DeePhys: A machine learning-assisted platform for electrophysiological phenotyping of human neuronal networks.

Reproducible functional assays to study in vitro neuronal networks represent an important cornerstone in the quest to develop physiologically relevant cellular models of human diseases. Here, we introduce DeePhys, a MATLAB-based analysis tool for data-driven functional phenotyping of in vitro neuronal cultures recorded by high-density microelectrode arrays. DeePhys is a modular workflow that offers a range of techniques to extract features from spike-sorted data, allowing for the examination of functional phenotypes both at the individual cell and network levels, as well as across development. In addition, DeePhys incorporates the capability to integrate novel features and to use machine-learning-assisted approaches, which facilitates a comprehensive evaluation of pharmacological interventions. To illustrate its practical application, we apply DeePhys to human induced pluripotent stem cell-derived dopaminergic neurons obtained from both patients and healthy individuals and showcase how DeePhys enables phenotypic screenings.

Evaluation of Cellular Immune Response to Adeno-Associated Virus-Based Gene Therapy.

The number of approved or investigational late phase viral vector gene therapies (GTx) has been rapidly growing. The adeno-associated virus vector (AAV) technology continues to be the most used GTx platform of choice. The presence of pre-existing anti-AAV immunity has been firmly established and is broadly viewed as a potential deterrent for successful AAV transduction with a possibility of negative impact on clinical efficacy and a connection to adverse events. Recommendations for the evaluation of humoral, including neutralizing and total antibody based, anti-AAV immune response have been presented elsewhere. This manuscript aims to cover considerations related to the assessment of anti-AAV cellular immune response, including review of correlations between humoral and cellular responses, potential value of cellular immunogenicity assessment, and commonly used analytical methodologies and parameters critical for monitoring assay performance. This manuscript was authored by a group of scientists involved in GTx development who represent several pharma and contract research organizations. It is our intent to provide recommendations and guidance to the industry sponsors, academic laboratories, and regulatory agencies working on AAV-based GTx viral vector modalities with the goal of achieving a more consistent approach to anti-AAV cellular immune response assessment.

2021 White Paper on Recent Issues in Bioanalysis: TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccine Assays; Immunogenicity of Biotherapeutics and Novel Modalities; Integrated Summary of Immunogenicity Harmonization).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.

Characterization of AAV-mediated dorsal root ganglionopathy.

Recent studies in non-human primates administered recombinant adeno-associated viruses (rAAVs) have shown lesions in the dorsal root ganglia (DRG) of unknown pathogenesis. In this study, rAAV9s manufactured using different purification methods alongside a non-expressing Null AAV9 vector was administered to groups of cynomolgus monkeys followed by neuropathological evaluation after 4 weeks. Lesions, including neuronal degeneration, increased cellularity, and nerve fiber degeneration, were observed in the DRG, regardless of purification methods. Animals did not develop any neurological signs throughout the study, and there was no loss of function observed in neuro-electrophysiological endpoints or clear effects on intraepidermal nerve fiber density. However, magnetic resonance imaging (MRI) of animals with axonopathy showed an increase in short tau inversion recovery (STIR) intensity and decrease in fractional anisotropy. In animals administered the Null AAV9 vector, DRG lesions were not observed despite vector DNA being detected in the DRG at levels equivalent to or greater than rAAV9-treated animals. This study further supports that DRG toxicity is associated with transgene overexpression in DRGs, with particular sensitivity at the lumbar and lumbosacral level. The data from this study also showed that the nerve fiber degeneration did not correlate with any functional effect on nerve conduction but was detectable by MRI.

Electrophysiological Phenotype Characterization of Human iPSC-Derived Neuronal Cell Lines by Means of High-Density Microelectrode Arrays.

