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 in vivo. In our process to develop an AAV amiR targeting SOD1, we performed a preclinical characterization of two pri-miRNA scaffolds, miR155 and miR30a, sharing the same guide strand sequence. We report that, while the miR155-based vector, compared with the miR30a-based vector, leads to a higher level of the amiR and more robust suppression of SOD1 in vitro and in vivo, it also presents significantly greater risks for CNS-related toxicities in vivo. Despite miR30a-based vector showing relatively lower potency, it can significantly delay the development of ALS-like phenotypes in SOD1-G93A mice and increase survival in a dose-dependent manner. These data highlight the importance of scaffold selection in the pursuit of highly efficacious and safe amiRs for RNA interference gene therapy.

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. An analytical assay with high sensitivity is essential for the detection of residual iodixanol to ensure the safety of AAV products. We developed a liquid chromatography-mass spectrometry method with the limit of quantification of 0.01 µg/mL for residual iodixanol measurement in AAVs. The method also demonstrated linearity over four orders of magnitude that allows quantifying a high iodixanol concentration in in-process samples with excellent recovery and accuracy. In addition, we further explored a highly efficient purification method for removal of the residual iodixanol, to minimize the safety concern from iodixanol as a process impurity.

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. Therefore, a method to quantify target mRNA repression is necessary for measuring vector potency both in vitro and in vivo. Here, we report the development of a one-step reverse-transcription droplet digital PCR (RT-ddPCR) method to analyze expression of AAV vectors and the potency of AAV-RNAi vectors. This one-step RT-ddPCR method simplifies the workflow, allows for duplexing reactions, and enables absolute quantification of transcripts without standard materials. With a gene augmentation vector, we demonstrate the application of RT-ddPCR in quantifying vector expression in vitro and in non-human primate (NHP) samples. This novel method is demonstrated to be precise and linear within the range of 0.05-25 ng of RNA input. Using an AAV-RNAi vector, we further demonstrate the utility of this RT-ddPCR method in quantifying potency. Orthogonal potency assays, including ELISA and functional readout, correlate well with RT-ddPCR results. Therefore, one-step RT-ddPCR can be implemented in the analytical and pharmacological characterization of AAV vectors.