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Immunogenicity Assessment of AAV-based Gene Therapies: An IQ Consortium Industry White Paper
No full textImmunogenicity is critical to AAV-based gene therapy during discovery and development. The cross industry working groups organized by International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) have focused on various aspects of critical questions related to AAV-based gene therapy. This white paper summarizes the immunogenicity potential and risks associated with recombinant AAV-based GTs based on current clinical experiences and provides a framework for bioanalytical approaches related to the immunogenicity assessment of rAAV-based GTs. The paper provides a comprehensive overview of immunogenicity risks associated with rAAV GTs, as well as describes the methodologies for monitoring pre-existing and treatment boosted/or induced immune responses (humoral vs cellular). In addition, we discuss current clinical mitigation strategies deployed to reduce immunogenicity upon administration of rAAV-based G
Chemokine-like receptor 1 plays a critical role in modulating the regenerative and contractile properties of muscle tissue.
No full textMusculoskeletal diseases are a leading contributor to mobility disability worldwide. Since the majority of patients with musculoskeletal diseases present with associated muscle weakness, treatment approaches typically comprise an element of resistance training to restore physical strength. The health-promoting effects of resistance exercise are mediated via complex, multifarious mechanisms including modulation of systemic and local inflammation. Here we investigated whether targeted inhibition of the chemerin pathway, which largely controls inflammatory processes via chemokine-like receptor 1 (CMKLR1), can improve skeletal muscle function. Using genetically modified mice, we demonstrate that blockade of CMKLR1 transiently increases maximal strength during growth, but lastingly decreases strength endurance. In-depth analyses of the underlying long-term adaptations revealed microscopic alterations in the number of Pax7-positive satellite cells, as well as molecular changes in genes governing myogenesis and calcium handling. Taken together, these data provide evidence of a critical role for CMKLR1 in regulating skeletal muscle function by modulating the regenerative and contractile properties of muscle tissue. CMKLR1 antagonists are increasingly viewed as therapeutic modalities for a variety of diseases (e.g., psoriasis, metabolic disorders, and multiple sclerosis). Our findings thus have implications for the development of novel drug substances that aim at targeting the chemerin pathway for musculoskeletal or other diseases
Evaluation of Ambient Sound, Vibration, and Light in Rodent Housing Rooms
No full textExcessive sound, vibration, and light are detrimental to rodent welfare, yet these parameters are rarely recorded in vivaria. Whether housing environments exceed the suggested thresholds and which specific factors may alter these parameters is generally unknown. The goal of this study was to determine how environmental factors may alter sound, vibration, and light at the room and cage levels. Measurements were made using an ultrasonic microphone, accelerometer, and light sensor. Measurement sites were 1) in open air at a central location in 64 rooms located in 9 buildings, and 2) inside an empty mouse or rat cage containing chow, water, and bedding and located on an animal transfer station (n = 51) or housing rack (n = 102). Information collected for each transfer station and rack measurement included the year of manufacture, the species on the rack, and the number of cages on the rack. For each location, a baseline measurement was taken with the transfer station turned off, followed by another measurement after the transfer station was turned on. In general, many factors influenced ambient sound, vibration, and light, indicating that values are not uniform across rodent rooms in the same institution or across cages in a single room. Sound peaks capable of startling rodents were measured in association with hallway ultrasonic motion sensors and during cage change. Vibration and light intensity were generally low when cages were located on the rack. In contrast, active transfer stations had more vibration and light intensity, reaching levels that were potentially stressful for rodents. These data reflect the ambient sound, vibration, and light that rodents experience during normal facility operations. These patterns may extend to other locations, but given the variability in all parameters, the data highlight the need for institutions to conduct their own monitoring
Activation of GPR116/ADGRF5 by its tethered ligand relies on key amino acids in extracellular loop 2 of the transmembrane region
No full textAdhesion GPCRs are activated via a tethered agonist, revealed upon ligand binding to the N-terminal extracellular domains. The mechanistic details of this mode of activation has not been deciphered. We set out to investigate the physiologic importance of autocatalytic cleavage upstream of the agonistic peptide sequence and characterize tethered agonist-mediated activation of GPR116 in vitro and in vivo. A transgenic mouse expressing a non-cleavable GPR116 mutant phenocopies the pulmonary surfactant phenotype of GPR116 knock-out mice, demonstrating that tethered agonist-mediated receptor activation is indispensable for function in vivo. Using site-directed mutagenesis and species swapping approaches we identified key amino acids for GPR116 activation in the tethered agonist sequence and in extracellular loop 2/transmembrane 6 (ECL2/TM6) that are conserved in human and mouse. We further highlight residues in TM7 that mediate a stronger signaling in mouse versus human GPR116. We recapitulate these findings in a model supporting tethered agonist:ECL2 interactions for GPR116 activation
T cells in the skin: lymphoma and inflammatory skin disease
No full textT cells are established contributors to the pathogenesis of atopic dermatitis (AD) and psoriasis, yet whether they are the key drivers or simply unwitting participants remains incompletely understood. Conversely, malignant T cells are the undisputed culprits of cutaneous T cell lymphoma (CTCL), a group of diseases that share key clinical, histopathological and molecular features with ISD. Here, we compare the pathogenesis of ISD and CTCL and discuss the resulting insights. Recurrent, skin-limited disease implicates skin-resident T cells (TRM) in both ISD and CTCL. In CTCL, malignant T cells recruit benign T cells into inflammatory skin lesions, a disease-amplifying function also proposed for pathogenic T cells in ISD. Mechanistically, cytokines produced by malignant T cells in CTCL and by pathogenic T cells in ISD, respectively, are likely both necessary and sufficient to drive skin inflammation and pruritus, which in turn promotes skin barrier dysfunction and dysbiosis. Therapies for ISD target T cell effector functions but do not address the chronicity of disease while treatments for CTCL target malignant T cells but not primarily the symptoms of the disease. By integrating our understanding of ISD and CTCL, important insights into pathogenesis and therapy can be made which may improve the lives of sufferers of both disease groups
Aptamer Proteomics for Biomarker Discovery in Heart Failure with Reduced Ejection Fraction.
