Five types of conversion risks
Species barrier
Differences in receptors, glycans, endothelial barriers, and immune background can cause mouse enrichment results to disappear in NHP or human systems. The solution is to add human cells/organoids, second lines and cross-species validation as early as possible.
Production adaptation
Highly transducing candidates may be poorly assembled, have low titers, have many empty shells, or be unstable in the scale-up process. Yield, VP ratio, genome integrity and freeze-thaw stability thresholds should be set in the early stage of screening.
Library bias
Systematic deviations exist between plasmid libraries, virus input libraries, and tissue recovery libraries. Input libraries must be sequenced and bottlenecks estimated with biological replicates and negative tissue.
Off-target enrichment
High exposure to liver, spleen or endothelium can produce strong enrichment masking target cells. RNA expression, cell sorting, and non-target organs should be analyzed in parallel.
Intellectual Property
Sequences, insertion sites, screening methods and uses may be individually protected; sequence searches and free implementation evaluations should be completed before candidates enter amplification.
Safety risks are not a simple ranking of "low natural capsid, high engineered capsid". Dosage, receptor distribution, expression cassette toxicity, impurities, and manufacturing processes all combine to modify risk. High-dose systemic administration and CSF administration particularly require validation that incorporates hepatotoxicity, complement/hematological events, DRG pathology, and non-target tissue expression. [18-2 to 18-5]
References for this chapter
[18-1] Hordeaux J, et al. Neurotropic properties of AAV-PHP.B are limited to C57BL/6J mice. Mol Ther. 2018;26:664-668. [H]
[18-2] Hinderer C, et al. Severe toxicity following high-dose intravenous AAV-SMN in NHP and piglets. Hum Gene Ther. 2018;29:285-298. DOI:10.1089/hum.2018.015. PMID:29378426. [H]
[18-3] Hordeaux J, et al. AAV-induced dorsal root ganglion pathology. Hum Gene Ther. 2020;31:808-818. DOI:10.1089/hum.2020.167. [H]
[18-4] Shen W, et al. rAAV immunogenicity, toxicity, and durability in 255 clinical trials. Front Immunol. 2022;13:1001263. [H]
[18-5] Lek A, et al. Death after high-dose rAAV9 gene therapy in Duchenne muscular dystrophy. N Engl J Med. 2023;389:1203-1210. DOI:10.1056/NEJMoa2307798. [N]
[18-6] Park S, et al. DoE optimization of AAV2/AAV9 production and full-capsid ratio. Biotechnol J. 2024;19:e2300667. DOI:10.1002/biot.202300667. [N]