The development of novel methods for the targeting and manipulation of neural circuits in vivo

Neural networks are at the core of the brain’s ability to compute complex responses to our external environment. Clinically, network dysfunction is emerging as a key component of several psychiatric and neurodegenerative disorders such as Alzheimer’s disease or schizophrenia. However, our ability to precisely and safely manipulate neural networks for research and deliver network-specific therapy remains limited. To address this problem, our lab recently developed a monosynaptically restricted Self-Inactivating Rabies virus (SiR) which enables the targeting of neural circuits without cytotoxicity. To expand the scope of SiR we further developed the technology in two directions: A) By incorporating the CRISPR/CAS9 gene-editing machinery into the SiR genome to successfully edit endogenous loci in vitro and in vivo. B) By designing an improved second generation SiR virus (SiR 2.0) which applies the same SiR technology to a challenge rabies strain (CVS-N2C). SiR 2.0 demonstrates increased neurotropism, increased trans-synaptic transfer efficiency and markedly decreased immunogenicity compared to the SiR 1.0 vector. These advancements expand the scope of SiR viruses to be used in the genome-editing of circuits in vivo. A combined SiR 2.0 CAS9 virus, in physiology, allows us to investigate the roles of genes within circuits in the brain function of live animals. For therapy, it paves the way for the rabies virus’ potential use to edit disease-related genes in dysfunctional circuits. Despite the circuit-basis of many neurological disorders, existing gene therapy vectors are not circuit specific. In addition, the practical difficulties of delivering therapeutic agents at high doses into the central nervous system exacerbates our inability to achieve high therapeutic loads into affected circuits. In contrast, a SiR 2.0 CAS9 virus would, following injection into peripheral organs, trans-synaptically spread into desired circuits of the central nervous system that are affected in neurological disease (e.g. networks demonstrating pathological protein propagation in neurodegenerative disorders) and edit disease-related genes. Lastly, our interest in network-level pathological protein propagation also led us to investigate the biology behind this observation. Due to additional evidence that a significant number of other proteins in physiology also show interneuronal movement, we hypothesised that perhaps this is an overlooked phenomena in neurobiology which could have key implication

Analysis and optimization of equitable US cancer clinical trial center access by travel time

Importance: Racially minoritized and socioeconomically disadvantaged populations are currently underrepresented in clinical trials. Data-driven, quantitative analyses and strategies are required to help address this inequity. Objective: To systematically analyze the geographical distribution of self-identified racial and socioeconomic demographics within commuting distance to cancer clinical trial centers and other hospitals in the US. Design, Setting, and Participants: This longitudinal quantitative study used data from the US Census 2020 Decennial and American community survey (which collects data from all US residents), OpenStreetMap, National Cancer Institute–designated Cancer Centers list, Nature Index of Cancer Research Health Institutions, National Trial registry, and National Homeland Infrastructure Foundation-Level Data. Statistical analyses were performed on data collected between 2006 and 2020. Main Outcomes and Measures: Population distributions of socioeconomic deprivation indices and self-identified race within 30-, 60-, and 120-minute 1-way driving commute times from US cancer trial sites. Map overlay of high deprivation index and high diversity areas with existing hospitals, existing major cancer trial centers, and commuting distance to the closest cancer trial center. Results: The 78 major US cancer trial centers that are involved in 94% of all US cancer trials and included in this study were found to be located in areas with socioeconomically more affluent populations with higher proportions of self-identified White individuals (+10.1% unpaired mean difference; 95% CI, +6.8% to +13.7%) compared with the national average. The top 10th percentile of all US hospitals has catchment populations with a range of absolute sum difference from 2.4% to 35% from one-third each of Asian/multiracial/other (Asian alone, American Indian or Alaska Native alone, Native Hawaiian or Other Pacific Islander alone, some other race alone, population of 2 or more races), Black or African American, and White populations. Currently available data are sufficient to identify diverse census tracks within preset commuting times (30, 60, or 120 minutes) from all hospitals in the US (N = 7623). Maps are presented for each US city above 500 000 inhabitants, which display all prospective hospitals and major cancer trial sites within commutable distance to racially diverse and socioeconomically disadvantaged populations. Conclusion and Relevance: This study identified biases in the sociodemographics of populations living within commuting distance to US-based cancer trial sites and enables the determination of more equitably commutable prospective satellite hospital sites that could be mobilized for enhanced racial and socioeconomic representation in clinical trials. The maps generated in this work may inform the design of future clinical trials or investigations in enrollment and retention strategies for clinical trials; however, other recruitment barriers still need to be addressed to ensure racial and socioeconomic demographics within the geographical vicinity of a clinical site can translate to equitable trial participant representation

Węcowski Marek. Sympozion czyli wspуlne picie. Początki greckiej biesiady arystokratycznej (IX—VII wiek p. n. e.)

Рецензія на: Węcowski Marek. Sympozion czyli wspólne picie. Początki greckiej biesiady arystokratycznej (IX—VII wiek p. n. e.). — (Parnassus. Seria naukowa pod red. dr. M. Staniszewskiego). — Warszawa: wydawnictwo Naukowe Sub Lupa, 2011. — 402 s

Genomic stability of self-inactivating rabies.

