Engineering AAV receptor footprints for gene therapy
Introduction
The last decade has witnessed the transition of Adeno-associated viruses (AAV) from gene transfer vectors to clinically viable, therapeutic candidates. Discovered as a contaminant in Adenovirus preparations during the mid 1960s [1], at first AAV failed to garner much clinical interest due to its complete lack of pathogenicity [2]. AAV is helper-dependent, meaning its replication relies on the presence of factors from a helper virus, such as adenovirus or herpes simplex virus [3, 4]. As a member of the Dependovirus genus of Parvoviridae, AAV has a T = 1 icosahedral capsid, ∼25 nm in diameter. The 4.7 kb single-stranded DNA genome contains two open reading frames: Rep and Cap [5]. Rep encodes four alternatively spliced proteins for viral genome packaging and replication, while Cap, encodes the capsid structural proteins VP1, VP2, and VP3 along with the Assembly Activating Protein (AAP) [5, 6, 7]. VP1, VP2, and VP3 share a common beta barrel domain, but differ in their N-terminal extension [5, 6, 7].
The AAV genome is flanked by inverted terminal repeats (ITRs), which are crucial for packaging, replication, and integration into the host genome. The ITRs represent the only essential cis-acting element of the AAV genome, enabling the incorporation of different transgenes between the ITRs, which can then be packaged into AAV capsids to generate recombinant AAV vectors (rAAV) [8]. Recombinant AAVs can be generated via transfection of plasmids containing the ITR-flanked transgene, an AAV capsid gene, and necessary helper genes, followed by harvest of transfected cells and purification of virus [5, 6, 7]. The application of rAAVs in gene therapy applications has rapidly mushroomed over the past decade. During this time, significant improvements to rAAV technology have been made with the goal of improving gene transfer efficiency, altering tropism and/or reducing antigenicity [9, 10]. Structural studies of AAV capsids combined with rational mutagenesis as well as combinatorial protein engineering strategies combined with directed evolution have yielded several lab-derived AAV strains [9, 10]. Here, we specifically review AAV capsid engineering strategies focused on exploiting knowledge of AAV capsid structure and interactions with host cell surface glycan receptors. Potential future studies focused on re-engineering other AAV–receptor footprints are also discussed.
Section snippets
AAV and host receptors
Several distinct AAV serotypes and variants have been discovered and isolated from different animal species including humans over the past decade [9, 11, 12, 13, 14]. It is now well known that different AAV isolates can utilize a variety of cell surface glycans for cell surface binding, including heparan sulfate (HS), N-linked sialic acids (SIA), O-linked sialic acids or mucins (MUC), or galactose (GAL) [5, 15, 16, 17, 18, 19]. Some naturally occurring AAV serotypes recognize two different
Structural determinants of AAV glycan interactions
The VP3 capsid protein subunit of AAV consists of 8 antiparallel beta strands forming a barrel. The beta strands are interconnected by variable loops, which are localized on the capsid surface. The VP1, VP2 and VP3 monomers assemble with twofold, threefold, and fivefold axes of symmetry to create the 60-subunit AAV capsid [5, 7, 34, 35]. The twofold axis of symmetry is characterized by depressions, where the variable loops form the barrier between these depressions [5]. The variable loops also
Re-engineering AAV capsid glycan interactions
A thorough structural understanding of AAV capsid glycan interactions has enabled rational manipulation of glycan footprints on the AAV capsid surface. This re-engineering approach has yielded novel, synthetic AAV strains with potential applications in therapeutic gene transfer. Specifically, structure-inspired design has been utilized to abrogate capsid binding to glycan receptors, alter binding affinity, and more recently engineer orthogonal glycan receptor interactions. Structural diagrams
Conflict of interest
Aravind Asokan is a co-founder at Stridebio LLC and an inventor on patents owned by UNC-CH.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We would like to acknowledge funding support from the NIH (R01HL089221; P01HL112761 awarded to AA and training grant T32GM007092 to VM).
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