Development of a Rapid Adeno-Associated Virus (AAV) Identity Testing Platform through Comprehensive Intact Mass Analysis of Full-Length AAV Capsid Proteins.

Adeno-associated viruses (AAVs) are commonly used as vectors for the delivery of gene therapy targets. Characterization of AAV capsid proteins (VPs) and their post-translational modifications (PTMs) have become a critical attribute monitored to evaluate product quality. Liquid chromatography-mass spectrometry (LC-MS) analysis of intact AAV VPs provides both quick and reliable serotype identification as well as proteoform information on each VP. Incorporating these analytical strategies into rapid good manufacturing practice (GMP)-compliant workflows containing robust, but simplified, data processing methods is necessary to ensure effective product quality control (QC) during production. Here, we present a GMP-compliant LC-MS workflow for the rapid identification and in-depth characterization of AAVs. Hydrophilic interaction liquid chromatography (HILIC) MS with difluoroacetic acid as a mobile phase modifier is utilized to achieve the intact separation and identification of AAV VPs and their potential proteoforms. Peptide mapping is performed to confirm PTMs identified during intact VP analysis and for in-depth PTM characterization. The intact separations platform is then incorporated into a data processing workflow developed using GMP-compliant software capable of rapid AAV serotype identification and, if desired, specific serotype PTM monitoring and characterization. Such a platform provides product QC capabilities that are easily accessible in a regulatory setting.

Mini-review: Equipment evaluation for process scalability and readiness for current Good Manufacturing Practices in cell therapy workflows.

Cell therapies present a promising treatment for a variety of diseases and are a rapidly growing market. This facilitates the need for robust biomanufacturing processes that can be implemented early during process establishment which enables scalable and reproducible manufacturing. Historically, cell therapy has used equipment originally repurposed from biologics, where the supernatant is harvested at the end of production and not the cells. Unlike biologics, cell therapy requires the preservation of cell phenotype and potency, as well as the functional recovery of the cells for the final formulation. These traditional equipment platforms have been widely adopted and, in many cases, successfully. However, given that cell therapy processes are complex, equipment specifically designed for the intended application will add immense value by producing products that are pure, potent and stable. New equipment better suited for cell therapy is being introduced to improve efficiency and product quality compared with current systems, fill key gaps that exist in current workflows or address an emerging need in new paradigms. Integration of these new instruments in laboratories using current Good Manufacturing Practices to produce cell-based drug products and drug substances requires a risk-based approach to evaluate features based on suitability and compliance with regulatory requirements. The speed at which new equipment is evaluated and implemented into new workflows is critical to match the speed of therapeutic product innovations and manufacturing capabilities. Here, we outline a framework to evaluate new equipment and de-risk implementation based on a series of features, namely, hardware, software, consumables, and workflow compatibility for the intended use. A hypothetical evaluation of three cell processing workflows is used as an example to inform equipment deployment for early process establishment and translational use for current Good Manufacturing Practices-destined workflows.

Historical evaluation of the in vivo adventitious virus test and its potential for replacement with next generation sequencing (NGS).

The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) collected historical data from 20 biopharmaceutical industry members on their experience with the in vivo adventitious virus test, the in vitro virus test, and the use of next generation sequencing (NGS) for viral safety. Over the past 20 years, only three positive in vivo adventitious virus test results were reported, and all were also detected in another concurrent assay. In more than three cases, data collected as a part of this study also found that the in vivo adventitious virus test had given a negative result for a sample that was later found to contain virus. Additionally, the in vivo adventitious virus test had experienced at least 21 false positives and had to be repeated an additional 21 times all while using more than 84,000 animals. These data support the consideration and need for alternative broad spectrum viral detection tests that are faster, more sensitive, more accurate, more specific, and more humane. NGS is one technology that may meet this need. Eighty one percent of survey respondents are either already actively using or exploring the use of NGS for viral safety. The risks and challenges of replacing in vivo adventitious virus testing with NGS are discussed. It is proposed to update the overall virus safety program for new biopharmaceutical products by replacing in vivo adventitious virus testing approaches with modern methodologies, such as NGS, that maintain or even improve the final safety of the product.