Peer reviewed: TrueAcknowledgements: We thank Elena Williams for comments on the manuscript. We thank Jerome Boulanger for writing the script for the 3d-alignment of 2-photon recordings, Nicolas Alexandre for the help with the bioinformatic analysis of the NGS datasets, the Laboratory of Molecular Biology (LMB) workshops for the help with software and hardware development, and members of the Biological Service Group for their support with the in vivo work. This study was supported by the Medical Research Council (MC_UP_1201/2), the European Research Council (STG 677029 to MT), the European Union’s Horizon 2020 research and innovation program with the Marie Sklodowska-Curie fellowship to DdM (894697), the Cambridge Philosophical Society and St. Edmund’s College (University of Cambridge) with the Henslow Research Fellowship to AGR, the Rosetrees Trust with an MBPhD fellowship to HL (M598). For the purpose of open access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising. All data are stored on the LMB server. All materials described in this paper can be obtained upon reasonable request and for non-commercial purposes after signing a material transfer agreement (MTA) with the MRC.Transsynaptic viral vectors provide means to gain genetic access to neurons based on synaptic connectivity and are essential tools for the dissection of neural circuit function. Among them, the retrograde monosynaptic ΔG-Rabies has been widely used in neuroscience research. A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, allows the long term genetic manipulation of neural circuits. However, the high mutational rate of the rabies virus poses a risk that mutations targeting the key genetic regulatory element in the SiR genome could emerge and revert it to a canonical ΔG-Rabies. Such revertant mutations have recently been identified in a SiR batch. To address the origin, incidence and relevance of these mutations, we investigated the genomic stability of SiR in vitro and in vivo. We found that "revertant" mutations are rare and accumulate only when SiR is extensively amplified in vitro, particularly in suboptimal production cell lines that have insufficient levels of TEV protease activity. Moreover, we confirmed that SiR-CRE, unlike canonical ΔG-Rab-CRE or revertant-SiR-CRE, is non-toxic and that revertant mutations do not emerge in vivo during long-term experiments

Genomic stability of self-inactivating rabies

Transsynaptic viral vectors provide means to gain genetic access to neurons based on synaptic connectivity and are essential tools for the dissection of neural circuit function. Among them, the retrograde monosynaptic ΔG-Rabies has been widely used in neuroscience research. A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, allows the long term genetic manipulation of neural circuits. However, the high mutational rate of the rabies virus poses a risk that mutations targeting the key genetic regulatory element in the SiR genome could emerge and revert it to a canonical ΔG-Rabies. Such revertant mutations have recently been identified in a SiR batch. To address the origin, incidence and relevance of these mutations, we investigated the genomic stability of SiR in vitro and in vivo. We found that “revertant” mutations are rare and accumulate only when SiR is extensively amplified in vitro, particularly in suboptimal production cell lines that have insufficient levels of TEV protease activity. Moreover, we confirmed that SiR-CRE, unlike canonical ΔG-Rab-CRE or revertant-SiR-CRE, is non-toxic and that revertant mutations do not emerge in vivo during long-term experiments

Combining long-term circuit mapping and network transcriptomics with SiR-N2c

An exciting frontier in circuit neuroscience lies at the intersection between neural network mapping and single-cell genomics. Monosynaptic rabies viruses provide a promising platform for the merger of circuit mapping methods with -omics approaches. However, three key limitations have hindered the extraction of physiologically meaningful gene expression profiles from rabies-mapped circuits: inherent viral cytotoxicity, high viral immunogenicity and virus-induced alteration of cellular transcriptional regulation. These factors alter the transcriptional and translational profiles of infected neurons and their neighboring cells. To overcome these limitations we applied a self-inactivating genomic modification to the less immunogenic rabies strain, CVS-N2c, to generate a self-inactivating CVS-N2c rabies virus (SiR-N2c). SiR-N2c not only eliminates undesired cytotoxic effects but also substantially reduces gene expression alterations in infected neurons and dampens the recruitment of innate and acquired immune responses, thus enabling open-ended interventions on neural networks and their genetic characterization using single-cell genomic approaches

CamGFR v2: A New Model for Estimating the Glomerular Filtration Rate from Standardized or Non-standardized Creatinine in Patients with Cancer.

PURPOSE: Management of patients with cancer, specifically carboplatin dosing, requires accurate knowledge of glomerular filtration rate (GFR). Direct measurement of GFR is resource limited. Available models for estimated GFR (eGFR) are optimized for patients without cancer and either isotope dilution mass spectrometry (IDMS)- or non-IDMS-standardized creatinine measurements. We present an eGFR model for patients with cancer compatible with both creatinine measurement methods. EXPERIMENTAL DESIGN: GFR measurements, biometrics, and IDMS- or non-IDMS-standardized creatinine values were collected for adult patients from three cancer centers. Using statistical modeling, an IDMS and non-IDMS creatinine-compatible eGFR model (CamGFR v2) was developed. Its performance was compared with that of the existing models Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI), Modification of Diet in Renal Disease (MDRD), Full Age Spectrum (FAS), Lund-Malmö revised, and CamGFR v1, using statistics for bias, precision, accuracy, and clinical robustness. RESULTS: A total of 3,083 IDMS- and 4,612 non-IDMS-standardized creatinine measurements were obtained from 7,240 patients. IDMS-standardized creatinine values were lower than non-IDMS-standardized values in within-center comparisons (13.8% lower in Cambridge; P 20% error of calculated carboplatin dose for IDMS, 0.12 (0.09-0.14) and non-IDMS, 0.17 (0.15-0.2)], and least biased [median residual for IDMS, 0.73 mL/minute (-0.68 to 2.2) and non-IDMS, -0.43 mL/minute (-1.48 to 0.91)] eGFR model, particularly when eGFR was larger than 60 ml/minute. CONCLUSIONS: CamGFR v2 can utilize IDMS- and non-IDMS-standardized creatinine measurements and outperforms previous models. CamGFR v2 should be examined prospectively as a practice-changing standard of care for eGFR-based carboplatin dosing