Site Specific Modification of Adeno-Associated Virus Enables Both Fluorescent Imaging of Viral Particles and Characterization of the Capsid Interactome

Abstract

Adeno-associated viruses (AAVs) are attractive gene therapy vectors due to their low toxicity, high stability, and rare integration into the host genome. Expressing ligands on the viral capsid can re-target AAVs to new cell types, but limited sites have been identified on the capsid that tolerate a peptide insertion. Here, we incorporated a site-specific tetracysteine sequence into the AAV serotype 9 (AAV9) capsid, to permit labelling of viral particles with either a fluorescent dye or biotin. We demonstrate that fluorescently labelled particles are detectable in vitro, and explore the utility of the method in vivo in mice with time-lapse imaging. We exploit the biotinylated viral particles to generate two distinct AAV interactomes, and identify several functional classes of proteins that are highly represented: actin/cytoskeletal protein binding, RNA binding, RNA splicing/processing, chromatin modifying, intracellular trafficking and RNA transport proteins. To examine the biological relevance of the capsid interactome, we modulated the expression of two proteins from the interactomes prior to AAV transduction. Blocking integrin αVβ6 receptor function reduced AAV9 transduction, while reducing histone deacetylase 4 (HDAC4) expression enhanced AAV transduction. Our method demonstrates a strategy for inserting motifs into the AAV capsid without compromising viral titer or infectivity.

Materials promoting viral gene delivery

“…Strategies include amino acids mutations, peptide domain insertions and incorporation of chemical functional groups [83][84][85] as recently reviewed for AAV. [86][87][88] For example, introduction of unnatural amino acid residues such as azides on the capsid surface can give access to selective click chemistry.…”

“…[86][87][88] For example, introduction of unnatural amino acid residues such as azides on the capsid surface can give access to selective click chemistry. 89,90 Further, genetically modified viruses can enhance TE, 91,92 broaden the tropism, 63,93 enable fluorescent imaging, 85 escape neutralizing antibodies, 94,95 generate stimuli responsive vectors, 96 e.g. by light 97,98 or enzymes, 99 and target cells.…”

Positron emission tomography imaging of novel AAV capsids maps rapid brain accumulation

“…The insertion site is located in the N-terminal disordered region of capsid proteins VP1 and VP2. A similar modification was made in AAV9 and was shown to be accessible by labeling reagents without compromising viral integrity 24 .…”

“…2e), directly related to the ratio of protein abundance for the three VPs, 1:1:10 (VP1, VP2, VP3). AAV9-TC was generated by site-specific insertion of the HRWCCPGCCKTF peptide motif at the VP1/VP2 interface at the 139 th amino acid 24 . As a result, the gel image from autoradiography of 64 Cu-AAV9-TC showed the VP2 band as the major radiolabeled VP whereas the protein staining (blue) of 64 Cu-AAV9-TC was similar to the ratio of each VP (VP1:VP2: VP3, 1:1:10) (right column image of Fig.…”

“…PET imaging has not previously been applied for systemic AAV tracking. Surface modification of AAVs has previously focused on tagging fluorophores [19][20][21][22][23][24] to PEG 25,26 , or adding peptides 27,28 , antibodies 23 , or small molecules 29 to the capsid. Given that most earlier generations of AAV and other viral therapies were not designed for specific organ targeting, imaging studies labeled multiple lysines on the capsid with a lesser impact on organ-specific endothelial targeting and transduction 30 .…”