What you should know about rAAV gene therapy

Curing a disease with one injection: the dream, the hope, the goal of medicine. Gene therapy brings this vision to reality by harnessing viruses into therapeutic tools. Among them, adeno-associated viruses (AAVs) are the most used: genetically modified AAVs, named recombinant AAVs (rAAVs), are already used in six gene therapies approved for medical use. Over 200 clinical trials are ongoing.

AAV, a virus reprogrammed to cure diseases

Gene therapy inserts genetic instructions into a patient to correct a mutation responsible for a genetic disorder. Thanks to genetic engineering, researchers have co-opted AAVs (along with adenoviruses, herpes simplex viruses and lentiviruses) into delivering these instructions. Researchers have swapped the genes that allow AAVs to jump from person to person with genes to treat diseases. In other words, the virus has been genetically reprogrammed into a vector for gene transfer. The gene supplemented is referred to as transgene.

Biology of AAVs

AAVs were discovered in the 1960s as contaminants in cell cultures infected by adenoviruses, a coexistence to which they owe their name.

AAVs consist of a protein shell (capsid) wrapped around the viral genome, a single strand of DNA long approximately 4,700 bases (4.7 kb). The genome is capped at both ends by palindromic repetitive sequences folded into T-shaped structures, the Inverted Tandem Repeats (ITRs). Sandwiched between the ITRs, four genes are found. They determine capsid components (cap) and capsid assembly (aap), genome replication (rep) and viral escape from infected cells (maap) (Figure 1, top panel).

The replacement of these four genes with a transgene of therapeutic use and its expression by infected cells (transduction) lie at the heart of gene therapy mediated by rAAVs.

Transgene transfer by rAAVs

Researchers favour rAAVs as vectors because AAVs are safe (they are not linked to any disease and do not integrate into the genome), they can maintain the production of a therapeutic gene for over ten years and infect a wide range of tissues.

In an rAAV, the ITRs are the only viral element preserved. The four viral genes are replaced by a therapeutic transgene, and regulatory sequences to maximise its expression. Therefore, an rAAV contains the coding sequence of the transgene, an upstream promoter to induce transcription and a downstream regulatory sequence (poly-A tail) to confer stability to the mRNA molecules produced (Figure 1, bottom panel).

Steps of rAAV production

Based on the disease, rAAVs can be administered into the blood, an organ, a muscle or the fluid bathing the central nervous system (cerebrospinal fluid).

rAAVs dock on target cells via a specific interaction between the capsid and proteins on the cell surface that serve as viral receptors and co-receptors. The capsid mainly dictates which cell types will be infected (cell tropism).

Upon binding, the cell engulfs the virus into membrane vesicles (endosomes) typically used to digest and recycle material. The rAAVs escape the endosomes, avoiding digestion, and enter the nucleus, where the capsid releases the single-strand DNA (ssDNA) genome, a process known as uncoating. The ITRs direct the synthesis of the second strand to reconstitute a double-strand DNA (dsDNA), the replication of the viral genome and the concatenation of individual genomes into larger, circular DNA molecules (episomes) that can persist in the host cell for years.

Nuclear proteins transcribe the transgene into mRNAs; mRNAs are exported in the cytoplasm where they are translated into proteins. The rAAV has achieved successful transduction: the transgene can start exerting its therapeutic effects.

A simplified overview of rAAV transduction is presented in Figure 2.

The triumphs of rAAV gene therapies

rAAV gene therapies are improving lives and saving patients. Unsurprisingly, the most remarkable examples of this come from the drugs already approved.

Roctavian is an rAAV gene therapy for haemophilia A, a life-threatening bleeding disorder in which the blood does not clot properly because the body cannot produce the coagulation Factor VIII. In a phase III clinical trial, Roctavian reduced bleeding rates by 85% and most treated patients (128 out of 134) no longer needed regular administration of Factor VIII, the standard therapy for the disease, for up to two years after treatment. Similar impressive results were noted for the rAAV Hemgenix, a gene therapy for haemophilia B (a bleeding disorder caused by the absence of the coagulation Factor IX). Hemgenix reduced bleeding rates by 65% and most treated patients (52 out of 54) no longer needed regular administration of Factor IX, for up to two years.

The benefits of Zolgensma are even more awe-inspiring. Zolgensma is an rAAV gene therapy for spinal muscular atrophy (SMA), a genetic disorder in which neurons in the spinal cord die causing muscles to waste away irreversibly. The life expectancy of SMA patients can be as short as two years, therefore timing is critical. As a consequence, Zolgensma had to be tested in neonates: babies with the most severe form of SMA were dosed with the drug before six weeks of age and symptoms onset (SPRINT study). After 14 months, all 14 treated babies were alive and breathing without a ventilator, whilst only a quarter of untreated babies did. After 18 months, all 14 could sit without help, an impossible feat without Zolgensma.

These and other resounding achievements are fuelling research on rAAVs gene therapies.

Current limitations

Scientists still have some significant hurdles to overcome:

●       Packaging capacity: AAVs can fit in their capsids relatively short DNA sequences, which do not allow the replacement of many long genes associated with genetic disorders,

●       Immunogenicity: 30-60% of individuals have antibodies against AAVs, which block rAAVs and prevent transduction,

●       Tissue specificity: rAAVs often infect tissues which are not the intended target (e.g., inducing the expression for a transgene to treat a neurological disease in the liver rather than in neurons).

Gene therapies, not only those delivered by rAAVs, face an additional challenge, this one only partially of a technological nature: their price tags. Their prices – rAAVs range from $850,000 (£690,000) to $3,500,000 (£2,850,000) – make them inaccessible for most patients. A cautionary tale is already out there: Glybera, the first rAAV gene therapy approved for medical use, albeit only in Europe (2012), was discontinued in 2017 because too expensive. Research is likely to reduce the exorbitant manufacturing costs, but the time may have come to reconsider our healthcare systems. 

Notes

1. One non-viral vector exists, but its development lags behind the viral vector.

2. Glybera for treating lipoprotein lipase deficiency, Luxturna for Leber congenital amaurosis, Zolgensma for spinal muscular atrophy, Roctavian for haemophilia A, Hemgenix for haemophilia B, and Elevidys for Duchenne muscular dystrophy.

Written by Matteo Cortese, PhD

Related articles: Germline gene therapy (GGT) / A potential treatment for HIV / Rabies