Recent advances in the field of cellular reprogramming have opened a route to studying the fundamental mechanisms underlying common neurological disorders. High-density microelectrode-arrays (HD-MEAs) provide unprecedented means to study neuronal physiology at different scales, ranging from network through single-neuron to subcellular features. In this work, HD-MEAs are used in vitro to characterize and compare human induced-pluripotent-stem-cell-derived dopaminergic and motor neurons, including isogenic neuronal lines modeling Parkinson's disease and amyotrophic lateral sclerosis. Reproducible electrophysiological network, single-cell and subcellular metrics are used for phenotype characterization and drug testing. Metrics, such as burst shape and axonal velocity, enable the distinction of healthy and diseased neurons. The HD-MEA metrics can also be used to detect the effects of dosing the drug retigabine to human motor neurons. Finally, it is shown that the ability to detect drug effects and the observed culture-to-culture variability critically depend on the number of available recording electrodes.

Versatile live-cell activity analysis platform for characterization of neuronal dynamics at single-cell and network level.

Chronic imaging of neuronal networks in vitro has provided fundamental insights into mechanisms underlying neuronal function. Current labeling and optical imaging methods, however, cannot be used for continuous and long-term recordings of the dynamics and evolution of neuronal networks, as fluorescent indicators can cause phototoxicity. Here, we introduce a versatile platform for label-free, comprehensive and detailed electrophysiological live-cell imaging of various neurogenic cells and tissues over extended time scales. We report on a dual-mode high-density microelectrode array, which can simultaneously record in (i) full-frame mode with 19,584 recording sites and (ii) high-signal-to-noise mode with 246 channels. We set out to demonstrate the capabilities of this platform with recordings from primary and iPSC-derived neuronal cultures and tissue preparations over several weeks, providing detailed morpho-electrical phenotypic parameters at subcellular, cellular and network level. Moreover, we develop reliable analysis tools, which drastically increase the throughput to infer axonal morphology and conduction speed.

Correction to: Recommendations for the Development of Cell-Based Anti-Viral Vector Neutralizing Antibody Assays.

The first author's name was published incorrectly as "Gorovits Boris". The correct name is "Boris Gorovits".

Recommendations for the Development of Cell-Based Anti-Viral Vector Neutralizing Antibody Assays.

Viral vector-based gene therapies (GTx) have received significant attention in the recent years and the number of ongoing GTx clinical trials is increasing. A platform of choice for many of these studies is adeno-associated virus (AAV). All humans may be exposed to natural AAV infections and could mount an immune response against the virus. Consequently, there can be a high prevalence of pre-existing anti-AAV immunity. This presents a potential limitation for AAV-based GTx due to the potential for AAV-specific antibodies to reduce the efficacy of the GTx. Therefore, appropriate assessment of potential subjects enrolled in these studies should include evaluation for the presence and degree of anti-AAV immunity, including anti-AAV neutralizing antibodies (NAb). Recommendations for the development and validation of cell-based anti-AAV NAb detection methods, including considerations related to selection of appropriate cell line, surrogate vector/reporter gene, assay matrix and controls, and methodologies for calculating assay cut-point are discussed herein. General recommendations for the key assay validation parameters are provided as well as considerations for the development of NAb diagnostic tests. This manuscript is produced by a group of scientists involved in GTx therapeutic development representing various companies. It is our intent to provide recommendations and guidance to industrial and academic laboratories working on viral vector based GTx modalities with the goal of achieving a more consistent approach to anti-AAV NAb assessment.

AAV8-vectored suprachoroidal gene transfer produces widespread ocular transgene expression.

There has been great progress in ocular gene therapy, but delivery of viral vectors to the retinal pigmented epithelium (RPE) and retina can be challenging. Subretinal injection, the preferred route of delivery for most applications, requires a surgical procedure that has risks. Herein we report a novel gene therapy delivery approach, suprachoroidal injection of AAV8 vectors, which is less invasive and could be done in an outpatient setting. Two weeks after suprachoroidal injection of AAV8.GFP in rats, GFP fluorescence covered 18.9% of RPE flat mounts and extended entirely around sagittal and transverse sections in RPE and photoreceptors. After 2 suprachoroidal injections of AAV8.GFP, GFP fluorescence covered 30.5% of RPE flat mounts. Similarly, widespread expression of GFP occurred in nonhuman primate and pig eyes after suprachoroidal injection of AAV8.GFP. Compared with subretinal injection in rats of RGX-314, an AAV8 vector expressing an anti-VEGF Fab, suprachoroidal injection of the same dose of RGX-314 resulted in similar expression of anti-VEGF Fab and similar suppression of VEGF-induced vascular leakage. Suprachoroidal AAV8 vector injection provides a noninvasive outpatient procedure to obtain widespread transgene expression in retina and RPE.

