Biology subjects

Amin, P.

Publications and source records attributed to Amin, P..

6 recordsLinked to original sources

bioRxiv2026

Syndecan-4 exerts canonical heparan sulfate-dependent and noncanonical heparan sulfate-independent functions that regulate Aβ amyloid homeostasis

Background Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) are components of the amyloid deposits in Alzheimers disease (AD) and other amyloidoses. HS and HSPGs are canonically thought to facilitate amyloid deposition by accelerating the aggregation of amyloidogenic proteins and impairing their clearance in a HS-dependent manner. Methods Leveraging insights from large-scale proteomic data, we focused on Syndecan-4 (Sdc4), the most increased transmembrane HSPG in the AD brain and in the brain of A{beta} amyloid depositing mice. We used proximity ligation assays (PLA) to evaluate the association of Sdc4 with A{beta} in situ and assessed the impacts of the Sdc4 ectodomain on A{beta} aggregation in vitro. Overexpression studies in cells, hiPSC-derived neurons, and mouse organotypic brain slice cultures (OBSCs) coupled with structure-function studies were used to investigate impacts on A{beta} production and APP processing. Finally, effects of overexpression of Sdc4 in vivo in the CRND8 amyloid deposition model were evaluated. Results Consistent with canonical roles, PLA demonstrated a spatial association of Sdc4 with amyloid deposits, and in vitro, the Sdc4 ectodomain accelerated A{beta} fibril formation in a HS-dependent manner. Unexpectedly, Sdc4 overexpression reduced A{beta} production in CHO cells, hiPSC-derived neurons, and OBSCs. These effects were accompanied by dramatic decreases in the levels of sAPP and C83 and increased immature APP in the cell. Sdc4 promoted altered APP localization into detergent resistant membrane domains and increased APP association with ATG5+/LC3+/Cathepsin D+ vesicles. Structure-function studies revealed that the transmembrane region mediates these effects in a glycosaminoglycan-independent manner. Sdc4 overexpression in the brain of APP mice significantly reduced amyloid deposition at an early age. Conclusions Sdc4 exerts paradoxical and mechanistically distinct effects that could impact AD pathogenesis differentially, potentially promoting A{beta} fibrillization extracellularly while suppressing APP processing and A{beta} production. Such data challenge the prevailing view that increased levels of HSPGs in AD are always pro-amyloidogenic and identify Sdc4 as a previously unrecognized regulator of amyloid homeostasis in AD.

neuroscience↗

bioRxiv2024

Breaking Free: Development of Circular AAV Cargos for Targeted Seamless Integration in the Liver

Recent advancements in gene insertion have shifted from DNA repair-dependent mechanisms to more precise approaches, enhancing safety and predictability for editing outcomes. Integrase-mediated programmable genomic integration (I-PGI) utilizes a DNA cargo to insert transgenes in a targeted, unidirectional manner. In vivo, where nuclear delivery of DNA is challenging, adeno-associated virus (AAV) can act as the cargo vector. While I-PGI does not require DNA double-stranded breaks (DSBs) for activity, linear DNA cargo, like AAV, stimulates DNA end joining activity after integration. To mitigate potential risks from DSBs with linear viral cargo, we developed two circular genome types capable of seamless gene insertion in non-dividing cells. We first harnessed the orthogonal property of large serine integrases to produce circle-AAV (cAAV) from linear viral genomes in cells. cAAV demonstrated faithful seamless cargo integration in primary human hepatocytes (PHH) and robust DSB-free insertion structures in vivo. We then investigated the delivery of packaged circular AAV cargo (AAV.AD), which eliminates the need for enzymatic manipulation in the cell. AAV.AD proved to be a viable cargo for I-PGI, exhibiting functional integration in PHH and in vivo, that resulted in seamless insertion structures. Together, these findings provide the first reported evidence of DSB-free programmable genomic integration using integrase and AAV cargo, addressing a previously unrecognized challenge in the field.

bioengineering↗

bioRxiv2024

Curative levels of endogenous gene replacement achieved in non-human primate liver using programmable genomic integration

