Biology subjects

Shay, T. F.

Publications and source records attributed to Shay, T. F..

5 recordsLinked to original sources

bioRxiv2024

Adeno-Associated Viral Tools to Trace Neural Development and Connectivity Across Amphibians

The development, evolution, and function of the vertebrate central nervous system (CNS) can be best studied using diverse model organisms. Amphibians, with their unique phylogenetic position at the transition between aquatic and terrestrial lifestyles, are valuable for understanding the origin and evolution of the tetrapod brain and spinal cord. Their metamorphic developmental transitions and unique regenerative abilities also facilitate the discovery of mechanisms for neural circuit remodeling and replacement. The genetic toolkit for amphibians, however, remains limited, with only a few species having sequenced genomes and a small number of transgenic lines available. In mammals, recombinant adeno-associated viral vectors (AAVs) have become a powerful alternative to genome modification for visualizing and perturbing the nervous system. AAVs are DNA viruses that enable neuronal transduction in both developing and adult animals with low toxicity and spatial, temporal, and cell-type specificity. However, AAVs have never been shown to transduce amphibian cells efficiently. To bridge this gap, we established a simple, scalable, and robust strategy to screen AAV serotypes in three distantly-related amphibian species: the frogs Xenopus laevis and Pelophylax bedriagae, and the salamander Pleurodeles waltl, in both developing larval tadpoles and post-metamorphic animals. For each species, we successfully identified at least two AAV serotypes capable of infecting the CNS; however, no pan-amphibian serotype was identified, indicating rapid evolution of AAV tropism. In addition, we developed an AAV-based strategy that targets isochronic cohorts of developing neurons - a critical tool for parsing neural circuit assembly. Finally, to enable visualization and manipulation of neural circuits, we identified AAV variants for retrograde tracing of neuronal projections in adult animals. Our findings expand the toolkit for amphibians to include AAVs, establish a generalizable workflow for AAV screening in non-canonical research organisms, generate testable hypotheses for the evolution of AAV tropism, and lay the foundation for modern cross-species comparisons of vertebrate CNS development, function, and evolution.

neuroscience↗

bioRxiv2024

Human cell surface-AAV interactomes identify LRP6 as blood-brain-barrier transcytosis receptor and immune cytokine IL3 as AAV9 binder

Adeno-associated viruses (AAVs) are foundational gene delivery tools for basic science and clinical therapeutics. However, lack of mechanistic insight, especially for engineered vectors created by directed evolution, can hamper their application. Here, we adapted an unbiased human cell microarray platform to determine the extracellular and cell surface interactomes of natural and engineered AAVs. We identified a naturally-evolved and serotype-specific interaction between the AAV9 capsid and human interleukin 3 (IL3), with possible roles in host immune modulation, as well as lab-evolved low-density-lipoprotein-receptor-related-protein 6 (LRP6) interactions specific to engineered capsids that cross the blood-brain barrier in non-human primates after intravenous administration. The unbiased cell microarray screening approach also allowed us to identify off-target tissue binding interactions of engineered brain-enriched AAV capsids that may inform vectors peripheral organ tropism and side effects. These results allow confident application of engineered AAVs in diverse organisms and unlock future target-informed engineering of improved viral and non-viral vectors for non-invasive therapeutic delivery to the brain.

bioengineering↗

bioRxiv2023

APPRAISE: Fast, accurate ranking of engineered proteins by receptor binding propensity using structural modeling

Deep learning-based methods for protein structure prediction have achieved unprecedented accuracy. However, the power of these tools to guide the engineering of protein-based therapeutics remains limited due to a gap between the ability to predict the structures of candidate proteins and the ability to assess which of those proteins are most likely to bind to a target receptor. Here we bridge this gap by introducing Automated Pairwise Peptide-Receptor AnalysIs for Screening Engineered proteins (APPRAISE), a method for predicting the receptor binding propensity of engineered proteins. After generating models of engineered proteins competing for binding to a target using an established structure-prediction tool such as AlphaFold-Multimer or ESM-Fold, APPRAISE performs a rapid (under 1 CPU second per model) scoring analysis that takes into account biophysical and geometrical constraints. As a proof-of-concept, we demonstrate that APPRAISE can accurately classify receptor-dependent vs. receptor-independent adeno-associated viral vectors and diverse classes of engineered proteins such as miniproteins targeting the SARS-CoV-2 spike, nanobodies targeting a G-protein-coupled receptor, and peptides that specifically bind to transferrin receptor or PD-L1. APPRAISE is accessible through a web-based notebook interface using Google Colaboratory (https://tiny.cc/APPRAISE). With its accuracy, interpretability, and generalizability, APPRAISE promises to expand the utility of protein structure prediction and accelerate protein engineering for biomedical applications.

bioengineering↗

bioRxiv2023

Primate-conserved Carbonic Anhydrase IV and murine-restricted Ly6c1 are new targets for crossing the blood-brain barrier

The blood-brain barrier (BBB) presents a major challenge to delivering large molecules to study and treat the central nervous system (CNS). This is due in part to the scarcity of effective targets for BBB crossing, the identification of which is the crucial first step of drug development. Here, we leveraged a panel of adeno-associated viruses (AAVs) previously identified through directed evolution for improved BBB transport to reverse engineer protein targets for enhanced BBB crossing. We identify both murine-restricted Ly6c1 and primate-conserved carbonic anhydrase IV (Car4; CA4) as novel receptors for crossing the BBB. We demonstrate how these receptors can unlock new experimental and computational target-focused engineering strategies by creating the enhanced Ly6c1-binding vector AAV-PHP.eC and by applying AlphaFold2-enabled in silico methods to rank capsids against identified receptors and generate capsid-receptor binding models. Here, with Car4, we add a completely new receptor to the very short list currently available for crossing the BBB in humans and, with Ly6c1, we validate a pipeline for receptor-targeted engineering. The identification of Car4/CA4 and structural insights from computational modeling provide new paths toward human brain-penetrant chemicals (drugs) and biologicals (including gene delivery).

bioengineering↗

bioRxiv2023

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds and rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and ex vivo human brain slices although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. Vasculature-secreted Hevin (a synaptogenic protein) rescued synaptic deficits in a mouse model.

bioengineering↗