Single-Cell Electrical Stimulation Using CMOS-Based High-Density Microelectrode Arrays.

Non-invasive electrical stimulation can be used to study and control neural activity in the brain or to alleviate somatosensory dysfunctions. One intriguing prospect is to precisely stimulate individual targeted neurons. Here, we investigated single-neuron current and voltage stimulation in vitro using high-density microelectrode arrays featuring 26,400 bidirectional electrodes at a pitch of 17.5 µm and an electrode area of 5 × 9 µm2. We determined optimal waveforms, amplitudes and durations for both stimulation modes. Owing to the high spatial resolution of our arrays and the close proximity of the electrodes to the respective neurons, we were able to stimulate the axon initial segments (AIS) with charges of less than 2 pC. This resulted in minimal artifact production and reliable readout of stimulation efficiency directly at the soma of the stimulated cell. Stimulation signals as low as 70 mV or 100 nA, with pulse durations as short as 18 µs, yielded measurable action potential initiation and propagation. We found that the required stimulation signal amplitudes decreased with cell growth and development and that stimulation efficiency did not improve at higher electric fields generated by simultaneous multi-electrode stimulation.

Automatic spike sorting for high-density microelectrode arrays.

High-density microelectrode arrays can be used to record extracellular action potentials from hundreds to thousands of neurons simultaneously. Efficient spike sorters must be developed to cope with such large data volumes. Most existing spike sorting methods for single electrodes or small multielectrodes, however, suffer from the "curse of dimensionality" and cannot be directly applied to recordings with hundreds of electrodes. This holds particularly true for the standard reference spike sorting algorithm, principal component analysis-based feature extraction, followed by k-means or expectation maximization clustering, against which most spike sorters are evaluated. We present a spike sorting algorithm that circumvents the dimensionality problem by sorting local groups of electrodes independently with classical spike sorting approaches. It is scalable to any number of recording electrodes and well suited for parallel computing. The combination of data prewhitening before the principal component analysis-based extraction and a parameter-free clustering algorithm obviated the need for parameter adjustments. We evaluated its performance using surrogate data in which we systematically varied spike amplitudes and spike rates and that were generated by inserting template spikes into the voltage traces of real recordings. In a direct comparison, our algorithm could compete with existing state-of-the-art spike sorters in terms of sensitivity and precision, while parameter adjustment or manual cluster curation was not required. NEW & NOTEWORTHY We present an automatic spike sorting algorithm that combines three strategies to scale classical spike sorting techniques for high-density microelectrode arrays: 1) splitting the recording electrodes into small groups and sorting them independently; 2) clustering a subset of spikes and classifying the rest to limit computation time; and 3) prewhitening the spike waveforms to enable the use of parameter-free clustering. Finally, we combined these strategies into an automatic spike sorter that is competitive with state-of-the-art spike sorters.

Causal evidence for retina-dependent and -independent visual motion computations in mouse cortex.

How neuronal computations in the sensory periphery contribute to computations in the cortex is not well understood. We examined this question in the context of visual-motion processing in the retina and primary visual cortex (V1) of mice. We disrupted retinal direction selectivity, either exclusively along the horizontal axis using FRMD7 mutants or along all directions by ablating starburst amacrine cells, and monitored neuronal activity in layer 2/3 of V1 during stimulation with visual motion. In control mice, we found an over-representation of cortical cells preferring posterior visual motion, the dominant motion direction an animal experiences when it moves forward. In mice with disrupted retinal direction selectivity, the over-representation of posterior-motion-preferring cortical cells disappeared, and their responses at higher stimulus speeds were reduced. This work reveals the existence of two functionally distinct, sensory-periphery-dependent and -independent computations of visual motion in the cortex.

