Adeno-associated virus (AAV) is a diminutive yet remarkably adaptable viral entity that generally poses no pathogenic threat yet has acquired growing relevance in scientific inquiry. Despite the relative simplicity of its virus genome, AAV exhibits intricate biological activities, thus making it an invaluable resource in molecular biology and genetic research. This review addresses the genome's architecture, its mode of interaction with host cells, and its prospective roles across various scientific investigations.
Summary of Adeno-Associated Virus
AAV is part of the Parvoviridae family and falls under the Dependoparvovirus genus. Known for its diminutive size, AAV possesses a non-enveloped, icosahedral capsid roughly 25 nm in diameter. Its genome, a single-stranded DNA molecule of about 4.7 kilobases, encodes two main regions: Rep, which is involved in replication and integration, and Cap, which codes for the structural proteins of the capsid. Flanking this genome are inverted terminal repeats (ITRs) that are vital for both replication and packaging.
In contrast to many pathogenic viruses, AAV does not cause disease on its own in humans or animals. Its ability to replicate depends on the presence of a helper virus, such as adenovirus or herpes simplex virus (HSV). Without these co-infections, AAV can enter a latent state-either persisting as an episome or integrating into specific genomic locations, notably at the AAVS1 site on human chromosome 19. This unique behavior, combined with its low immunogenic profile, sets AAV apart from other viral vectors.
The infection process of AAV is relatively direct and well-documented. Initially, the virus attaches to cell surface receptors-such as heparan sulfate proteoglycans, sialic acid, or integrins-depending on its serotype. Following receptor-mediated endocytosis, AAV is transported within the cell, escapes from the endosome, and eventually reaches the nucleus. Here, the single-stranded DNA genome is converted into a double-stranded template by host cell mechanisms, a prerequisite for transcription and replication.
Replication relies entirely on the helper virus, which provides necessary factors for viral DNA replication and capsid packaging. In productive infections, helper virus proteins support the generation and assembly of new AAV particles, resulting in the release of progeny virions. Without a helper virus, AAV persists either in an episomal form or through site-specific integration, maintaining a long-term presence without inducing cytopathic effects.
Because of its minimal pathogenicity, low immunogenicity, and capacity for sustained gene expression, AAV has become a crucial tool in molecular biology and genetic research. Engineered AAV vectors are widely used for gene delivery, functional genomics, and therapeutic interventions, effectively targeting various tissues including the central nervous system, liver, muscle, and retina. This adaptability has fostered their extensive application in both preclinical and clinical studies.
Additionally, AAV's ability to mediate transduction across species has broadened its utility in comparative genomics and translational medicine. Innovations in capsid engineering, such as the creation of synthetic and hybrid forms, have further refined tissue specificity, enhanced transduction efficiency, and reduced immune responses. These advancements continue to reinforce AAV's role as a primary vector in gene therapy, neuroscience, and regenerative medicine.
In conclusion, AAV is a distinctive viral system that offers significant advantages for both scientific research and biomedical applications despite its small genome and dependency on helper viruses. Its non-pathogenic nature, stable persistence in host cells, and versatility as a gene delivery tool make it an invaluable asset for both research and therapeutic development.
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Structure and Composition of the AAV Genome
Although the AAV genome spans roughly 4.7 kilobase pairs, it possesses several features of considerable scientific significance. This viral genetic material is single-stranded DNA, contained within an icosahedral capsid formed by 60 subunits. The main sections of the genome, termed Rep and Cap, fulfill distinct and critical roles.
(1) Rep Region
The Rep segment governs the replication cycle of AAV by encoding four proteins-Rep78, Rep68, Rep52, and Rep40-responsible for synthesizing viral DNA. These proteins can also facilitate integration into the host genome, a trait that distinguishes AAV from many other viruses.
(2) Cap Region
In contrast, the Cap segment encodes the virus's structural proteins, crucial for packaging and propagation. Within this region, three protein components (VP1, VP2, and VP3) collaborate to enable successful host-cell entry and subsequent delivery of the AAV genome.
Two inverted terminal repeats (ITRs) appear at the ends of the genome. These sequences are indispensable for both replication and packaging, as well as for preserving the virus's stability within the host cell.
Figure 1.Structural Diagram of the AAV Genome.(S V Martini.2011)
AAV Integrate into the Host Genome
A distinctive feature of the AAV genome is its potential for integration into the host genome. Unlike many other viruses, AAV has the ability to integrate its genome into the host's DNA. Although this integration is not a guaranteed outcome of infection, it can occur under specific conditions, enabling AAV to persist within the host cell for extended periods.
Studies have shown that AAV integration tends to occur at specific sites within the host genome, particularly within certain regions of chromosomes, such as the p5 region on human chromosome 1. The Rep proteins are central to this integration process, as they are responsible for recognizing and binding to the ITR sequences, facilitating the insertion of the viral genome into the host genome.
While AAV is capable of integrating into the host genome, the frequency of this event is relatively low. As a result, AAV is considered to be a safer vector for gene delivery and genetic research, as it does not typically induce significant genomic mutations or adverse effects in the host cell.
