A3 - AAV Vectors - Capsid Engineering
971: Novel AAV Variants for Huntington’s Disease Gene Therapy
Type: Poster Session
Poster Board Number: 971
Presentation Details
Session Title: Thursday Posters: AAV Vectors - Capsid Engineering
Huntington’s disease (HD) affects a distributed network of brain areas including the basal ganglia and cerebral cortex. However, current state-of-the-art AAV capsids do not sufficiently target areas critical for HD therapy without large doses using prolonged surgery times. To overcome this challenge, we performed a screen of tens of millions of peptide-modified (PM) AAV capsid variants in adult rhesus macaques to identify novel variants that transduce relevant areas following a single focused low dose infusion. Our screen identified multiple variants with robust transduction across basal ganglia and cortical regions in rhesus. To test the across-species translatability of our PM-AAV screening approach, we took the top ~150 capsid variants identified after two rounds of screening in rhesus macaque and tested their performance in rhesus, African green monkey (AGM) and in mice. Capsid performance measured from next generation sequencing (NGS) results in rhesus and AGM was highly correlated, with a Pearson’s correlation of 0.85. Similarly, there was enrichment of top capsid variants in mouse medium spiny neurons (MSNs) when assessed by single cell sequencing methods.
One PM-AAV variant, DB-3, was consistently highly enriched in each round of selection. Biodistribution of AAV-DB-3 was characterized by native fluorescence of AAV-DB3.mTFP following relatively low dose injections in rhesus. We observed remarkably efficient transduction of basal ganglia regions and layer V/VI projection neurons across multiple cortical areas, including primary motor cortex.
A critical target for HD gene therapy are MSNs in the basal ganglia. We assessed cell-type tropism in the caudate and putamen using RNAscope™ fluorescence in situ hybridization with probes targeted to MSN marker genes. DB-3 demonstrated high selectivity for MSNs, transducing ~50% of MSNs in both regions and only 5-20% of non-MSN cell types. We also assessed DB-3 transduction outside the nervous system. In digital droplet PCR of liver samples, DB-3 transgene levels were below the threshold of detection.
To test whether DB-3 would exhibit similarly high transduction efficiency in human cells, we quantified transgene expression in neurons derived from human iPSCs using qPCR. At a dose of 5.0E3 vg/cell, DB-3 showed a ~5700-fold increase in transgene expression compared to the wild-type parental serotype. In summary, DB-3 exhibits remarkable tropism for therapeutically relevant HD-related brain regions and improved transduction efficiency in both human and NHP neurons at doses orders of magnitude lower than those currently in clinical use. DB-3 will be a powerful tool for the delivery of therapeutic transgenes for HD and other disorders affecting the brain.
Brian Lewandowski1, David E. Leib2, Yonghong Chen1, Paul T. Ranum2, Luis Tecedor1, Sakshi Arora1, Ashley Robbins3, Bryan P. Simpson2, Elena Lysenko1, Megan S. Keiser4, Xueyuan Liu5, Jang-Ho Cha2, Beverly L. Davidson1
1Children's Hospital of Philadelphia, Philadelphia, PA,2Latus Bio, Philadelphia, PA,3University of Pennsylvania, Philadelphia, PA,4Ohio State University Wexner Medical Center, Columbus, OH,5The Children's Hospital of Philadelphia, Philadelphia, PA"
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