Moyer, T. C. | bioRxiv Science

bioRxiv2024

Highly conserved brain vascular receptor ALPL mediates transport of engineered viral vectors across the blood-brain barrier

Delivery of systemically administered therapeutics to the central nervous system (CNS) is restricted by the blood-brain barrier (BBB). Bioengineered Adeno-Associated Virus (AAV) capsids have been shown to penetrate the BBB with great efficacy in mouse and non-human primate models, but their translational potential is often limited by species selectivity and undefined mechanisms of action. Here, we apply our RNA-guided TRACER AAV capsid evolution platform to generate VCAP-102, an AAV9 variant with markedly increased brain tropism following intravenous delivery in both rodents and primates. VCAP-102 demonstrates a similar CNS tropism in cynomolgus macaque, african green monkey, marmoset and mouse, showing 20- to 400-fold increased transgene expression across multiple brain regions relative to AAV9. We demonstrate that the enhanced CNS tropism of VCAP-102 results from direct interaction with alkaline phosphatase (ALPL), a highly conserved membrane-associated protein expressed on the brain vasculature. VCAP-102 interacts with human, primate and murine ALPL isoforms, and ectopic expression of ALPL is sufficient to initiate receptor-mediated transcytosis of VCAP-102 in an in vitro transwell model. Our work identifies VCAP-102 as a cross-species CNS gene delivery vector with a strong potential for clinical translation and establishes ALPL as a brain delivery shuttle capable of efficient BBB transport to maximize CNS delivery of biotherapeutics.

neuroscience↗

bioRxiv2017

Applying the auxin-inducible degradation (AID) system for rapid protein depletion in mammalian cells

The ability to deplete a protein of interest is critical for dissecting cellular processes. Traditional methods of protein depletion are often slow-acting, which can be problematic when characterizing a cellular process that occurs within a short period of time. Furthermore, these methods are usually not reversible. Recent advances to achieve protein depletion function by inducibly trafficking proteins of interest to an endogenous E3 ubiquitin ligase complex to promote ubiquitination and subsequent degradation by the proteasome. One of these systems, the auxin-inducible degron (AID) system, has been shown to permit rapid and inducible degradation of AID-tagged target proteins in mammalian cells. The AID system can control the abundance of a diverse set of cellular proteins, including those contained within protein complexes, and is active in all phases of the cell cycle. Here we discuss considerations for the successful implementation of the AID system and describe a protocol using CRISPR/Cas9 to achieve bi-allelic insertion of an AID degron in human cells. This method can also be adapted to insert other tags, such as fluorescent proteins, at defined genomic locations.

cell biology↗