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How Did Researchers Resolve Adverse Symptoms that Emerged with AAV-based RNA Interference Strategies for Ataxia Therapy?

Published on October 18, 2021 in Cornerstone Blog · Last updated 5 months 3 weeks ago


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Extended safety testing of adeno-associated virus vectors is critical.

The findings:

A team of researchers at the Raymond G. Perelman Center for Cellular and Molecular Therapeutics at Children’s Hospital of Philadelphia investigated the root cause of toxicity revealed in a study assessing the long-term safety and tolerability of a new therapeutic strategy to treat the gain-of-function disease spinocerebellar ataxia type 1 (SCA1). Their previous long-term work with animal models showed that using adeno-associated virus (AAV) vectors to deliver sequences that silence expression of the mutant gene Ataxin-1 (ATXN1) via RNA interference (RNAi) could prevent or reverse SCA1 disease phenotypes; however, when the study team extended their translational studies, they observed adverse side effects in the brain. Looking for the underlying factors behind these unexpected findings, the investigators determined that it wasn’t the RNAi per se, but rather the context of how the RNAi was expressed.

Why it matters:

This study highlights that extended safety testing of AAVs is critical, as researchers assess the first gene therapy approach for this class of hereditary ataxias that cause debilitating symptoms including muscle incoordination and gait unsteadiness, uncontrolled eye movements, difficulty swallowing, and abnormal speech. This data is significant for the community of investigators advancing AAV-based RNAi therapies to treat central nervous system disorders and other human diseases so that they can guide the creation and testing of new constructs and prove them effective and safe in translational studies.

Who conducted the study:

The first authors of the study include research scientist Megan Keiser, PhD; and postdoctoral research fellows Paul Ranum, PhD, and Carolyn Yrigollen, PhD, all of the Raymond G. Perelman Center for Cellular and Molecular Therapeutics. Beverly Davidson, PhD, is the senior author and director of the Center. Dr. Davidson also is chief scientific strategy officer for CHOP Research Institute.

How they did it:

The study team conducted a series of investigations using several next generation sequencing (NGS) methodologies on the vector itself and the treated tissues. They ascertained that the inverted terminal repeats (ITR) can transcribe non-desired sequences in certain vector configurations, and that these transcripts or the proteins they express could cause neuropathology. Upon re-engineering, using NGS data as a guide, the researchers completely negated the aberrant transcription, and there were no observed neurological symptoms or pathologies.

Quick thoughts:

“We uncovered a complete surprise that is relevant to the broader community of researchers working to reduce the expression of disease genes by vector-based RNA interference,” Dr. Davidson said. “Our sponsors at the NIH agreed that it was important to understand the cause of toxicity, resolve that toxicity, and continue to move forward and contribute our findings to advance the field. We were fortunate to work with a committed team at CHOP and the University of Pennsylvania focused on ascertaining what was going wrong, so that others in the field of AAV-based RNAi can use this knowledge for safer vector designs.”

What’s next:

The next steps for the research team are to continue with the new RNAi expression cassette with renewed confidence in the AAV vectors intended for human therapies to treat SCA1. They will resume performing dose-response analyses. Also, other researchers now know RNA-seq after delivery is a valuable way to predict ITR promoter activity in preclinical models.

Where the study was published:

Nature Medicine

Where to learn more:

Find out more about the ongoing research by the Raymond G. Perelman Center for Cellular and Molecular Therapeutics.