Respiratory gene therapy is hindered by the inefficiency of current delivery vectors. Among available vectors, adeno-associated virus 6 (AAV6) and AAV9 have shown promise for lung targeting. AAV9 has demonstrated effective intranasal delivery. A novel capsid, AAV.CPP.16, derived from AAV9, which exhibits enhanced lung tropism across species. Idiopathic pulmonary fibrosis (IPF) is a progressive and chronic lung disease that affects more than 3 million people globally. IPF triggers maladaptive epithelial responses and fibrosis. The current treatments merely slow the progression of the disease. Hence, more targeted therapies are needed. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a significant threat, particularly to immunocompromised individuals, as viral mutations compromise vaccine efficacy. Non-immune-based strategies offer promising alternatives for the prevention and treatment of coronavirus disease 2019 (COVID-19).
The recent study published in the Cell Reports Medicine systemically explores the role of AAV.CPP.16 gene vector for intranasal therapy in pulmonary and respiratory diseases. Cell lines, including human nasal epithelial cells (RPMI 2650, CCL-30) and human embryonic kidney cells (HEK293T, CRL-3216), were used in this study. Adult male and female C57BL/6J mice aged >8 weeks were included. Various analysis techniques were employed, including in vitro AAV transduction assays, lung histology, Western blotting, enzyme-linked immunosorbent assay (ELISA), SARS-CoV-2 genome sequence analysis, and quantitative real-time polymerase chain reaction (qRT-PCR). All statistical analyses were performed using GraphPad Prism software.
RPMI 2650 (CCL-30) nasal cells were treated with AAV at a multiplicity of infection (MOI) of 1 × 10⁵ for four days. It was observed that AAV.CPP.16 produced green fluorescent protein (GFP) fluorescence 3 times higher compared to AAV6 (p = 0.0023) and 6 times higher than AAV9 (p = 0.0006), with no significant difference between AAV6 and AAV9. AAVs-CAG-GFP 1 × 10¹¹ vg was intranasally administered to C57BL/6J mice for evaluation of AAV.CPP.16 in mice. After 3 weeks, fluorescence imaging showed that AAV.CPP.16 induced 2.7-fold and 1.8-fold stronger GFP expression in the nasal cavity compared to AAV9 and AAV6, respectively. It also significantly enhanced transduction in the trachea and lungs compared to both AAV6 and AAV9. There was no significant difference in transduction observed between AAV6 and AAV9.
Using MUC5A and α-tublin antibody markers, AAV.CPP.16 demonstrated superior transduction of airway cells, targeting 42.7% of goblet cells and 71% of club cells, compared to AAV6 and AAV9, with enhanced targeting of ciliated cells observed. AAV.CPP.16 demonstrated significantly increased lung transduction, targeting 13.6% of receptor for advanced glycation end-products (RAGE)-positive alveolar type I (ATI) cells, compared to 2.8% for AAV6 and 1.9% for AAV9. It also transduced ciliated bronchial cells more effectively than AAV6 and AAV9. Intranasal delivery of AAV.CPP.16 in rhesus macaques (Macaca mulatta) showed superior respiratory transduction over AAV9 with 2.5 to 10.5-fold higher GFP signals and greater targeting of goblet, club, and ATI cells. No adverse effects or off-target expression in the spleen, brain, and heart were observed, though minor kidney and liver transduction occurred.
In a bleomycin-induced IPF model, a single intranasal dose of CPP.16-IPF trap preserved lung architecture and reduced fibrosis, offering a targeted gene therapy alternative to continuous pirfenidone treatment. This trap gene therapy reduced vascular endothelial growth factor alpha (VEGFα) and transforming growth factor beta 1 (TGF-β1) levels in fibrotic lungs compared to untreated IPF lungs. Pooled 4x guide ribonucleic acids (gRNAs) designed using conserved RNA-dependent RNA polymerase (Rdrp) regions and validated efficient in vitro suppression of Rdrp transcripts by 44 to 58% with minimal predicted off-target effects. In SARS-CoV-2, AAV treatment reduced Ad5-mediated Rdrp expression by 50-60% in human nasal cells. Additionally, in mice, intranasal AAV-CasRx significantly lowered Rdrp transcription in the nostrils, lungs, and trachea.
This study’s limitations include the absence of a wild-type AAV12 and proper control groups, as well as unclear airway targeting mechanisms of AAV.CPP.16, reliance on a non-genetic IPF model, and untested antiviral efficacy against live SARS-CoV-2. In conclusion, these study findings support the use of AAV.CPP.16 as a potent vector for gene therapy against IPF and COVID-19 conditions.
Reference: Yang Z, Yao Y, Chen X, et al. Cross-species tropism of AAV.CPP.16 in the respiratory tract and its gene therapies against pulmonary fibrosis and viral infection. Cell Rep Med. 2025:102144. doi:10.1016/j.xcrm.2024.102144