The ability to efficiently place a large piece of DNA in a specific genomic location has been a goal for the gene therapy field since its inception; however, despite significant advances in gene editing technology, this had yet to be achieved. Here we describe two methods of programmable genomic integration (PGI) that overcome some of the limitations of current approaches. Using a combination of clinically validated delivery technologies (LNP, AAV), we demonstrate the ability to specifically integrate large (>2 kb) DNA sequences into endogenous introns in the liver of non-human primates (NHP). PGI was effective across multiple genomic locations and transgenes, and insertion led to expression from the endogenous promoter. PGI was highly efficient, achieving expression in >50% of liver cells after a single course of treatment, which would be curative for most monogenic recessive liver diseases. This is the first report of clinically curative level of gene insertion at endogenous loci in NHP.

bioengineering↗

bioRxiv2024

Large Serine Integrase Off-target Discovery and Validation for Therapeutic Genome Editing

While numerous technologies for the characterization of potential off-target editing by CRISPR/Cas9 have been described, the development of new technologies and analytical methods for off-target recombination by Large Serine Integrases (LSIs) are required to advance the application of LSIs for therapeutic gene integration. Here we describe a suite of off-target recombination discovery technologies and a hybrid capture validation approach as a comprehensive framework for off-target characterization of LSIs. HIDE- Seq (High-throughput Integrase-mediated DNA Event Sequencing) is a PCR-free unbiased genome-wide biochemical assay capable of discovering sites with LSI- mediated free DNA ends (FDEs) and off-target recombination events. Cryptic-Seq is a PCR-based unbiased genome-wide biochemical or cellular-based assay that is more sensitive than HIDE-Seq but is limited to the discovery of sites with off-target recombination. HIDE-Seq and Cryptic-Seq discovered 38 and 44,311 potential off-target sites respectively. 2,455 sites were prioritized for validation by hybrid capture NGS in LSI- edited K562 cells and off-target integration was detected at 52 of the sites. We benchmarked the sensitivity of our LSI off-target characterization framework against unbiased whole genome sequencing (WGS) on LSI-edited samples, and off-target integration was detected at 5 sites with an average genome coverage of 40x. This reflects a greater than 10-fold increase in sensitivity for off-target detection compared to WGS, however only 4 of the 5 sites detected by WGS were also validated by hybrid capture NGS. The dissemination of these technologies will help advance the application of LSIs in therapeutic genome editing by establishing methods and benchmarks for the sensitivity of off-target detection.

genomics↗

bioRxiv2024

Feature Importance in Predicting Clinical Outcome: Statistics vs. Explainable Artificial Intelligence

At the time of diagnosis for cancer patients, a wide array of data can be gathered, ranging from clinical information to multiple layers of omics data. Determining which of these data are most informative is crucial, not only for advancing biological understanding but also for clinical and economic considerations. This process facilitates the selection of the most significant markers, enhancing patient stratification and informing treatment recommendations. In this paper, we start with 89 features extracted from multiomics and clinical data and aim to identify the most important ones in predicting response to neoadjuvant chemotherapy (NAC) using different explainable Artificial Intelligence (XAI) models and statistics. Our results show that XAI methods consistently recover important features that are missed by statistics and vice versa, hinting towards the need for complementary implementation of these methods. Furthermore, we find that a myriad of features, from mutations to immune infiltration, affect the response to NAC in breast tumors.

bioinformatics↗

bioRxiv2018

In vivo, regulated reconstitution of spindle checkpoint arrest and silencing through chemical-induced dimerisation

Chemical-induced dimerisation (CID) uses small molecules to control specific protein-protein interactions. Here, we employ CID dependent on the plant hormone abscisic acid (ABA) to reconstitute spindle checkpoint signalling in fission yeast. The spindle checkpoint signal usually originates at unattached or inappropriately attached kinetochores. These are complex, multi-protein structures with several important functions. To bypass kinetochore complexity, we take a reductionist approach to study checkpoint signalling. We generate a synthetic checkpoint arrest ectopically by inducing hetero-dimerisation of the checkpoint proteins Mph1Mps1 and Spc7KNL1. These proteins are engineered such that they cant localise to kinetochores, and only form a complex in the presence of ABA. Using this novel assay we are able to checkpoint arrest a synchronous population of cells within 30 minutes of ABA addition. This assay allows for detailed genetic dissection of checkpoint activation and importantly it also provides a valuable tool for studying checkpoint silencing. To analyse silencing of the checkpoint and the ensuing mitotic exit, we simply wash-out the ABA from arrested cells. We show here that silencing is critically-dependent on PP1Dis2 recruitment to Mph1Mps1-Spc7KNL1 signalling platforms.

cell biology↗