Biology subjects

Tachida, Y.

Publications and source records attributed to Tachida, Y..

3 recordsLinked to original sources

bioRxiv2024

Systematic empirical evaluation of individual base editing targets: validating therapeutic targets in USH2A and comparison of methods

Base editing shows promise for the correction of human mutations at a higher efficiency than other repair methods and is especially attractive for mutations in large genes that are not amenable to gene augmentation therapy. Here, we demonstrate a comprehensive workflow for in vitro screening of potential therapeutic base editing targets for the USH2A gene and empirically validate the efficiency of adenine and cytosine base editor/guide combinations for correcting 35 USH2A mutations. Editing efficiency and bystander edits are compared between different target templates (plasmids versus transgenes) and assays (Next generation sequencing versus Sanger), as well as comparisons between unbiased empirical results and computational predictions. Based on these observations, practical assay recommendations are discussed. Finally, a humanized knock-in mouse model was created with the best-performing target, the nonsense mutation c.11864G>A p.(Trp3955*). Split-intein AAV9 delivery of editing reagents resulted in the restoration of USH2A protein and a correction rate of 65 {+/-} 3% at the mutant base pair and of 52 {+/-} 3% excluding bystander amino acid changes. This efficiency compares favorably to a prior genome editing strategy tested in the retina that completed a clinical trial and demonstrates the effectiveness of this overall strategy to identify and test base editing reagents with the potential for human therapeutic applications.

genetics↗

bioRxiv2022

Non-classical glycosylation determines intracellular trafficking of APP and Aβ production

A primary pathology of Alzheimers disease (AD) is A{beta} deposition in brain parenchyma and blood vessels, the latter being called cerebral amyloid angiopathy (CAA). Parenchymal amyloid plaques presumably originate from neuronal A{beta} precursor protein (APP), but vascular amyloid deposits origins remain unclear. Endothelial APP expression in APP-knock-in mice was recently shown to expand CAA pathology, highlighting endothelial APPs importance. Furthermore, two types of endothelial APP--with and without O-glycans--have been biochemically identified, but only the former is cleaved for A{beta} production, indicating the critical relationship between APP O-glycosylation and processing. Here, we analyzed APP glycosylation and its intracellular trafficking in neurons and endothelial cells. Although protein glycosylation is generally believed to precede cell surface trafficking, which was true for neuronal APP, we unexpectedly observed that APP lacking O-glycans is externalized to the endothelial cell surface and transported back to the Golgi apparatus, where it then acquires O-glycans. Knockdown of genes encoding enzymes initiating APP O-glycosylation significantly reduced A{beta} production, suggesting this non-classical glycosylation pathway contributes to CAA pathology and is a novel therapeutic target.

neuroscience↗

bioRxiv2020

Endothelial expression of human APP leads to cerebral amyloid angiopathy in mice

The deposition of amyloid {beta} (A{beta}) in blood vessels of the brain, known as cerebral amyloid angiopathy (CAA), is observed in more than 90% of Alzheimers disease (AD) patients. The presence of such CAA pathology is not as evident, however, in most mouse models of AD, thereby making it difficult to examine the contribution of CAA to the pathogenesis of AD. Since blood levels of soluble amyloid precursor protein (sAPP) in rodents are less than 1% of those in humans, we hypothesized that endothelial APP expression would be markedly lower in rodents, thus providing a reason for the poorly expressed CAA pathology. Here we generated mice that specifically express human APP770 in endothelial cells. These mice exhibited an age-dependent robust deposition of A{beta} in brain blood vessels but not in the parenchyma. Crossing these animals with APP knock-in mice led to an expanded CAA pathology as evidenced by increased amounts of amyloid accumulated in the cortical blood vessels. These results show that both neuronal and endothelial APP contribute cooperatively to vascular A{beta} deposition, and suggest that this mouse model will be useful for studying disease mechanisms underlying CAA and for developing novel AD therapeutics.

neuroscience↗