Adeno-associated virus serotype 9 (AAV9) has enhanced capsid-associated tropism for cardiac muscle and the ability to cross the blood-brain barrier compared to other AAV serotypes. To help identify the structural features facilitating these properties, we have used cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction (cryo-reconstruction) and X-ray crystallography to determine the structure of the AAV9 capsid at 9.7- and 2.8-Å resolutions, respectively. The AAV9 capsid exhibits the surface topology conserved in all AAVs: depressions at each icosahedral two-fold symmetry axis and surrounding each five-fold axis, three separate protrusions surrounding each three-fold axis, and a channel at each five-fold axis. The AAV9 viral protein (VP) has a conserved core structure, consisting of an eight-stranded, β-barrel motif and the αA helix, which are present in all parvovirus structures. The AAV9 VP differs in nine variable surface regions (VR-I to -IX) compared to AAV4, but at only three (VR-I, VR-II, and VR-IV) compared to AAV2 and AAV8. VR-I differences modify the raised region of the capsid surface between the two-fold and five-fold depressions. The VR-IV difference produces smaller three-fold protrusions in AAV9 that are less “pointed” than AAV2 and AAV8. Significantly, residues in the AAV9 VRs have been identified as important determinants of cellular tropism and transduction and dictate its antigenic diversity from AAV2. Hence, the AAV9 VRs likely confer the unique infection phenotypes of this serotype.

Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY−F+T−V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY−F+T−V) −hGRK1−GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.

Vectors based on adeno-associated virus (AAV) serotype 9 are candidates for in vivo gene delivery to many organs, but the receptor(s) mediating these tropisms have yet to be defined. We evaluated AAV9 uptake by glycans with terminal sialic acids (SAs), a common mode of cellular entry for viruses. We found, however, that AAV9 binding increased when terminal SA was enzymatically removed, suggesting that galactose, which is the most commonly observed penultimate monosaccharide to SA, may mediate AAV9 transduction.

The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of AAV2 vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. Removal of several critical tyrosine (Y) and serine (S) residues on the AAV2 capsid has been shown to significantly increase transduction efficiency compared with the wild-type (WT) vectors. In the present study, site-directed mutagenesis of each of the 17 surface-exposed threonine (T) residues was conducted, and the transduction efficiency of four of these mutants, T455V, T491V, T550V, and T659V, was observed to increase up to 4-fold in human HEK293 cells in vitro. The most critical Y, S, and T mutations were subsequently combined, and the quadruple-mutant (Y444+500+730F+T491V) AAV2 vector was identified as the most efficient. This vector increased the transduction efficiency ∼24-fold over the WT AAV2 vector, and ∼2–3-fold over the previously described triple-mutant (Y444+500+730F) vector in a murine hepatocyte cell line, H2.35, in vitro. Similar results were obtained in murine hepatocytes in vivo following tail vein injection of the Y444+500+730F+T491V scAAV2 vector, and whole-body bioluminescence imaging of C57BL/6 mice. The increase in the transduction efficiency of the Y-T quadruple-mutant over that of the Y triple-mutant correlated with an improved nuclear translocation of the vectors, which exceeded 90%. These observations suggest that further optimization of the AAV2 capsid by targeting amino acid residues involved in phosphorylation may not be possible. This study has thus led to the generation of a novel Y444+500+730F+T491V quadruple-mutant AAV2 vector with potential for use in liver-directed human gene therapy.

b Adeno-associated virus serotype 9 (AAV9) vectors show promise for gene therapy of a variety of diseases due to their ability to transduce multiple tissues, including heart, skeletal muscle, and the alveolar epithelium of the lung. In addition, AAV9 is unique compared to other AAV serotypes in that it is capable of surpassing the blood-brain barrier and transducing neurons in the brain and spinal cord. It has recently been shown that AAV9 uses galactose as a receptor to transduce many different cell types in vitro, as well as cells of the mouse airway in vivo. In this study, we sought to identify the specific amino acids of the AAV9 capsid necessary for binding to galactose. By site-directed mutagenesis and cell binding assays, plus computational ligand docking studies, we discovered five amino acids, including N470, D271, N272, Y446, and W503, which are required for galactose binding that form a pocket at the base of the protrusions around the icosahedral 3-fold axes of symmetry. The importance of these amino acids for tissue tropism was also confirmed by in vivo studies in the mouse lung. Identifying the interactions necessary for AAV9 binding to galactose may lead to advances in vector engineering.A deno-associated virus (AAV) vectors show great potential for use in gene therapy applications due to their ability to transduce many different types of tissues and provide stable, long-term gene expression (41). Numerous serotypes of AAV have been characterized, and more than 120 other variants have been isolated from both human and nonhuman primate tissues (13-15, 27, 35, 35a). Each of these variants has its own unique properties providing a large pool of potential vectors for gene therapy. Different AAV serotypes display distinctive tissue tropism and transduction efficiency. For example, AAV1 demonstrates efficient muscle transduction, AAV6 shows promise in targeting the conducting airway epithelium of the lung, and AAV8 provides impressive liver transduction (6,15,18,24,46).The three-dimensional structure of the capsid of many AAV serotypes has been determined by X-ray crystallography (7,16,22,28,29,47,48). The core of the virion consists of a highly conserved eight stranded ␤-barrel motif with large loops inserted between these ␤-strands that contain defined variable regions (VRs) (16,28,29). The VRs are present at the surface of the capsid structure and are therefore readily available for cellular interactions. The high diversity of the VRs between AAV serotypes likely defines their respective phenotypes in regard to tropism, transduction efficiency, and antigenicity. Most of the VRs are located at or near the icosahedral 3-fold axes of symmetry of the capsid, forming protrusions around these axes (16,28,29).Receptor binding is a proximal step necessary for AAV vector transduction. Interaction with cell surface receptors allows internalization of the virion and subsequent trafficking to the nucleus where gene expression occurs (8). Receptors for many AAV serotypes have been identified. AAV2 has been shown ...

