Vincenzo Di Cerbo, Ph.D.
Lead Scientist – Technology and Process Innovation Department
Cell and Gene Therapy Catapult
London, United Kingdom
AAV particles are too small for conventional microscopy, but may be detected through cryogenic transmission electron microscopy (cryoTEM), as shown in this figure. Three recombinant AAV (rAAV) species can be identified in this analysis. Fully packaged rAAV particles present a high inner density and no distinction between the outer shell and the core (black arrow). Empty rAAVs are characterized by a distinctive outer shell and low inner density (white arrow). Partly filled rAAVs show an intermediate inner density and a detectable outer shell (dashed black arrow). Ice contaminations, occasionally present in the analysis, have a distinctive pattern (dashed white arrow) and should not be confused with rAAVs.
What?
Adeno-associated viruses were initially discovered as a contamination of simian adenovirus preparations. As their name suggests, they require a helper function provided by adeno, herpes, or papilloma viruses for replication (1). AAV particles form a ~25 nm icosahedral capsid encapsulating a ~4.7 kb single-stranded DNA genome. The wild-type genome comprises two main open reading frames (ORFs) for rep proteins, responsible for genome replication and packaging, and cap proteins, essential for capsid structure formation. Flanking these ORFs, inverted terminal repeat (ITR) sequences play a key role in AAV replication and packaging. In gene therapy, native ORFs are fully replaced by an expression cassette encoding therapeutic genes that compensate for defective or pathological ones .
Why?
Three features make rAAVs amenable to gene therapy: broad tropism, non-pathogenicity, and low immunogenicity. With 12 identified natural serotypes and novel synthetic species generated through capsid engineering, rAAV tropism is expanding across various organs and tissues (3). Wild-type AAVs are naturally occurring in ~80% of the human population without symptoms, which, together with their overall inability to integrate into the human host genome, underscores their non-pathogenicity. Moreover, rAAVs infusions reportedly elicit a relatively low immunological response, contributing to their proven long-term therapeutic effects .
However, they also present limitations. Their small genome size restricts their cargo capacity to relatively small therapeutic genes, limiting gene replacement strategies for certain indications. The high prevalence of natural AAV infections may result in pre-existing immunity, which has prompted research in synthetic capsids solutions to overcome the presence of neutralizing antibodies . Despite overall safety, rAAV toxicity linked to both innate and adaptive immune responses, particularly for treatments requiring a high dose, has been reported . Additionally, pre-clinical studies in animal models have shown evidence of rAAV integration in the host genome leading to hepatocarcinoma in some mice models . Whilst no adverse events due to integration have been reported in clinical studies, long-term genotoxicity remains a frequently debated topic requiring ongoing monitoring (9).
Who?
rAAVs are the predominant viral vector modality from early-stage programs to commercialization, holding 45% of market shares . They are currently employed in ~350 clinical trials , whereas 7 AAV-based therapies have received regulatory approval in US and Europe to date.
When?
The first rAAV clinical trial was carried out in the early 90s to treat cystic fibrosis , yet it took nearly 20 years for the first rAAV therapy, Glybera, treating familial lipoprotein lipase deficiency, to gain European Medicines Agency (EMA) market authorization in 2012. However, due to its high cost and the extremely small patient population, the product was withdrawn from the market in 2017 despite its clinical efficacy . Around this time, a new product, Luxturna, was approved by the Food and Drug Administration (FDA) and EMA to treat a retinal dystrophy caused by RPE65 gene mutations . Between 2022-2023, 4 rAAV-based therapies have been approved in Europe or USA.
How?
Cost-efficient manufacturability of rAAV vectors remains a major challenge in the field . Innovations in upstream and downstream processing, along with analytics, are key to unlocking the current manufacturing capacity. Scaling-up rAAV production is costly and represents a significant barrier to commercially viable gene therapies, particularly those requiring higher doses, such as for systemic delivery or high prevalence diseases . An estimated 100-1000-fold increase in yield is necessary to overcome the current industry bottleneck .
Did you know that…
Glybera, dubbed “the world’s first million-dollar drug”, was the most expensive treatment at the time. Consequently, reimbursement negotiations turned out unsuccessful, and eventually only one patient obtained treatment following authorization. The remaining doses post-market withdrawal were administered for a nominal €1 fee . Recent rAAV gene therapies have seen prices soar to $3.5 million .
References
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