Dorsal root ganglion toxicity after AAV intra-CSF delivery of a RNAi expression construct into non-human primates and mice.

Dorsal root ganglion (DRG) toxicity has been consistently reported as a potential safety concern after delivery of adeno-associated viruses (AAVs) containing gene-replacement vectors but has yet to be reported for RNAi-based vectors. Here, we report DRG toxicity after AAV intra-CSF delivery of an RNAi expression construct-artificial microRNA targeting superoxide dismutase 1 (SOD1)-in non-human primates (NHPs) and provide evidence that this can be recapitulated within mice. Histopathology evaluation showed that NHPs and mice develop DRG toxicity after AAV delivery, including DRG neuron degeneration and necrosis and nerve-fiber degeneration that were associated with increases in cerebrospinal fluid (CSF) and serum phosphorylated neurofilament heavy chain (pNF-H).

Efficacy and safety of a SOD1-targeting artificial miRNA delivered by AAV9 in mice are impacted by miRNA scaffold selection.

Toxic gain-of-function mutations in superoxide dismutase 1 (SOD1) contribute to approximately 2%-3% of all amyotrophic lateral sclerosis (ALS) cases. Artificial microRNAs (amiRs) delivered by adeno-associated virus (AAV) have been proposed as a potential treatment option to silence SOD1 expression and mitigate disease progression. Primary microRNA (pri-miRNA) scaffolds are used in amiRs to shuttle a hairpin RNA into the endogenous miRNA pathway, but it is unclear whether different primary miRNA (pri-miRNA) scaffolds impact the potency and safety profile of the expressed amiR .

Development of a one-step RT-ddPCR method to determine the expression and potency of AAV vectors.

Robust assays to quantify adeno-associated virus (AAV) vector expression and potency are essential for gene therapy development. These assays inform the efficacy, safety, and pharmacodynamic profiles of AAV development candidates. Additionally, for gene downregulation strategies such as RNAi, knockdown of endogenous genes reflects the mechanism of action of such development candidates.

Development and Application of a Liquid Chromatography-Mass Spectrometry Method for Residual Iodixanol Quantification in AAV-Based Gene Therapy Product Development.

Adeno-associated viruses (AAVs) are nonenveloped viruses that have become popular gene transfer vectors to deliver DNA to target cells in clinical gene therapy. Iodixanol-based density gradient is one of the widely used purification methods for serotype-independent AAVs. However, residual iodixanol in AAV could be a safety concern, and further purification to remove this process-related impurity is typically needed.