The 10th GCC Closed Forum: rejected data, GCP in bioanalysis, extract stability, BAV, processed batch acceptance, matrix stability, critical reagents, ELN and data integrity and counteracting fraud.

The 10th Global CRO Council (GCC) Closed Forum was held in Orlando, FL, USA on 18 April 2016. In attendance were decision makers from international CRO member companies offering bioanalytical services. The objective of this meeting was for GCC members to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at this closed forum included reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, biomarker assay validation, processed batch acceptance criteria, electronic laboratory notebooks and data integrity, Health Canada's Notice regarding replicates in matrix stability evaluations, critical reagents and regulatory approaches to counteract fraud. In order to obtain the pharma perspectives on some of these topics, the first joint CRO-Pharma Scientific Interchange Meeting was held on 12 November 2016, in Denver, Colorado, USA. The five topics discussed at this Interchange meeting were reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, processed batch acceptance criteria and electronic laboratory notebooks and data integrity. The conclusions from the discussions of these topics at both meetings are included in this report.

9th GCC closed forum: CAPA in regulated bioanalysis; method robustness, biosimilars, preclinical method validation, endogenous biomarkers, whole blood stability, regulatory audit experiences and electronic laboratory notebooks.

The 9th GCCClosed Forum was held just prior to the 2015 Workshop on Recent Issues in Bioanalysis (WRIB) in Miami, FL, USA on 13 April 2015. In attendance were 58 senior-level participants, from eight countries, representing 38 CRO companies offering bioanalytical services. The objective of this meeting was for CRO bioanalytical representatives to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues selected at this year's closed forum include CAPA, biosimilars, preclinical method validation, endogenous biomarkers, whole blood stability, and ELNs. A summary of the industry's best practices and the conclusions from the discussion of these topics is included in this meeting report.

Structures of Neural Correlation and How They Favor Coding.

The neural representation of information suffers from "noise"-the trial-to-trial variability in the response of neurons. The impact of correlated noise upon population coding has been debated, but a direct connection between theory and experiment remains tenuous. Here, we substantiate this connection and propose a refined theoretical picture. Using simultaneous recordings from a population of direction-selective retinal ganglion cells, we demonstrate that coding benefits from noise correlations. The effect is appreciable already in small populations, yet it is a collective phenomenon. Furthermore, the stimulus-dependent structure of correlation is key. We develop simple functional models that capture the stimulus-dependent statistics. We then use them to quantify the performance of population coding, which depends upon interplays of feature sensitivities and noise correlations in the population. Because favorable structures of correlation emerge robustly in circuits with noisy, nonlinear elements, they will arise and benefit coding beyond the confines of retina.

Congenital Nystagmus Gene FRMD7 Is Necessary for Establishing a Neuronal Circuit Asymmetry for Direction Selectivity.

Neuronal circuit asymmetries are important components of brain circuits, but the molecular pathways leading to their establishment remain unknown. Here we found that the mutation of FRMD7, a gene that is defective in human congenital nystagmus, leads to the selective loss of the horizontal optokinetic reflex in mice, as it does in humans. This is accompanied by the selective loss of horizontal direction selectivity in retinal ganglion cells and the transition from asymmetric to symmetric inhibitory input to horizontal direction-selective ganglion cells. In wild-type retinas, we found FRMD7 specifically expressed in starburst amacrine cells, the interneuron type that provides asymmetric inhibition to direction-selective retinal ganglion cells. This work identifies FRMD7 as a key regulator in establishing a neuronal circuit asymmetry, and it suggests the involvement of a specific inhibitory neuron type in the pathophysiology of a neurological disease.

A method for electrophysiological characterization of hamster retinal ganglion cells using a high-density CMOS microelectrode array.

