Adeno-Associated Virus Vectors: Principles, Practices, and Prospects in Gene Therapy

Abstract

Gene therapy offers promising potential as an efficacious and long-lasting therapeutic option for genetic conditions, by correcting defective mutations using engineered vectors to deliver genetic material to host cells. Among these vectors, adeno-associated viruses (AAVs) stand out for their efficiency, versatility, and safety, making them one of the leading platforms in gene therapy. The enormous potential of AAVs has been demonstrated through their use in over 225 clinical trials and the FDA's approval of six AAV-based gene therapy products, positioning these vectors at the forefront of the field. This review highlights the evolution and current applications of AAVs in gene therapy, focusing on their clinical successes, ongoing developments, and the manufacturing processes required for the rapid commercial growth anticipated in the AAV therapy market. It also discusses the broader implications of these advancements for future therapeutic strategies targeting more complex and multi-systemic conditions and biological processes such as aging. Finally, we explore some of the major challenges currently confronting the field.

Keywords: adeno-associated virus (AAV); clinical trials; gene therapy; vector manufacturing.

Figure 1

AAV vector structure. (A) The wild-type (WT) AAV genome contains the rep (replication) and cap (capsid) genes, along with regulatory elements like polyadenylation (PolyA). These are flanked by two inverted terminal repeats (ITRs), which form T-shaped hairpin structures critical for genome replication and packaging. (B) In single-stranded recombinant AAVs (rAAV), the rep and cap genes are removed and replaced with a transgene expression cassette that includes a promoter, additional enhancers or a Kozak consensus sequence (yellow box), gene of interest, and PolyA, flanked by ITRs. The foreign DNA packaging capacity is approximately 4.5 kb. (C) In self-complementary rAAVs (scAAVs), the 5′ ITR is mutated, allowing the single-stranded genome to fold into a double-stranded structure, reducing the genome size to 2.2 kb but increasing the transduction efficiency. rAAVs can be tailored at the capsid and promoter levels, where the capsid’s serotype determines the tissue tropism, while the promoter drives tissue-specific or widespread transgene expression.

Figure 2

AAV transduction pathway. Main steps in the AAV transduction pathway: The rAAV capsid binds to cell surface receptors, triggering the internalization of the virus via endocytosis, which forms endosomes. Following endosomal escape, the AAV is transported into the nucleus and uncoats. The single-stranded AAV genome converts to double-stranded DNA (except in scAAV, which skips this step for faster transduction). The AAV DNA is then transcribed into mRNA, which exits the nucleus and is translated into therapeutic proteins.

Figure 3

A schematic overview of AAV vector manufacturing. Upstream process: Cells are expanded and transferred into a production bioreactor (50–200 L) based on the scale of AAVs required. A triple transfection process with three plasmids (transgene, packaging, and helper) is then performed to produce AAV vectors designed to deliver therapeutic genes. A few days later, the cells are lysed to release both full and empty AAV vectors, as well as other cellular components. Downstream process: Purification methods are employed, including affinity chromatography and ion exchange chromatography, which are crucial for separating full AAV capsids from empty ones, ensuring high purity and potency. Tangential flow filtration (TFF) is often used for concentration and buffer exchange, ensuring sterility. The final gene therapy product is packaged into vials and undergoes detailed characterization, including assessments of the viral genome titer, purity, and stability to ensure safe, clinical-grade AAV vectors.

Figure 4

Advancements in gene therapy for aging. Schematic representation of recent advancements in gene therapy targeting aging and age-related diseases. Top panel: AAV vectors for the delivery of therapeutic genes are being explored for various aging-related targets, for example, TERT to extend the lifespan and klotho to treat age-related diseases and improve cognition. APOE (for Alzheimer’s) and VEGF (for vascular health) are also under investigation. The AAV-mediated delivery of PGC-1α4 and NT-3 has demonstrated the restoration of muscle function in sarcopenia in mouse models, while AAV8-based therapies targeting longevity-associated genes (FGF21, α-klotho, and sTGFβR2) have enhanced the health span and longevity in multiple age-related disease models. Bottom panel: Emerging strategies like partial reprogramming, using the AAV-mediated delivery of Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc; OSKM), aim to rejuvenate cells, tissues, and organisms, showing the potential to reverse aging-related symptoms and improve longevity. Gene therapy approaches for targeting age-related diseases and phenotypes are still largely in the preclinical stages (animal models and early testing), with clinical trials expected to follow.