Adeno-associated virus (AAV) vectors are one of the most interesting and impactful biomedical technologies in development. They pose an interesting biological puzzle through their life cycle, adaptability and surprisingly complex interactions with a myriad of host cell factors on the cell surface, in the cytosol and in the nucleus. AAV vectors are highly impactful as they are the leading gene delivery platform spearheading multiple preclinical and clinical successes involving gene replacement, silencing and editing. Recent achievements include the micro-dystrophin gene replacement therapy for Duchenne muscular dystrophy (1), the now commercially approved AAV1 (Glybera, 2)- and AAV2 (Luxturna, 3)-based platforms, but also Cas9 reconstitution by PTS (intein-mediated protein trans-splicing) (4,5).

AAV as a Virus

AAV is found in humans and non-human primates and is not known to cause any human diseases. It has a single-stranded DNA genome of about 4.7 kb enclosed in an icosahedral protein capsid roughly 26 nm in diameter (6). The viral capsid is made up of three types of protein subunits, VP1, VP2 and VP3, repeated to a total of 60 copies in a 1:1:10 ratio, respectively. The viral genome houses three genes and is flanked by two inverted terminal repeats (ITRs) that largely serve as origins of replication and packaging signals. Through alternative splicing and translation from different start codons, multiple proteins can be synthesized from the same viral gene. The rep gene encodes four proteins required for viral replication. The cap gene encodes the three protein subunits that construct the capsid. A third gene is hidden within the cap coding sequence but in a different reading frame, and it encodes the assembly activating protein (AAP) which stimulates virion assembly (7, 8). AAV can integrate into the genome of human cells in a locus designated AAVS1 on chromosome 19 to establish latency (9).

AAV as a Vector for Gene Delivery

To achieve their potential as vectors for in vivo gene delivery, recombinant AAV (rAAV) genomes contain therapeutic gene expression cassettes in place of AAV protein-coding sequences. The ITRs are the only viral sequences shared between wild-type and recombinant AAVs, as they are essential for genome replication and packaging during vector production. Removal of the viral coding sequences increases the rAAV packaging capacity, being able to accommodate genomes up to 5 kb, and decreases their immunogenicity and cytotoxicity when delivered in vivo (10).

Many ongoing studies investigate AAV biology, the molecular interactions between the capsid and target cell surface receptors as well as the downstream events following internalization, culminating with second-strand synthesis and eventual integration (11-13). Prevalent serotypes (classified based on their cell surface antigens) are presumed to recognize distinct cell receptors and thereby display different cell type and tissue type preferences, or tropism profiles (13). Upon binding to cell surface receptors, rAAV particles are internalized into endosomes where they undergo pH-dependent structural changes and traffic through the cytosol along the cytoskeletal network (14).

After entering the nucleus through the nucleus pore complex, the viral capsid is uncoated to release the genome (15). Identifying the key host factors and underlying mechanisms regulating intracellular trafficking would greatly benefit recombinant AAV (rAAV) transduction efficiency. Before it can be transcribed, the single-stranded rAAV genome must be converted to a double-stranded form. Second-strand synthesis is initiated by the self-primed 3’-ITR, and the assembly of double-stranded genomes can be aided further by delivering virions packaged with complementary rAAV strands (16, 17). Replication of the rAAV genome and assembly of virion particles are dependent on host cell factors and other elements provided by helper viruses, such as adenovirus (AdV) and herpes simplex virus (HSV).

Vector Design

We are witnessing a new stage in the development of viral vector-based gene therapies, with technologies such as high-throughput sequencing/screening and advanced computational tools that greatly support capsid directed evolution and in silico approaches (18-21). Additionally, techniques such as error-prone PCR and capsid shuffling are commonly employed to generate capsid libraries for screening (18-22). Ongoing work in rAAV genome design, on the other hand, is leading with strategies such as micro-gene, dual-vector, or PTS-mediated protein reconstitution that enable the use of larger transgenes (5, 23-25).

Although gene replacement therapies have enjoyed the most clinical success, gene addition can address some of the most urgent unmet medical needs, such as heart failure and infectious diseases—by supplying growth factors, tuning signaling pathways, etc. Editing strategies, on the other hand, can silence or mutate target genes or introduce new ones at specific genomic loci. These are fascinating avenues to follow, and they bring real hope for effective, safe and cost-efficient gene therapy in the near future.

But what are the key challenges for producing clinical-grade vectors? Some of the main questions addressed by ongoing investigations include the following: Are recombinant baculovirus platforms better than stable eukaryotic cell lines for achieving large-scale production? Are ex vivo potency assays using human explants applicable for different targeted tissues? Are they a good replacement for animal models? Particularly intriguing is also the interaction with the host immune system. Would autologous Treg cells co-delivered in vivo with the gene therapy efficiently modulate the rAAV-triggered immune response? These and similar interrogation points are actively investigated to achieve safe and affordable clinical-grade vector production.

References

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Author: Sebastian Florescu, PhD

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Published online: 12 April 2020