De La Cruz, D. | bioRxiv Science

bioRxiv2024

Metabolic engineering improves transduction efficiency and downstream vector isolation by altering the lipid composition of extracellular vesicle-enclosed AAV.

Adeno-associated viruses (AAV) are promising vectors for gene therapy due to their efficacy in vivo. However, there is room for improvement to address key limitations such as the pre-existing immunity to AAV in patients, high-dose toxicity, and relatively low efficiency for some cell types. This study introduces a metabolic engineering approach, using knockout of the enzyme phosphatidylserine synthase 1 (PTDSS1) to increase the abundance of extracellular vesicle-enclosed AAV (EV-AAV) relative to free AAV in the supernatant of producer cells, simplifying downstream purification processes. The lipid-engineered HEK293T-{Delta}PTDSS1 cell line achieved a 42.7-fold enrichment of EV-AAV9 compared to free AAV9 in the supernatant. The rational genetic strategy also led to a 300-fold decrease of free AAV in supernatant compared to wild-type HEK293T. The membrane-engineered EV-AAV9 (mEV-AAV9) showed unique envelope composition alterations, including cholesterol enrichment and improved transduction efficiency in human AC16 cardiomyocytes by 1.5-fold compared to conventional EV-AAV9 and by 11-fold compared to non-enveloped AAV9. Robust in-vivo transduction four weeks after intraparenchymal administration of mEV-AAV9 was observed in the murine brain. This study shows promise in the potential of lipid metabolic engineering strategies to improve the efficiency and process development of enveloped gene delivery vectors.

bioengineering↗

bioRxiv2023

In vivo selection in non-human primates identifies superior AAV capsids for on-target CSF delivery to spinal cord

Systemic administration of adeno-associated virus (AAV) vectors for spinal cord gene therapy has challenges including toxicity at high doses and pre-existing immunity that reduces efficacy. Intrathecal delivery of AAV vectors into the cerebral spinal fluid (CSF) can avoid many of the issues of systemic delivery, although achieving broad distribution of the vector and transgene expression throughout the spinal cord is challenging and vector entry to the periphery occurs, sometimes initiating hepatotoxicity. Here we performed two rounds of in vivo biopanning in non-human primates (NHPs) with an AAV9 peptide display library injected intrathecally and performed insert sequencing on DNA isolated from either whole tissue (conventional selection), isolated nuclei, or nuclei from transgene-expressing cells. A subsequent barcoded pool of candidates and AAV9 was compared at the DNA (biodistribution) and RNA (expression) level in spinal cord and liver of intrathecally injected NHPs. Most of the candidates displayed enhanced biodistribution compared to AAV9 at all levels of spinal cord ranging from 2 to 265-fold. Nuclear isolation or expression-based selection yielded 4 of 7 candidate capsids with enhanced transgene expression in spinal cord (up to 2.4-fold), while no capsid obtained by conventional selection achieved that level. Furthermore, several capsids displayed lower biodistribution to the liver of up to 1,250-fold, compared to AAV9, providing a remarkable on target/off target biodistribution ratio. These capsids may have potential for gene therapy programs directed at the spinal cord and the selection method described here should be useful in clinically relevant large animal models.

bioengineering↗