Patient-Customized Oligonucleotide Therapy for a Rare Genetic Disease.

Genome sequencing is often pivotal in the diagnosis of rare diseases, but many of these conditions lack specific treatments. We describe how molecular diagnosis of a rare, fatal neurodegenerative condition led to the rational design, testing, and manufacture of milasen, a splice-modulating antisense oligonucleotide drug tailored to a particular patient. Proof-of-concept experiments in cell lines from the patient served as the basis for launching an "N-of-1" study of milasen within 1 year after first contact with the patient. There were no serious adverse events, and treatment was associated with objective reduction in seizures (determined by electroencephalography and parental reporting). This study offers a possible template for the rapid development of patient-customized treatments. (Funded by Mila's Miracle Foundation and others.).

Who Stays at Home? The Politics of Social Distancing in Brazil, Mexico, and the United States during the COVID-19 Pandemic.

CONTEXT: Reductions in population mobility can mitigate COVID-19 virus transmission and disease-related mortality. But do social distancing policies actually change population behavior and, if so, what factors condition policy effects? METHODS: We leverage subnational variation in the stringency and timing of state-issued social distancing policies to test their effects on mobility across 109 states in Brazil, Mexico, and the United States. We also explore how conventional predictors of compliance, including political trust, socioeconomic resources, health risks, and partisanship, modify these policy effects. FINDINGS: In Brazil and the United States, stay-at-home orders and workplace closures reduced mobility, especially early in the pandemic. In Mexico, where federal intervention created greater policy uniformity, workplace closures produced the most consistent mobility reductions. Conventional explanations of compliance perform well in the United States but not in Brazil or Mexico, apart from those emphasizing socioeconomic resources. CONCLUSIONS: In addition to new directions for research on the politics of compliance, the article offers insights for policy makers on which measures are likely to elicit compliance. Our finding that workplace closure effectiveness increases with socioeconomic development suggests that cash transfers, stimulus packages, and other policies that mitigate the financial burdens of the pandemic may help reduce population mobility.

SCAI position statement on the performance of percutaneous coronary intervention in ambulatory surgical centers.

The Centers for Medicare & Medicaid Services (CMS) began reimbursement for percutaneous coronary intervention (PCI) performed in ambulatory surgical centers (ASC) in January 2020. The ability to perform PCI in an ASC has been made possible due to the outcomes data from observational studies and randomized controlled trials supporting same day discharge (SDD) after PCI. In appropriately selected patients for outpatient PCI, clinical outcomes for SDD or routine overnight observation are comparable without any difference in short-term or long-term adverse events. Furthermore, a potential for lower cost of care without a compromise in clinical outcomes exists. These studies provide the framework and justification for performing PCI in an ASC. The Society for Cardiovascular Angiography and Interventions (SCAI) supported this coverage decision provided the quality and safety standards for PCI in an ASC were equivalent to the hospital setting. The current position paper is written to provide guidance for starting a PCI program in an ASC with an emphasis on maintaining quality standards. Regulatory requirements and appropriate standards for the facility, staff and physicians are delineated. The consensus document identified appropriate patients for consideration of PCI in an ASC. The key components of an ongoing quality assurance program are defined and the ethical issues relevant to PCI in an ASC are reviewed.

Oceanic Microplankton Do Not Adhere to the Latitudinal Diversity Gradient.

A latitudinal biodiversity gradient has captivated ecologists for years, and has become a widely recognized pattern in biogeography, manifest as an increase in biodiversity from the poles to the tropics. Oceanographers have attempted to discern whether these distribution patterns are shared with marine biota, and a lively debate has emerged concerning the global distribution of microbes. Limitations in sampling resolution for such large-scale assessments have often prohibited definitive conclusions. We evaluated microbial planktonic communities along a ~ 15,400-km Pacific Ocean transect with DNA from samples acquired every 2 degrees of latitude within a 3-month period between late August and early November 2003. Next-generation sequencing targeting the Bacteria, Archaea, and Eukarya yielded ~ 10.8 million high-quality sequences. Beta-analysis revealed geographic patterns of microbial communities, primarily the Bacteria and Archaea domains. None of the domains exhibited a unimodal pattern of alpha-diversity with respect to latitude. Bacteria communities increased in richness from Arctic to Antarctic waters, whereas Archaea and Eukarya communities showed no latitudinal or polar trends. Based on our analyses, environmental factors related to latitude thought to influence various macrofauna may not define microplankton diversity patterns of richness in the global ocean.