Knowledge of neuronal cell types in the mammalian retina is important for the understanding of human retinal disease and the advancement of sight-restoring technology, such as retinal prosthetic devices. A somewhat less utilized animal model for retinal research is the hamster, which has a visual system that is characterized by an area centralis and a wide visual field with a broad binocular component. The hamster retina is optimally suited for recording on the microelectrode array (MEA), because it intrinsically lies flat on the MEA surface and yields robust, large-amplitude signals. However, information in the literature about hamster retinal ganglion cell functional types is scarce. The goal of our work is to develop a method featuring a high-density (HD) complementary metal-oxide-semiconductor (CMOS) MEA technology along with a sequence of standardized visual stimuli in order to categorize ganglion cells in isolated Syrian Hamster (Mesocricetus auratus) retina. Since the HD-MEA is capable of recording at a higher spatial resolution than most MEA systems (17.5 µm electrode pitch), we were able to record from a large proportion of RGCs within a selected region. Secondly, we chose our stimuli so that they could be run during the experiment without intervention or computation steps. The visual stimulus set was designed to activate the receptive fields of most ganglion cells in parallel and to incorporate various visual features to which different cell types respond uniquely. Based on the ganglion cell responses, basic cell properties were determined: direction selectivity, speed tuning, width tuning, transience, and latency. These properties were clustered to identify ganglion cell types in the hamster retina. Ultimately, we recorded up to a cell density of 2780 cells/mm(2) at 2 mm (42°) from the optic nerve head. Using five parameters extracted from the responses to visual stimuli, we obtained seven ganglion cell types.

Visual coding with a population of direction-selective neurons.

The brain decodes the visual scene from the action potentials of ∼20 retinal ganglion cell types. Among the retinal ganglion cells, direction-selective ganglion cells (DSGCs) encode motion direction. Several studies have focused on the encoding or decoding of motion direction by recording multiunit activity, mainly in the visual cortex. In this study, we simultaneously recorded from all four types of ON-OFF DSGCs of the rabbit retina using a microelectronics-based high-density microelectrode array (HDMEA) and decoded their concerted activity using probabilistic and linear decoders. Furthermore, we investigated how the modification of stimulus parameters (velocity, size, angle of moving object) and the use of different tuning curve fits influenced decoding precision. Finally, we simulated ON-OFF DSGC activity, based on real data, in order to understand how tuning curve widths and the angular distribution of the cells' preferred directions influence decoding performance. We found that probabilistic decoding strategies outperformed, on average, linear methods and that decoding precision was robust to changes in stimulus parameters such as velocity. The removal of noise correlations among cells, by random shuffling trials, caused a drop in decoding precision. Moreover, we found that tuning curves are broad in order to minimize large errors at the expense of a higher average error, and that the retinal direction-selective system would not substantially benefit, on average, from having more than four types of ON-OFF DSGCs or from a perfect alignment of the cells' preferred directions.

Recommendations for the development and validation of confirmatory anti-drug antibody assays.

Identification and characterization of anti-drug antibodies is a critical component of biopharmaceutical drug development. The tiered approach for immunogenicity testing consists of screening, confirmatory, and characterization assays. Herein, we provide recommendations for confirmatory assays by expanding upon published guidance and present common practices across the industry. The authors recommend scientific approaches for development and validation of confirmatory assays using competition methods in ligand-binding assays, along with statistical formulae for routine use and validation. The paper will assist in understanding the confirmatory assay, and carefully implementing validation criteria a priori, as well as during sample analysis. These approaches represent the authors' current knowledge and practices, with the aim that more uniform practices will be applied across the industry.

A network comprising short and long noncoding RNAs and RNA helicase controls mouse retina architecture.

Brain regions, such as the cortex and retina, are composed of layers of uniform thickness. The molecular mechanism that controls this uniformity is not well understood. Here we show that during mouse postnatal development the timed expression of Rncr4, a retina-specific long noncoding RNA, regulates the similarly timed processing of pri-miR-183/96/182, which is repressed at an earlier developmental stage by RNA helicase Ddx3x. Shifting the timing of mature miR-183/96/182 accumulation or interfering with Ddx3x expression leads to the disorganization of retinal architecture, with the photoreceptor layer being most affected. We identify Crb1, a component of the adhesion belt between glial and photoreceptor cells, as a link between Rncr4-regulated miRNA metabolism and uniform retina layering. Our results suggest that the precise timing of glia-neuron interaction controlled by noncoding RNAs and Ddx3x is important for the even distribution of cells across layers.