Revolution of AAV in Drug Discovery: From Delivery System to Clinical Application

Abstract

Adeno-associated virus (AAV) is a non-enveloped DNA virus infecting a wide variety of species, tissues, and cell types, which is recognized as a safe and effective method for delivering therapeutic transgenes. AAV vector is the most popular viral gene delivery system in clinical delivery systems with unique and multiple advantages, such as tissue tropism, transduction specificity, long-lasting gene expression, low immune responses, and without host chromosome incorporation. Till now, four AAV-based gene therapy drugs have already been approved by the US Food and Drug Administration (FDA) or European Medicines Agency (EMA). Despite the success of AAV vectors, there are still some remaining challenges that limit further usage, such as poor packaging capacity, low organ specificity, pre-existing humoral immunity, and vector dose-dependent toxicity. In the present review, we address the different approaches to optimize AAV vector delivery system with a focus on capsid engineering, packaging capacity, and immune response at the clinical level. The review further investigates the potential of manipulating AAV vectors in preclinical applications and clinical translation, which emphasizes the challenges and prospects in viral vector selection, drug delivery strategies, immune reactions in cancer, neurodegenerative disease, retinal disease, SARS-CoV-2, and monkeypox. Finally, it forecasts future directions and potential challenges of artificial intelligence (AI), vaccines, and nanobodies, which emphasizes the need for ethical and secure approaches in AAV application.

Keywords: AAV; clinical application; delivery system; drug discovery.

Figure 1

Schematic overview of adeno‐associated virus (AAV) packaging and engineering. The diagram illustrates key components and processes involved in AAV production, including packaging capacity, producing yield, and transducing safety. The figure also shows advanced techniques like capsid engineering, genome editing, and the integration of artificial intelligence in AAV research.

Figure 2

The strategies for optimizing AAV capsid engineering. AAV capsids could be engineered through various methods, including rational design, directed evolution, and machine learning. Rational Design focuses on targeted modifications based on structural or functional insights. Directed Evolution (bottom) presents a high‐throughput mutant library. Machine learning is employed to analyze variant patterns and optimize screening efficiency. These diversified AAV capsids are then validated and tested within a chosen system of interest, In Vitro and In Vivo.

Figure 3

The strategies for enhancing AAV packaging capacity. To accommodate large transgenes, there are several strategies, such as overlapping, trans‐splicing, hybrid, and sequential homologous recombination.

Figure 4

The strategies for reducing AAV immune response. The host antiviral immune responses directed against AAV capsid and transgene product could be resolved by several strategies, such as capsid engineering (rational design, directed revolution), surface tethering (polymer conjugation, peptide targeting), viral load (liposome‐AAV, exosome‐AAV), dual‐AAV dosing, immunosuppressive, molecular imprinting (epitope mapping, neutralizing antibody).

Figure 5

Combination therapy of AAV with miRNA/ICI/ACT could boost the cancer drug discovery by inhibiting tumor initiation, growth, and metastasis. The figure illustrates key pathways in cancer development: viral infection outcomes in normal vs. affected cells (DNA repair vs. integration), genomic instability in precancerous states with mRNA targets, oncogenic transformation with mRNA degradation, immune checkpoint markers, and CAR T‐cell immunotherapy.

Figure 6

AAV‐mediated gene therapy could treat neurodegenerative diseases by overcoming blood–brain barrier, reprogramming capsid proteins, and selecting various serotypes. This figure illustrates the use of AAV vectors to model neurodegenerative disorders, highlighting key tau mutants (P301S, P301L, P301L/S320F, A152T, R406W) associated with Alzheimer's pathology, along with characteristic neuropathological features including amyloid β plaques, neurofibrillary tangles, α‐synuclein aggregates, and associated neurodegeneration. It further depicts neuroinflammatory components (activated microglia, reactive astrocytes, BBB leakage) in Alzheimer's/Parkinson's patients, and presents various AAV serotypes (AAV1/2 to AAV2/9) employed for modeling α‐synuclein pathology in Parkinson's disease.

Figure 7

AAV‐based gene therapy clinical applications for retinal diseases. There are several breakthroughs in retinal diseases, such as Achromatopsia, Choroideremia, Leber congenital amaurosis 2, Leber hereditary optic neuropathy, Neovascular/age‐related macular degeneration, Retinitis pigmentosa, X‐linked retinitis pigmentosa, and X‐linked retinoschisis, demonstrating the versatility of AAV serotypes (AAV2, AAV5, AAV8) for ocular gene delivery.

Figure 8

AAV‐based COVID‐19 vaccines target SARS‐CoV2 S protein RBD. The figure illustrates key functional domains of the SARS‐CoV‐2 spike protein, including the receptor binding domain (RBD) that interacts with human ACE2 receptors and the N‐terminal domain (NTD). Also depicted are AAV‐based vaccines targeting RBD, including AAV6‐RBD, AAV9‐RBD, ssAAV5‐RBD, and scAAV5‐RBD.

Figure 9

The distributions of AAV clinical trials in different diseases (A), periods (B), and areas (C). The data are summarized from ClinicalTrials.gov. The figure categorizes gene therapy targets into four major groups: ocular diseases (e.g., Achromatopsia, Leber Congenital Amaurosis), cancer and metabolic disorders (e.g., AADC deficiency, Duchenne Muscular Dystrophy), infectious diseases (COVID‐19, HIV) with vaccine development applications, CNS and systemic disorders (e.g., Alzheimer's, Hemophilia, Cystic Fibrosis). The figure outlines the historical progression of AAV gene therapy across four key phases: (1) Early Development (1990s–2000s): Proof‐of‐concept studies focused on monogenic diseases (hemophilia, retinal disorders); (2) Expansion Phase (2000s–2010s): Diversification into neurological, metabolic and cardiovascular applications; (3) Breakthrough Era (2010s–2020s): FDA approvals of Luxturna (2017) and Zolgensma (2019); (4) Current Trends (2020s‐current): Therapeutic area distribution (40% neurological) and clinical phase breakdown (50% Phase I/II trials).

Figure 10

The Future direction of AAV‐mediated gene therapy will be accelerated and boosted by the application of artificial intelligence, vaccines, and nanobodies. The figure highlights three cutting‐edge therapeutic platforms: (1) Artificial Intelligence applications (ChatGPT, Deepseek) for AAV capsid optimization (CapsidMap, AAV Edge); (2) Vaccine technologies including mRNA platforms and novel delivery systems (nanocarriers, cellular vehicles); (3) Nanobody‐based therapies combined with checkpoint inhibitors, adoptive T cells, and CRISPR/Cas9 gene editing.