Stability of the adeno-associated virus 8 reference standard material.

Adeno-associated virus (AAV) vectors are extensively used for gene therapy clinical trials. Accurate and standardized titration methods are essential for characterizing and dosing AAV-based drugs and thus to assess their safety and efficacy. To this end, the Reference Standard Materials (RSM) working group generated standards for AAV serotype 2 and serotype 8. The AAV8RSM (ATCC® VR-1816™) was deposited to the American Type Culture Collection in 2014 and is available to the scientific community. Here, three independent laboratories of the RSM working group provide stability data of the AAV8RSM 2 years after the initial characterization and after container relabeling performed at the ATCC. The AAV8RSM showed constant titers across experimental conditions: 1.48 ± 0.62 × 1012 vector genome (vg)/ml, 9.38 ± 11.4 × 108 infectious units (IU)/ml and 5.76 ± 2.39 × 1011 total particles (p)/ml as determined by qPCR, TCID50 and ELISA, respectively. Additionally, the AAV8RSM capsid protein integrity assessed by SDS-PAGE was equivalent to the original analyses. In conclusion, the AAV8RSM titers remained stable for two years under appropriate storage conditions ( <-70° C). The use of RSM is strongly recommended and endorsed by regulatory agencies to normalize laboratory internal controls and to provide accurate titration of AAV vectors lots.

Surveillance of Microsporidia and Protozoan Pathogens in Pensacola Florida: A One-year Study.

Pathogens and the potential risk they present to public health in recreational waters are of continual public concern. The focus of this study was a year-long sampling campaign to document the presence of Microsporidia and protozoan pathogens in the Bayou Texar waterway in Pensacola, Florida. We used biofilms as sentinel indicators for trapping pathogens in five different locations in Pensacola, Florida. Of the 34 biofilm samples, 16 were positive for pathogens. Of these samples, 13 were positive for Enchephalitozoon spp. (mostly E. cuniculi), 11 were positive for Enterocytozoon bieneusi, and two were positive for Cryptosporidium parvum. The data demonstrate that Microsporidia were easily recovered and primarily present in water during summer months.

Biofilms for Monitoring Presence of Microsporidia in Environmental Water.

The development of molecular methodologies for targeting pathogens such as the Microsporidia has greatly improved our monitoring capabilities and initiatives. This study analyzed samples collected from five locations in Pensacola, Florida, USA for the presence of Microsporidian pathogens. To circumvent various impediments associated with water collection and filtration, we utilized biofilms as sentinels for detection of Microsporidia. We implemented membrane-dissolution and sample purification in a single confined step followed by real-time PCR to confirm pathogen presence. The results of this study demonstrate that microsporidia are present in environmental water sites in the Florida panhandle and that biofilms may serve as another alternative mode to circumvent filtration methods for their detection.

Case manager as therapy extender for cognitive behavior therapy of serious mental illness: a case report.

Cognitive behavior therapy (CBT) is an evidence-based intervention for individuals with serious mental illness and potentiates standard medication management. Americans receiving publicly funded treatment for serious mental illnesses have limited access to CBT and hence we need to devise innovative ways of providing access to this important intervention. We present a case of a man who had severe disability, was medication resistant, and diagnosed with Obsessive Compulsive Disorder and Major Depressive Disorder. After being home bound for many years he was provided CBT utilizing his existing case manager as a therapy extender. The specific roles of the primary therapist and case manager as well as the improvement in quality of life of the individual are delineated. This case report opens up the possibility of further studying case managers as therapy extenders for treating serious mental illnesses.

Patient-centered medical home impact on health plan members with diabetes.

