lumbar-intrathecal-injection-sod1-asos-for-precise-cns-targeting

Lumbar Intrathecal Injection of SOD1-ASOs for Precise CNS Targeting and Predictive Efficacy in Human SOD1-G93A ALS Mice

Intrathecal (IT) administration of central nervous system (CNS)-targeted therapeutics offers a minimally invasive route for direct drug delivery into the ...

Biogen

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  and . 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  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.

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

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). RNA-sequencing analysis of DRGs showed that dysregulated pathways were preserved between NHPs and mice, including increases in innate/adaptive immune responses and decreases in mitochondrial- and neuronal-related genes, following AAV treatment. Finally, endogenous miR-21-5p was upregulated in DRGs of AAV-treated NHPs and mice. Increases in miR-21-5p were also identified within the CSF of NHPs, which significantly correlated with pNF-H, implicating miR-21-5p as a potential biomarker of DRG toxicity in conjunction with other molecular analytes. This work highlights the importance of assessing safety concerns related to DRG toxicity when developing RNAi-based AAV vectors for therapeutic purposes.

Article	Year
Development of a one-step RT-ddPCR method to determine the expression and potency of AAV vectors. Molecular therapy. Methods & clinical development\| PubMed ID: 34589555	2021
Dorsal root ganglion toxicity after AAV intra-CSF delivery of a RNAi expression construct into non-human primates and mice. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 39563026	2025

Twinkle Chowdhury

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