Figure 2.Integration Process of the AAV Genome.(Li, C.,et.al,2020)
Research and Applications of the AAV Genome
The unique properties of the AAV genome make it a critical tool in molecular biology. Engineered AAV vectors are widely used to deliver exogenous genes into host cells. This gene delivery capability has applications in studying gene function, gene knockout, and gene replacement experiments.
In the field of neuroscience, AAV vectors have been extensively utilized to study the role of specific genes in neurodegenerative diseases. Compared to other viral vectors, AAV exhibits high efficiency in gene transduction, with stable gene expression, particularly in brain and muscle cells. This makes AAV an ideal vector for investigating gene function and disease mechanisms.
AAV vectors are also utilized in cancer research, immunology, and the development of models for genetic disorders. In many studies, AAV has been used to deliver oncogenes or tumor suppressor genes, helping scientists uncover the molecular mechanisms behind tumorigenesis and identify new therapeutic targets.
Cross-Species Evolution of AAV Capsids for Enhanced Kidney Transduction. Chronic kidney disease (CKD) affects a significant proportion of the global population, creating an urgent need for efficient gene-transfer strategies. AAVs offer a promising solution due to their relatively low immunogenicity and ability to deliver therapeutic genes. However, effective transduction of kidney tissues has been challenging, prompting the development of novel AAV variants with improved tropism.
In a recent study (Rosales et al., 2024), researchers engineered AAV capsid libraries by introducing mutations in a critical region of the AAV9 capsid. These libraries were then "cycled" through multiple kidney models-including mice, pigs, non-human primates, and human kidney organoids-to select variants capable of robust renal transduction. High-throughput sequencing was used to identify and enrich capsid variants showing enhanced kidney tropism, particularly in proximal tubules.
Two variants, AAV.k13 and AAV.k20, demonstrated marked improvements in transgene expression within kidney tissues compared with the parental AAV9 capsid. Importantly, these variants retained the ability to transduce across species, indicating broad applicability. Delivery via intravenous, arterial, or ureteral routes all resulted in superior renal gene transfer, highlighting their potential in treating CKD or improving transplant outcomes.
Figure 3. AAV capsid libraries were progressively enriched through various vivo and ex vivo kidney model systems. (Alan Rosales, et.al,2025)
Despite its potential, challenges remain in optimizing AAV for broader applications. Enhancing AAV delivery efficiency, ensuring genome stability in host cells, and minimizing immune responses are ongoing research priorities.
Challenges and Future Directions
Although AAV offers numerous advantages, several obstacles hinder its broader application:
1. Limited Packaging Capacity: The AAV genome has a maximum capacity of approximately 4.7 kb for foreign DNA, restricting its utility for delivering large genetic sequences.
2. Immunogenic Concerns: While AAV exhibits relatively low immunogenicity, some individuals carry pre-existing antibodies that may diminish the efficacy of AAV-based interventions.
3. High Manufacturing Expenses: Large-scale production of AAV vectors with high purity remains costly, posing a barrier to widespread implementation.
Ongoing research seeks to mitigate these challenges. Innovations in genetic engineering focus on strategies such as modifying inverted terminal repeat (ITR) sequences to expand vector capacity and refining capsid proteins to improve tissue specificity while minimizing immunogenicity.
Conclusion
Despite its compact and structurally simple genome, adeno-associated virus exhibits exceptional adaptability, making it a critical resource in molecular biology and genetic studies. Its unique genetic architecture, combined with its capacity to integrate into the host genome under certain conditions, distinguishes it as an invaluable model for investigating viral-host dynamics, gene regulation, and cellular processes. Furthermore, its ability to remain in host cells without eliciting significant immune reactions enhances its stability and dependability as a research instrument.
As research on AAV advances, innovations in capsid engineering, genome editing, and vector refinement are anticipated to improve its efficacy and expand its utility. Addressing obstacles such as limited vector capacity, tissue specificity, and immune reactions will further amplify its potential across various scientific fields. Additionally, its role in functional genomics, evolutionary studies, and comparative virology underscores its growing importance in both basic and applied research contexts.
Through continued investigation, AAV is expected to assume an even more central role in unraveling genetic mechanisms, refining gene delivery methods, and advancing understanding in virology and molecular genetics. Future research is likely to uncover new insights, solidifying its position as an indispensable tool in contemporary biological science.
References:
- Martini SV, Rocco PR, Morales MM. Adeno-associated virus for cystic fibrosis gene therapy. Braz J Med Biol Res. 2011;44(11):1097-1104. doi:10.1590/s0100-879x2011007500123
- Li, C., Samulski, R.J. Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet 21, 255–272 (2020). https://www.nature.com/articles/s41576-019-0205-4
- Rosales, A., Blondel, L.O., Hull, J. et al. Evolving adeno-associated viruses for gene transfer to the kidney via cross-species cycling of capsid libraries. Nat. Biomed. Eng (2025). https://www.nature.com/articles/s41551-024-01341-0
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