bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.04.14.536951

All-in-one AAV-delivered epigenome-editing platform: proof-of-concept and therapeutic implications for neurodegenerative disorders

Kantor, B.·Odonovan, B.·Rittiner, J.·Lindner, N.·Dong, W.·Zhang, A.·Nicholls, P.·Chiba-Falek, O.
View original source ↗Export metadata ↓

Abstract

Safely and efficiently controlling gene expression is a long-standing goal of biomedical research, and the recently discovered bacterial CRISPR/Cas system can be harnessed to create powerful tools for epigenetic editing. Current state-of-the-art systems consist of a deactivated-Cas9 nuclease (dCas9) fused to one of several epigenetic effector motifs/domains, along with a guide RNA (gRNA) which defines the genomic target. Such systems have been used to safely and effectively silence or activate a specific gene target under a variety of circumstances. Adeno-associated vectors (AAVs) are the therapeutic platform of choice for the delivery of genetic cargo; however, their small packaging capacity is not suitable for delivery of large constructs, which includes most CRISPR/dCas9-effector systems. To circumvent this, many AAV-based CRISPR/Cas tools are delivered in two pieces, from two separate viral cassettes. However, this approach requires higher viral payloads and usually is less efficient. Here we develop a compact dCas9-based repressor system packaged within a single, optimized AAV vector. The system uses a smaller dCas9 variant derived from Staphylococcus aureus (Sa). A novel repressor was engineered by fusing the small transcription repression domain (TRD) from MeCP2 with the KRAB repression domain. The final dSaCas9-KRAB-MeCP2(TRD) construct can be efficiently packaged, along with its associated gRNA, into AAV particles. Using reporter assays, we demonstrate that the platform is capable of robustly and sustainably repressing the expression of multiple genes-of-interest, both in vitro and in vivo. Moreover, we successfully reduced the expression of ApoE, the stronger genetic risk factor for late onset Alzheimers disease (LOAD). This new platform will broaden the CRISPR/dCas9 toolset available for transcriptional manipulation of gene expression in research and therapeutic settings.

Explore related subjects

bioengineeringPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Kantor, B., Odonovan, B., Rittiner, J., Lindner, N., Dong, W., Zhang, A., Nicholls, P., Chiba-Falek, O.. 2023-04-16. All-in-one AAV-delivered epigenome-editing platform: proof-of-concept and therapeutic implications for neurodegenerative disorders. https://doi.org/10.1101/2023.04.14.536951

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Synergy-informed bilevel optimization reveals joint-specific benefits for realistic exoskeleton torque prediction

Simulation-driven optimization can guide exoskeleton control design, but its utility is limited by the accuracy with which muscle coordination is predicted. Strict effort-minimization formulations predict non-physiological muscle shutdown under high exoskeleton assistance, leading to peak assistive torques that exceed values reported in human-in-the-loop experiments. We evaluated the accuracy of synergy-informed simulations at predicting assistance across four single-joint exoskeleton configurations: ankle plantarflexion, knee extension, hip flexion, and hip abduction. We implemented a bilevel optimization to predict a parameterized assistive torque that minimizes metabolic cost, with assisted muscle dynamics estimated via either effort-minimization simulations (minimizing muscle effort only) or synergy-informed simulations (minimizing muscle effort constrained to a synergy structure). We used experimental data from five unimpaired adults walking at their preferred speed and compared results descriptively between the two formulations within each subject. Assistance based on effort-minimization simulations shut down agonist muscle activations and overestimated peak assistive torque compared with experimental values. In contrast, constraining muscle controls to a synergy structure prevented non-physiological muscle shutdown and yielded assistive torques closer to those measured in human-in-the-loop experiments. This benefit was not observed equally across all assisted degrees of freedom or subjects, emerging only where no single synergy could be suppressed to unload the assisted muscles. Synergy weights can therefore be interpreted as joint-specific predictors of when simulations can reliably inform wearable robot design.

bioengineering↗
bioRxiv2026

Engineered microvessel networks -- Longitudinal transcriptomic profiling validates chip-grown tissues as a robust model for studying vasculature

Vascularised Organ-on-Chip (OoC) models are promising New Approach Methodologies (NAMs), but their translation requires validation for defined Contexts of Use (CoU). Here, we combine microfluidic microvessel networks (MVNs) with longitudinal bulk and single-cell RNA sequencing to determine how cell provenance influences engineered vascular phenotype. Stromal cell identity produced distinct network architectures and transcriptional trajectories, demonstrating that support cells, commonly treated as interchangeable, represent an important CoU variable. Primary human pancreatic microvascular endothelial cells failed to form stable perfusable networks, but supplementation with 10% Human Umbilical Vein Endothelial Cells (HUVECs) or Blood Outgrowth Endothelial Cells (BOECs) rescued self-assembly. Single-cell transcriptomics identified specialised endothelial populations associated with vascular morphogenesis, including tip, stalk, phalanx, endothelial to mesenchymal transition (EndMT) and pericyte-like endothelial cells. Failed networks lacked coherent endothelial specialisation, whereas rescued networks restored stabilising cell states and revealed FLRT2--UNC5B signalling within pericyte-like HUVECs, recapitulating a pathway implicated in angiogenic development in vivo. These findings establish cell provenance as a critical determinant of microvascular NAM performance and demonstrate how transcriptomic validation can define and strengthen the biological relevance of microvessel-on-chip models for specific CoUs.

bioengineering↗
bioRxiv2026

Benchmarking foundation models for resting-state functional magnetic resonance imaging time series in cognitive aging research

Foundation models trained on large neuroimaging datasets could help smaller studies investigate cognition from brain recordings. However, whether the foundation model-derived representations improve assessment of current cognition, prediction of follow-up cognition, or characterization of cognitive change remains unclear. We evaluated nine configurations from five model families that process regional resting-state functional magnetic resonance imaging time series, using 3,103 scans from 1,653 participants across two observational and five intervention aging cohorts. Primary frozen representations showed no consistent superiority over conventional functional connectivity for concurrent cognitive associations or diagnostic classification. Changing the readout improved selected concurrent cognitive associations without updating model weights, indicating that performance depended partly on how representations were extracted. Additional post-training and embedding alignment yielded gains in selected settings but did not consistently improve performance across outcomes. In the primary longitudinal analyses, imaging representations did not establish added value beyond baseline clinical measures. These findings show that readout choice influences concurrent cognitive associations, while the added value of these representations for longitudinal outcomes remains unestablished.

bioengineering↗