OBJECTIVE: To compare costs and utilization for patients with diabetes enrolled in patient-centered medical home (PCMH) practices and non-PCMH practices. DESIGN: Commercial Health Maintenance Organization members with diabetes who enrolled between 2008 and 2011 in 26 Pennsylvania-based PCMH practices that were recognized by the National Committee for Quality Assurance in 2009 were compared with similar patients in 97 non-PCMH primary care practices. A difference-in-differences longitudinal research design was used to analyze differences between both groups on per-member, per-month costs and utilization. The statistical models controlled for baseline practice and patient-level characteristics through 2-step propensity score matching. The regression analysis on program effect further controlled for within-practice variation. Sensitivity analyses were also conducted on patients with type 1 and type 2 diabetes separately, and a third analysis was limited to diabetic patients enrolled in practices within Philadelphia. RESULTS: Adoption of the PCMH reduced overall medical costs for diabetic patients by 21% in year 1. This reduction was driven largely by inpatient costs, which fell by 44%. Reductions in emergency department visits, outpatient costs, and specialist visits were also seen in subsequent years among patients enrolled in PCMH practices. Additional sensitivity analyses indicated that adoption of the PCMH model yielded similar results when analyzing patients with type 2 diabetes as well as for diabetic patients enrolled in PCMH practices located within the city of Philadelphia. CONCLUSIONS: The cost of care for patients with diabetes can be reduced by securing care at a PCMH practice. Immediate results were seen in reduction of inpatient costs, which indicate that these patients enrolled in PCMH practices were using less costly inpatient services.

Virtual Scribes and Physician Time Spent on Electronic Health Records.

Importance: Time on the electronic health record (EHR) is associated with burnout among physicians. Newer virtual scribe models, which enable support from either a real-time or asynchronous scribe, have the potential to reduce the burden of the EHR and EHR-related documentation. Objective: To characterize the association of use of virtual scribes with changes in physicians' EHR time and note and order composition and to identify the physician, scribe, and scribe response factors associated with changes in EHR time upon virtual scribe use. Design, Setting, and Participants: Retrospective, pre-post quality improvement study of 144 physicians across specialties who had used a scribe for at least 3 months from January 2020 to September 2022, were affiliated with Brigham and Women's Hospital and Massachusetts General Hospital, and cared for patients in the outpatient setting. Data were analyzed from November 2022 to January 2024. Exposure: Use of either a real-time or asynchronous virtual scribe. Main Outcomes: Total EHR time, time on notes, and pajama time (5:30 pm to 7:00 am on weekdays and nonscheduled weekends and holidays), all per appointment; proportion of the note written by the physician and team contribution to orders. Results: The main study sample included 144 unique physicians who had used a virtual scribe for at least 3 months in 152 unique scribe participation episodes (134 [88.2%] had used an asynchronous scribe service). Nearly two-thirds of the physicians (91 physicians [63.2%]) were female and more than half (86 physicians [59.7%]) were in primary care specialties. Use of a virtual scribe was associated with significant decreases in total EHR time per appointment (mean [SD] of 5.6 [16.4] minutes; P < .001) in the 3 months after vs the 3 months prior to scribe use. Scribe use was also associated with significant decreases in note time per appointment and pajama time per appointment (mean [SD] of 1.3 [3.3] minutes; P < .001 and 1.1 [4.0] minutes; P = .004). In a multivariable linear regression model, the following factors were associated with significant decreases in total EHR time per appointment with a scribe use at 3 months: practicing in a medical specialty (-7.8; 95% CI, -13.4 to -2.2 minutes), greater baseline EHR time per appointment (-0.3; 95% CI, -0.4 to -0.2 minutes per additional minute of baseline EHR time), and decrease in the percentage of the note contributed by the physician (-9.1; 95% CI, -17.3 to -0.8 minutes for every percentage point decrease). Conclusions and Relevance: In 2 academic medical centers, use of virtual scribes was associated with significant decreases in total EHR time, time spent on notes, and pajama time, all per appointment. Virtual scribes may be particularly effective among medical specialists and those physicians with greater baseline EHR time.