We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus–receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery.

Adeno-associated virus (AAV) is one of the most promising viral gene transfer vectors that has been shown to effect long-term gene expression and disease correction with low toxicity in animal models, and is well tolerated in human clinical trials. The surface of the AAV capsid is an essential component that is involved in cell binding, internalization, and trafficking within the targeted cell. Prior to developing a gene therapy strategy that utilizes AAV, the serotype should be carefully considered since each capsid exhibits a unique tissue tropism and transduction efficiency. Several approaches have been undertaken in an effort to target AAV vectors to specific cell types, including utilizing natural serotypes that target a desired cellular receptor, producing pseudotyped vectors, and engineering chimeric and mosaic AAV capsids. These capsid modifications are being incorporated into vector production and purification methods that provide for the ability to scale-up the manufacturing process to support human clinical trials. Protocols for small-scale and large-scale production of AAV, as well as assays to characterize the final vector product, are presented here. The structures of AAV2, AAV4, and AAV5 have been solved by X-ray crystallography or cryo-electron microscopy (cryo-EM), and provide a basis for rational vector design in developing customized capsids for specific targeting of AAV vectors. The capsid of AAV has been shown to be remarkably stable, which is a desirable characteristic for a gene therapy vector; however, recently it has been shown that the AAV serotypes exhibit differential susceptibility to proteases. The capsid fragmentation pattern when exposed to various proteases, as well as the susceptibility of the serotypes to a series of proteases, provides a unique fingerprint for each serotype that can be used for capsid identity validation. In addition to serotype identification, protease susceptibility can also be utilized to study dynamic structural changes that must occur for the AAV capsid to perform its various functions during the virus life cycle. The use of proteases for structural studies in solution complements the crystal structural studies of the virus. A generic protocol based on proteolysis for AAV serotype identification is provided here.

Adeno-associated viruses (AAVsA deno-associated viruses (AAVs) are nonpathogenic, singlestranded packaging DNA dependoparvoviruses within the family Parvoviridae. As the genus name implies, wild-type (WT) AAV is dependent on helper viruses, such as those in the Adenoviridae and Herpesviridae families, for replication. For its safety and the availability of a wide variety of naturally occurring serotypes displaying various tissue tropisms, recombinant AAV (rAAV) has found wide utility as both a gene therapy vector and biotechnological tool (1). Capsid crystal structures also have been determined for many of the AAV serotypes (2-9), leading to a revolution in identifying the structural determinants of receptor attachment, tropism, and transduction efficiency and, by extension, the ability to modify the capsid to generate variants with desired transduction profiles.One of the major clinical applications of AAV is as a gene therapy vector for the treatment of blindness. The eye is a particularly well-suited organ for gene therapy due to its small size, compartmentalization, and immune-privileged status (10). Clinical trials for RPE65-Leber congenital amaurosis (LCA2) demonstrate the ability to deliver a therapeutic transgene to the retinal pigment epithelium (RPE), thereby restoring retinal function and visually evoked behavior to patients (11-13). However, the majority of inherited retinal diseases are caused by defects in photoreceptors, highlighting the need to identify serotypes capable of transducing this cell type. We and others have shown that various subretinally delivered AAV serotypes are capable of efficient transduction of photoreceptors in nonhuman primates (NHP) (14-19). However, subretinal injection of AAV2 under the fovea of some LCA2 patients led to central retinal thinning and loss of visual acuity (20). Similar decreases in retinal thickness were ob-