Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2024 Apr 3;9(1):78.
doi: 10.1038/s41392-024-01780-w.

Adeno-associated virus as a delivery vector for gene therapy of human diseases

Affiliations
Review

Adeno-associated virus as a delivery vector for gene therapy of human diseases

Jiang-Hui Wang et al. Signal Transduct Target Ther. .

Abstract

Adeno-associated virus (AAV) has emerged as a pivotal delivery tool in clinical gene therapy owing to its minimal pathogenicity and ability to establish long-term gene expression in different tissues. Recombinant AAV (rAAV) has been engineered for enhanced specificity and developed as a tool for treating various diseases. However, as rAAV is being more widely used as a therapy, the increased demand has created challenges for the existing manufacturing methods. Seven rAAV-based gene therapy products have received regulatory approval, but there continue to be concerns about safely using high-dose viral therapies in humans, including immune responses and adverse effects such as genotoxicity, hepatotoxicity, thrombotic microangiopathy, and neurotoxicity. In this review, we explore AAV biology with an emphasis on current vector engineering strategies and manufacturing technologies. We discuss how rAAVs are being employed in ongoing clinical trials for ocular, neurological, metabolic, hematological, neuromuscular, and cardiovascular diseases as well as cancers. We outline immune responses triggered by rAAV, address associated side effects, and discuss strategies to mitigate these reactions. We hope that discussing recent advancements and current challenges in the field will be a helpful guide for researchers and clinicians navigating the ever-evolving landscape of rAAV-based gene therapy.

PubMed Disclaimer

Conflict of interest statement

G.G. is a scientific co-founder of Voyager Therapeutics, Adrenas Therapeutics, and Aspa Therapeutics, and holds equity in these companies. G.G. is an inventor on patents with potential royalties licensed to Voyager Therapeutics, Aspa Therapeutics, and other biopharmaceutical companies. D.J. G. is a cofounder of Aspa Therapeutics and holds equity in the company. D.J.G. is an inventor of patents with potential royalties licensed to Aspa Therapeutics and other biopharmaceutical companies. The remaining authors declare no competing interests. G.G. is one of the Associate Editors of Signal Transduction and Targeted Therapy, but he was not involved in the editorial administration and review of the manuscript.