PCR-based detection of gene transfer vectors: application to gene doping surveillance.

Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR.

Integration frequency and intermolecular recombination of rAAV vectors in non-human primate skeletal muscle and liver.

The comprehensive characterization of recombinant adeno-associated viral (rAAV) integration frequency and persistence for assessing rAAV vector biosafety in gene therapy is severely limited due to the predominance of episomal rAAV vector genomes maintained in vivo. Introducing rAAV insertional standards (rAIS), we show that linear amplification-mediated (LAM)-PCR and deep sequencing can be used for validated measurement of rAAV integration frequencies. Integration of rAAV2/1 or rAAV2/8, following intramuscular (IM) or regional intravenous (RI) administration of therapeutically relevant vector doses in nine adult non-human primates (NHP), occurs at low frequency between 10(-4) and 10(-5) both in NHP liver and muscle, but with no preference for specific genomic loci. High resolution mapping of inverted terminal repeat (ITR) breakpoints in concatemeric and integrated vector genomes reveals distinct vector recombination hotspots, including large deletions of up to 3 kb. Moreover, retrieval of integrated rAAV genomes indicated approximately threefold increase in liver compared to muscle. This molecular analysis of rAAV persistence in NHP provides a promising basis for a reliable genotoxic risk assessment of rAAV in clinical trials.

Universal ddPCR-based assay for the determination of lentivirus infectious titer and lenti-modified cell vector copy number.

The translation of cell-based therapies from research to clinical setting requires robust analytical methods that successfully adhere to current good manufacturing practices and regulatory guidelines. Lentiviral vectors are commonly used for gene delivery to generate genetically modified therapeutic cell products. For some cell therapy products, standardized characterization assays for potency and safety have gained momentum. Translational applications benefit from assays that can be deployed broadly, such as for lentiviral vectors with various transgenes of interest. Development of a universal method to determine lentivirus infectious titer and vector copy number (VCN) of lenti-modified cells was performed using droplet digital PCR (ddPCR). Established methods relied on a ubiquitous lenti-specific target and a housekeeping gene that demonstrated comparability among flow cytometry-based methods. A linearized plasmid control was used to determine assay linearity/range, sensitivity, accuracy, and limits of quantification. Implementing this assay, infectious titer was assessed for various production runs that demonstrated comparability to the flow cytometry titer. The ddPCR assay described here also indicates suitability in the determination of VCN for genetically modified CAR-T cell products. Overall, the development of these universal assays supports the implementation of standardized characterization methods for quality control.

Assessing the suitability of cell counting methods during different stages of a cell processing workflow using an ISO 20391-2 guided study design and analysis.

Cell counting is a fundamental measurement for determining viable cell numbers in biomanufacturing processes. The properties of different cell types and the range of intended uses for cell counts within a biomanufacturing process can lead to challenges in identifying suitable counting methods for each potential application. This is further amplified by user subjectivity in identifying the cells of interest and further identifying viable cells. Replacement of traditionally used manual counting methods with automated systems has alleviated some of these issues. However, a single cell type can exhibit different physical properties at various stages of cell processing which is further compounded by process impurities such as cell debris or magnetic beads. These factors make it challenging to develop a robust cell counting method that offers a high level of confidence in the results. Several initiatives from standards development organizations have attempted to address this critical need for standardization in cell counting. This study utilizes flow-based and image-based methods for the quantitative measurement of cell concentration and viability in the absence of a reference material, based on the tools and guidance provided by the International of Standards (ISO) and the US National Institute of Standards and Technology (NIST). Primary cells were examined at different stages of cell processing in a cell therapy workflow. Results from this study define a systematic approach that enables the identification of counting methods and parameters that are best suited for specific cell types and workflows to ensure accuracy and consistency. Cell counting is a foundational method used extensively along various steps of cell and gene therapy. The standard used in this study may be applied to other cell and gene therapy processes to enable accurate measurement of parameters required to guide critical decisions throughout the development and production process. Using a framework that confirms the suitability of the cell counting method used can minimize variability in the process and final product.