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Leveraging patient-centric sampling for clinical drug development and decentralized clinical trials: promise to reality
No full textAs part of drug development, blood samples are often collected from clinical trial participants for assessment of pharmacokinetics (PK) and pharmacodynamics (PD). The current standard practice requires participants to travel to a clinical site to have blood drawn using a needle and vacutainer by a healthcare professional. However, a patient-centric sampling (PCS) approach could improve clinical trial participants’ experience of how, where, or when samples are collected without compromising the objective of trials and quality of bioanalytical data generated from the collected samples. Sample collection can be considered patient-centric if it is less invasive, less painful, requires lower blood volume, or if the collection could occur remotely, i.e. not at the clinical site, via self-collection by the trial participant, or with the help of a caregiver or at-home nurse. This novel approach has been strongly recommended by patient advocacy groups and encouraged as well by health regulatory authorities
Development, Validation, and Application of a Custom-Made Mini-Reaction Calorimeter for Thermal Safety Screening
No full textBefore scaling up to production, it is of high im-portant to evaluate the potential of a reaction of leading to an undesired thermal degradation event. The use of large amount of compounds is usually required for calorimetry study. In this work, we re-port the development of a ml-scale reaction calorim-eter for reaction screening applications. The set-up was designed using lab-equipment and character-ized and validated with routine experiments. The results were accurate that we were able to observe trends in the measured reaction enthalpies depend-ing on the structure of the compounds in reaction with a strong base. The use of the micro scale tailor-made calorimeter is seen as very valuable for poten-tially highly energetic reactions where no data are available and to perform multiple experiments no using considerable amount of valuable material
Automatic system dynamics characterization of a pharmaceutical continuous production line
No full textContinuous Manufacturing (CM) of drug products is a new approach in the pharmaceutical industry. In the presented paper, a GMP continuous wet granulation line for production of solid oral dosage forms was investigated in order to assess the system dynamics of the line and to define the best control and diversion strategy. The following steps were involved in the continuous process: dosing / feeding, blending, twin-screw wet granulation, fluid-bed drying, sieving and tableting. Two drug products with two different drug substances were compared during this study: one drug substance as model drug compound and one formulation of a currently evaluated commercial drug product. Several step tests in API concentration were performed in order to characterize the process flow and assess the process dynamics. API content was monitored in real time by Process Analytical Technologies (PAT) thanks to three Near Infrared (NIR) probes located along the process and measuring the API content after blender, after dryer and in the tablet press feed frame. The process parameter values were changed during production in order to detect the impact on the quality of the final product. An automatic residence time distribution (RTD) computation method has been developed in order automate the RTD calculation on the basis of process data to further define and monitor the system dynamics with the final aim of out of specification material diversion during the continuous production. The RTD has been seen as a process fingerprint: a change in the RTD values implies a change in the process
Models of electroporation and the associated transmembrane molecular transport should be revisited
No full textElectroporation has become a powerful tool for nonviral delivery of various biomolecules such as nucleic acids, proteins, and chemotherapeutic drugs to virtually any living cell by exposing the cell membrane to an intense pulsed electric field. Different multiphysics and multiscale models have been developed to describe the phenomenon of electroporation and predict molecular transport through the electroporated membrane. In this paper, we critically examine the existing mechanistic, single-cell models which allow spatially and temporally resolved numerical simulations of electroporation-induced transmembrane transport of small molecules by confronting them with different experimental measurements. Furthermore, we assess whether any of the proposed models is universal enough to describe the associated transmembrane transport in general for all the different pulse parameters and small molecules used in electroporation applications. We show that none of the tested models can be universally applied to the full range of experimental measurements. Even more importantly, we show that none of the models has been compared to sufficient amount of experimental data to confirm the model validity. Finally, we provide guidelines and recommendations on how to design and report experiments that can be used to validate an electroporation model and how to improve the development of mechanistic models