Oligonucleotide conjugated multi-functional adeno-associated viruses

Abstract

Recombinant adeno-associated viruses (AAVs) are among the most commonly used vehicles for in vivo gene delivery. However, their tropism is limited, and additionally their efficacy can be negatively affected by prevalence of neutralizing antibodies in sera. Methodologies to systematically engineer AAV capsid properties would thus be of great relevance. In this regard, we develop here multi-functional AAVs by engineering precision tethering of oligonucleotides onto the AAV surface, and thereby enabling a spectrum of nucleic-acid programmable functionalities. Towards this, we engineered genetically encoded incorporation of unnatural amino acids (UAA) bearing bio-orthogonal chemical handles onto capsid proteins. Via these we enabled site-specific coupling of oligonucleotides onto the AAV capsid surface using facile click chemistry. The resulting oligo-AAVs could be sequence specifically labeled, and also patterned in 2D using DNA array substrates. Additionally, we utilized these oligo conjugations to engineer viral shielding by lipid-based cloaks that efficaciously protected the AAV particles from neutralizing serum. We confirmed these 'cloaked AAVs' retained full functionality via their ability to transduce a range of cell types, and also enable robust delivery of CRISPR-Cas9 effectors. Taken together, we anticipate this programmable oligo-AAV system will have broad utility in synthetic biology and AAV engineering applications.

Figure 1

Engineering robust UAA incorporation into AAVs. (a) Schematic of approach for addition of an azide bearing UAA to the virus capsid and subsequent click-chemistry based chemical linking of an effector to the UAA. (b) Locations of the surface residues assayed for replacement with UAAs (VP1 residues numbered). (c) Relative infective titers of the AAV2 mutants in the presence and absence of 2 mM UAA, quantified via the transduction of HEK293T cells and subsequent mCherry exprssion (n = 3 independent replicates, cells transduced with equal volumes of virus) (Error bars are SEM). (d) Comparison of the viral titers of AAV2-N587UAA and AAV-DJ-N589UAA (Error bars are SEM). (e) Confirmation that UAA incorporation does not negatively affect AAV activity (n = 3 independent replicates; experiments performed in HEK293Ts at varying vector genome containing particles (vg)/cell) (Error bars are SEM).

Figure 2

Confirmation of UAA-AAV functionality, and site-specific oligonucleotide conjugation onto UAA-AAVs. (a) Coomassie stain of SDS-PAGE resolved capsid proteins of AAV-DJ and AAV-DJ-N589UAA. (b) Coomassie stain of SDS-PAGE resolved capsid proteins of AAV-DJ and AAV-DJ-N589UAA following treatment with an alkyne-oligonucleotide (10 kDa). (c) Coomassie stain and western blot of the non-denatured AAV-DJ and AAV-DJ-N589UAA following treatment with an alkyne-oligonucleotide, and probed with a complementary oligonucleotide-biotin conjugate followed by streptavidin-HRP. (d) Fluorophore pseudotyping of AAVs via Alexa594 DIBO alkyne: successful linking onto the virus confirmed via fluorescence visualization of the virus 2 hours post addition of AAVs onto HEK293T cells (scale bars: 250 μm). (e) Oligonucleotide pseudotyping of AAVs via alkyne-tagged oligonucleotide: selective capture on DNA spots (arrayed in a checkerboard fashion) of AAVs bearing corresponding complementary oligonucleotides, evidenced via specific viral transduction of HEK293T cells dispersed on those spots (scale bars: 250 μm).

Figure 3

Engineering ‘cloaked AAVs’ resistant to neutralization via sera, and their functional characterization via CRISPR-Cas9 genome editing. (a) Representation of a ‘cloaked AAVs’ resistant to antibody neutralization. (b) Relative activity of AAVDJ and AAVDJ-N589UAA viruses tethered to a range of small molecule and polymer moieties post exposure to pig serum assayed via AAV-mCherry based transduction of HEK293T cells (n = 4 independent replicates) (error bars are SEM). (c) AAVS1 editing rates (% NHEJ events) of AAV-DJ-N589UAA, AAV-DJ-N589UAA + oligo, and AAV-DJ-N589UAA + oligo + lipofectamine in HEK293T cells (1E + 5 vg/cell) (error bars are SEM).