Figures

Fig. 1
Fig. 1
Approved gene therapy products and delivery platforms. Gene therapy products are generally developed for 1) Ex vivo gene therapy where affected patient cells are isolated, genetically modified in cell culture, expanded, enriched, and reinfused into patients to function as a living drug (e.g., CAR-T cells, Lyfgenia, Casgevy) and 2) In vivo gene therapy which is administered directly to patients to achieve therapeutic effects (e.g., Gendicine, Kynamaro, Imlygic, Luxturna, Onpattro). Delivery platforms for gene therapy drugs are primarily categorized into two groups: viral and non-viral-based. Viral-based gene therapy utilizes viruses as gene delivery vectors, including AAV, adenovirus, retrovirus, lentivirus, and herpes simplex virus. Non-viral-based gene therapy includes antisense oligonucleotides, siRNAs, and cell-based CRISPR genome editing. CAR-T chimeric antigen receptor (CAR) T cell therapy, SMA spinal muscular atrophy, AADC aromatic L-amino acid decarboxylase deficiency DMD Duchenne muscular dystrophy, LCA2 Leber congenital amaurosis type 2, hATTR hereditary transthyretin amyloidosis. * indicates non-FDA-approved gene therapy. Figure created with Biorender.com
Fig. 2
Fig. 2
Historical milestones in AAV biology research and gene therapy development. Decades of studying AAV biology have led to crucial advances in understanding its structure, biology, vectorology, and gene therapy applications. Historical milestones in AAV research and development are summarized chronologically. These advancements paved the way for successful clinical trials and regulatory approvals for rAAV-based gene therapeutics to treat various human diseases. Figure created with Biorender.com
Fig. 3
Fig. 3
Strategies applied for the development of novel AAV variants. a Natural occurring AAV variants can be isolated from human and NHP tissues using high-cycle PCR and high-throughput sequencing. b Rational design utilizes knowledge of AAV biology to modify the relevant amino acids in AAV capsid to enhance the transduction capability or evade immune surveillance. c Directed evolution is an engineering approach to develop novel AAV variants with designated specificity, including random or defined peptide insertion, capsid shuffling, error-prone PCR, and saturation mutagenesis. d In silico approach utilizes known capsid sequences to reconstruct ancestral AAV sequences. Machine learning is being used for predicting the relationship between specific sequences in the AAV genome with packaging capabilities and tissue tropism by using large datasets of samples transduced with AAVs whose genome had been mutagenized. Figure created with Biorender.com
Fig. 4
Fig. 4
Current approaches to manufacture rAAV. Currently, two main platforms are used for rAAV manufacturing: transfection- and viral infection-based approaches. Plasmid transient transfection of HEK293 cells remains the most widely used method, while stable cell lines, baculovirus (BV) systems, and the HSV type I system offer scalable alternatives for large-scale production. The transfection-free helper virus system TESSA has been developed to produce high-yield rAAVs. Pharmaceutically inducible all-in-one producer cell lines may represent the next generation’s optimal manufacturing platform for rAAV-based drugs. BHK baby hamster kidney. Figure created with Biorender.com
Fig. 5
Fig. 5
Current clinical applications of rAAV in major human diseases. Clinical applications of rAAV across a spectrum of significant human diseases, including ocular, neurological, metabolic, hematological, neuromuscular, cardiovascular diseases, and oncology. Refer to Supplementary Table 1 for a comprehensive list of 238 clinical trials employing rAAV-based gene therapy for the aforementioned diseases. Figure created with Biorender.com
Fig. 6
Fig. 6
Immune responses in rAAV-based gene therapy. a Pre-existing AAV-specific antibodies can interact with rAAV and block its target cell entry. Bacteria-derived endopeptidase IdeS and its homologs can cleave the intact antibody into Fab and Fc fragments, thus reducing its half-life in circulation and eliminating Fc-mediated functions. Alternatively, the capsid can be modified or encapsulated by EVs to prevent antibody recognition. b Complement activation is observed when high doses of rAAV are administered. Though the exact mechanism remains unclear, a leading hypothesis is that rAAV adhering to the cell surface activates the classical pathway by binding to complement C1qs, which cleaves C4 and C2 to form the C3 convertase C4b2b. C3 is then cleaved and forms C5 convertase that cleaves C5 into C5b, which associates with C6–9 to form the membrane attack complex (MAC) that directly causes cell lysis, resulting in liver or kidney injury. Complement activation mediates endothelial damage, thrombotic microangiopathy (TMA), and atypical hemolytic uremic syndrome (aHUS). Cyclic peptide APL-9 is a C1 inhibitor (C1inh) and eculizumab is an inhibitor of C1, C3, and C5, which may suppress the complement activation cascade. c Innate receptors, including TLR2 for virus capsid, TLR9 for unmethylated CpG, and RIG-I/MDA5 for dsRNA, have been shown to promote inflammation. Through a series of signal transduction events, IRF3, IRF7, and NF-κB will become phosphorylated and translocate to the nucleus, where they activate type I interferons (IFN-I), interferon-stimulated genes (ISGs), and proinflammatory cytokines that mediate antiviral immunity and propagate inflammation. Many pathway components can be targeted by pharmacological inhibitors. In addition, the telomere-derived io2 sequence can suppress TLR9-mediated recognition of the transgene. d Transgene and capsid can be processed by the proteasome into peptides, which are presented by MHC-I or MHC-II molecules. CD8 and CD4 T cells that recognize those peptides will become activated. CD8 T cells may directly kill the rAAV-transduced cell, while CD4 T cells can help both CD8 T cells and B cells to enhance their effector function. B cells with specificity to the capsid can release antibodies that eliminate future possibilities of redosing. miRNA-binding sites engineered to the transgene can interact with cellular miRNA, leading to transcript degradation. In antigen-presenting cells, this design may prevent the presentation of transgene-derived peptides to T cells. In addition, anti-CD20 antibody rituximab and mTOR inhibitor rapamycin can be used to reduce adaptive responses. Figure created with Biorender.com

Similar articles

Cited by

References

    1. Wang D, Gao G. State-of-the-art human gene therapy: part II. Gene therapy strategies and clinical applications. Discov. Med. 2014;18:151–161. - PMC - PubMed
    1. Adams D, et al. Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis. N. Engl. J. Med. 2018;379:11–21. doi: 10.1056/NEJMoa1716153. - DOI - PubMed
    1. Wang J, et al. AAV-delivered suppressor tRNA overcomes a nonsense mutation in mice. Nature. 2022;604:343–348. doi: 10.1038/s41586-022-04533-3. - DOI - PMC - PubMed
    1. Wang D, Gao G. State-of-the-art human gene therapy: part I. Gene delivery technologies. Discov. Med. 2014;18:67–77. - PMC - PubMed
    1. Bulcha JT, Wang Y, Ma H, Tai PWL, Gao G. Viral vector platforms within the gene therapy landscape. Sig. Transduct. Target. Ther. 2021;6:53. doi: 10.1038/s41392-021-00487-6. - DOI - PMC - PubMed

Publication types