Biology subjects

Soares, C.

Publications and source records attributed to Soares, C..

5 recordsLinked to original sources

bioRxiv2024

Evaluation of subretinally delivered Cas9 ribonucleoproteins in murine and porcine animal models highlights key considerations for therapeutic translation of genetic medicines

Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species. Editing occurs in retinal pigmented epithelium (RPE) and photoreceptor cells, with favorable tolerability in both species. Using transgenic reporter strains, we determine that editing primarily occurs close to the site of administration, within the bleb region associated with subretinal injection. Our results show that subretinal administration of eRNPs in mice mediates base editing of up to 12% of the total neural retina, with an average rate of 7% observed at the highest dose tested. In contrast, a substantially lower editing efficiency was observed in minipigs; even with direct quantification of only the treated region, a maximum base editing rate of 1.5%, with an average rate of <1%, was observed. Our data highlight the importance of species consideration in translational studies for genetic medicines targeting the eye and provide an example of a lack of translation between small and larger animal models in the context of subretinal administration of Cas9 eRNPs.

pharmacology and toxicology↗

bioRxiv2021

SARS-CoV-2 variants impact RBD conformational dynamics and ACE2 accessibility

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has killed over 5 million people and is causing a devastating social and economic impact all over the world. The rise of new variants of concern (VOCs) represents a difficult challenge due to the loss vaccine and natural immunity, and increased transmissibility. All circulating VOCs contain mutations in the spike glycoprotein, which mediates fusion between the viral and host cell membranes, via its receptor binding domain (RBD) that binds to angiotensin-converting enzyme 2 (ACE2). In an attempt to understand the effect of RBD mutations in circulating VOCs, a lot of attention has been given to the RBD-ACE2 interaction. However, this type of analysis is limited, since it ignores more indirect effects, such as the conformational dynamics of the RBD itself. Observing that some VOCs mutations occur in residues that are not in direct contact with ACE2, we hypothesized that they could affect RBD conformational dynamics. To test this, we performed long atomistic (AA) molecular dynamics (MD) simulations to investigate the structural dynamics of wt RBD, and that of three circulating VOCs (alpha, beta, and delta). Our results show that in solution, wt RBD presents two distinct conformations: an "open" conformation where it is free to bind ACE2; and a "closed" conformation, where the RBM ridge blocks the binding surface. The alpha and beta variants significantly impact the open/closed equilibrium, shifting it towards the open conformation by roughly 20%. This shift likely increases ACE2 binding affinity. Simulations of the currently predominant delta variant RBD were extreme in this regard, in that a closed conformation was never observed. Instead, the system alternated between the before mentioned open conformation and an alternative "reversed" one, with a significantly changed orientation of the RBM ridge flanking the RBD. This alternate conformation could potentially provide a fitness advantage not only due to increased availability for ACE2 binding, but also by aiding antibody escape through epitope occlusion. These results support the hypothesis that VOCs, and particularly the delta variant, impact RBD conformational dynamics in a direction that simultaneously promotes efficient binding to ACE2 and antibody escape.

biophysics↗

bioRxiv2021

Amino acids 484 and 494 of SARS-CoV-2 spike are hotspots of immune evasion affecting antibody but not ACE2 binding

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, with the effect of mutation synergy still incompletely understood. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations, and confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and N501Y. Our analysis of synergic mutations provides a landscape for hotspots for immune evasion and for targets for therapies, vaccines and diagnostics. One-Sentence SummaryAmino acids in SARS-CoV-2 spike protein implicated in immune evasion are biased for binding to neutralizing antibodies but dispensable for binding the host receptor angiotensin-converting enzyme

immunology↗

bioRxiv2019

A synthetic likelihood solution to the silent synapse estimation problem

Functional features of populations of synapses are typically inferred from random electrophysiological sampling of small subsets of synapses. Are these samples unbiased? Here, we developed a biophysically constrained statistical framework for addressing this question and applied it to assess the performance of a widely used method based on a failure-rate analysis to quantify the occurrence of silent (AMPAR-lacking) synapses in neural networks. We simulated this method in silico and found that it is characterized by strong and systematic biases, poor reliability and weak statistical power. Key conclusions were validated by whole-cell recordings from hippocampal neurons. To address these shortcomings, we developed a simulator of the experimental protocol and used it to compute a synthetic likelihood. By maximizing the likelihood, we inferred silent synapse fraction with no bias, low variance and superior statistical power over alternatives. Together, this generalizable approach highlights how a simulator of experimental methodologies can substantially improve the estimation of physiological properties.

neuroscience↗

bioRxiv2019

Parsing out the variability of transmission at central synapses using optical quantal analysis

Properties of synaptic release dictates the core of information transfer in neural circuits. Despite decades of technical and theoretical advances, distinguishing bona fide information content from the multiple sources of synaptic variability remains a challenging problem. Here, we employed a combination of computational approaches with cellular electrophysiology, two-photon uncaging of MNI-Glutamate and imaging at single synapses. We describe and calibrate the use of the fluorescent glutamate sensor iGluSnFR and found that its kinetic profile is close to that of AMPA receptors, therefore providing several distinct advantages over slower methods relying on NMDA receptor activation (i.e., chemical or genetically encoded Calcium indicators). Using an array of statistical methods, we further developed, and validated on surrogate data, an expectation-maximization algorithm that, by biophysically constraining release variability, extracts the quantal parameters n (maximum number of released vesicles) and p (unitary probability of release) from single-synapse iGluSnFR-mediated transients. Together, we present a generalizable mathematical formalism which, when applied to optical recordings, paves the way to an increasingly precise investigation of information transfer at central synapses.

neuroscience↗