled through the American Board of Medical Subspecialties website. Other physician specialties that submitted SUI and POP procedures claims were tabulated. RESULTS: Six hundred twenty-nine FPMRS and 833 non-FPMRS providers submitted claims for SUI and POP procedures. The SUI procedures claims had the following provider specialty distribution: obstetrics and gynecology (OB/GYN)-FPMRS, 46.7%; urology, 26.3%; OB/GYN, 12.2%; and urology-FPMRS, 13.9%, with the remaining 0.9% being performed by other specialties. The POP procedures had the following specialty distribution: OB/GYN-FPMRS, 63.4%; OB/GYN, 16.7%; urology, 8.3%; and urology-FPMRS, 7.1%, with the remaining 4.5% being performed by other specialties.Provider distribution was compared between transvaginal mesh and sling insertion procedures to transvaginal mesh and sling removal procedures. The FPMRS providers claimed 63.6% of sling and transvaginal mesh insertion procedures and performed 84.9% of mesh and sling removal procedures. CONCLUSIONS: Medicare reimbursement data provides a unique insight into the distribution of provider specialties performing SUI-related and POP-related procedures in the Medicare population. The OB/GYN-FPMRS providers submitted the majority of claims for SUI and POP procedures from 2012 to 2014. The FPMRS providers are also performing the majority of mesh removal procedures.