One size fits all: reapproaching AAVs
Recent research has culminated in a viral vector delivery system that is capable of delivering larger genes to target cells and tissues, with greater effectiveness than conventional methods.
A research paper, conducted by a team from the University of Zurich (Switzerland), unveils a novel adeno-associated virus (AAV) gene vector technology. This approach addresses a significant limitation in conventional AAV delivery systems – the efficient transport of large genes – and holds the potential to greatly enhance the delivery of larger-sized genes, like CRISPR-Cas9 modules, for gene therapy.
Gene therapies are advanced therapeutics that replace or inactivate disease-causing genes or conversely activate silent genes whose inactivity is disease-causing. The delivery of gene therapies to specific cells, tissues and organs is achieved by either viral or non-viral vector delivery systems. The most common form is that of an AAV, favorable for its advantageous safety profile and high gene transfer efficiency.
However, these have associated challenges that necessitate improving current methods. AAVs have limited genome packaging capacities of 4.7-5.0 kilobases, which is too short for several types of therapeutically relevant genes, such as CRISPR-Cas9 modules.
This has typically been rectified by using dual AAVs, where the gene is fragmented into smaller lengths, packaged into vectors that co-target the same cells and therein reconstitute within the cell. Reconstitution can occur at different levels depending on the design of the dual AAV delivery system: the AAV genome level, at the mRNA level and at the protein level.
AAV genome-level and protein-level reconstitution is hindered by a number of challenges, including low reconstitution efficiency, the production of immunogenic material or pathogenic protein segments (inteins) and low flexibility in split site selection, that necessitate time and resource-draining screening processes.
Beyond the barrier: ultrasound allows AAV vectors into the brain
Researchers utilized ultrasound to temporarily open the blood-brain barrier and deliver gene therapy directly to the brain as part of efforts to find a cure for Parkinson’s disease.
The research team designed and investigated a novel dual AAV delivery system based on reconstitution via mRNA trans-splicing called REVeRT, to probe whether a strategy could be developed to overcome these limitations.
The team used split-fluorophore and split-luciferin reporter gene assays, which consist of two individual protein fragments that combine to form a bioluminescent molecule. They were used to assess whether REVeRT could deliver the genetic material to the same cell and allow successful recombination of the fragments. If both fragments are co-delivered and reconstituted, then bioluminescence is observed. The team confirmed success in in vitro studies on human embryonic kidney cells, in vivo studies on mice retinal tissue and within human retinal organoids.
Next, the team investigated the ability of the gene delivery system to alleviate several different models of disease and assess whether the system is functional in a therapeutic context. They used a molecular complex that activates the transcription of a gene, called dCas9, that had been split into fragments in an analogous way to the aforementioned reporter gene assays. Both models of Usher syndrome and retinitis pigmentosa were successfully treated, showing the functionality of REVeRT in a therapeutically relevant context.
This was further explored in models of ABCA4-related Stargardt disease, an extremely common form of macular degeneration. REVeRT was successful in co-delivering fragmented ABCA4 genes, which went on to successfully reconstitute and alleviate the disease. This not only confirmed the success of REVeRT as a delivery system but also unveiled a potential therapeutic avenue for ABCA-4 related Stargardt disease, of which there is no current therapy.
“The advantages of this method are increased efficiency and fewer side effects,” explains lead author Elvir Becirovic. “It is also more flexible than previous methods, as the large genes can be divided into two fragments at various points.”