AAV-ShD for Lung Macrophages
AAV Gene Therapy
The safety and efficacy of gene therapies rely on targeted transgene expression while minimizing off-target effects. Among the different AAV serotypes, AAV1, AAV2, AAV5, AAV9 and AAVrh.74 has been approved for gene therapies to treatment different rare diseases. However, those AAVs are always multiple targeting. For example,AAV9 is widely used for its strong gene expression across multiple organs, including the liver, brain, lungs, heart, muscles, and kidneys. Developing highly efficient, tissue-specific AAVs is the most promising approach to reduce AAV gene therapy dosage, enhance safety, and lower costs.
AAV-ShDs: Novel AAVs with Super High DNA
Utilizing our ATHENA-I and ATHENA-III capsid engineering platforms, we selected and evolved AAV capsids, leading to the discovery of novel variants with high DNA enrichment in the lungs, termed AAV-ShDs, and significant detargeting from the liver and other major organs in C57BL/6J and B6C3 mice. AAV-ShD capsids also exhibit high lung enrichment in non-human primates. These capsids showed over 100-fold DNA enrichment and about 22-fold RNA enrichment in the lung of macaques compared to AAV9. Moreover, AAV-ShDs can cross the BBB and efficiently transduce neural cells in both non-human primates and in vitro human cell models.
Generation of AAV-ShDs
Using our ATHENA-I and ATHENA-III capsid engineering platforms, we identified and evolved novel AAV variants with high DNA enrichment in the lungs and brain, leading to the discovery of AAV-ShD.
The ATHENA-I AAV Capsid Platform comprises a comprehensive library of over 1,000 distinct AAV capsids, each tagged with three unique DNA barcodes for evaluation using Barcode-seq technology. By comparing DNA or RNA barcode levels, researchers can pinpoint the most efficient AAV capsid variant for their specific application.
The ATHENA-III Platform is a DNA-shuffling AAV capsid library designed to create hybrid capsids with enhanced transduction efficiency and improved biological properties.
Building on NHP selection data from ATHENA-I, this platform enabled the discovery of AAV-ShD.
AAV-ShD specifically targets lung macrophages in mouse model
Among the engineered variants, the AAV-ShD, exhibited an RNA enrichment that was 2.56-fold relative to the parental AAV-ShD0 (4.0-fold of AAV9) in the lungs of C57BL/6 mice at the 2-week time point, while preserving its high lung vector DNA levels (548-fold vs AAV9). At 12 weeks post-injection, AAV-ShD exhibited a 38.9-fold enrichment in vector DNA and a 6.1-fold RNA enrichment relative to AAV9, suggesting that the high vector DNA abundance may contribute to sustained long-term expression when compared with earlier time points (e.g., 2 weeks vs. 12 weeks). Multilabel immunofluorescence and confocal imaging localized AAV-ShD transduction predominantly to F4/80⁺ macrophages within the alveolar septum, with minimal colocalization with CD68 or IBA1 and no detectable colocalization with epithelial (SFTPC, CC10), endothelial (CD31/CDH5/ZO1), fibroblasts (Vimentin), or smooth muscle cell (α‑SMA/Desmin) markers.
AAV-ShD has >100-fold DNA levels in NHP lung compared with AAV9
In cynomolgus macaques, AAV-ShD similarly demonstrated robust lung specificity, with ~100-fold higher lung vector DNA and ~22-fold higher lung RNA relative to parent AAV9. Notably, AAV-ShD also exhibited blood–brain barrier crossing activity in macaques, with ~385-fold higher brain vector DNA than AAV9. Overall, AAV-ShD produced markedly enhanced lung transduction while maintaining strong liver detargeting, highlighting its potential for targeted in vivo gene delivery.
