Intravitreal injection of a rationally designed AAV capsid library in non-human primate identifies variants with enhanced retinal transduction and neutralizing antibody evasion.

Adeno-associated virus (AAV) continues to be the gold standard vector for therapeutic gene delivery and has proven especially useful for treating ocular disease. Intravitreal injection (IVtI) is a promising delivery route because it increases accessibility of gene therapies to larger patient populations. However, data from clinical and non-human primate (NHP) studies utilizing currently available capsids indicate that anatomical barriers to AAV and pre-existing neutralizing antibodies can restrict gene expression to levels that are "sub-therapeutic" in a substantial proportion of patients. Here, we performed a combination of directed evolution in NHPs of an AAV2-based capsid library with simultaneous mutations across six surface-exposed variable regions and rational design to identify novel capsid variants with improved retinal transduction following IVtI. Following two rounds of screening in NHP, enriched variants were characterized in intravitreally injected mice and NHPs and shown to have increased transduction relative to AAV2. Lead capsid variant, P2-V1, demonstrated an increased ability to evade neutralizing antibodies in human vitreous samples relative to AAV2 and AAV2.7m8. Taken together, this study further contributed to our understanding of the selective pressures associated with retinal transduction via the vitreous and identified promising novel AAV capsid variants for clinical consideration.

Characterization of the Serpentine Adeno-Associated Virus (SAAV) Capsid Structure: Receptor Interactions and Antigenicity.

Adeno-associated viruses (AAVs) are being developed as clinical gene therapy vectors. One issue undermining their broad use in the clinical setting is the high prevalence of circulating antibodies in the general population capable of neutralizing AAV vectors. Hence, there is a need for AAV vectors that can evade the preexisting immune response. One possible source of human naive vectors are AAVs that do not disseminate in the primate population, and one such example is serpentine AAV (SAAV). This study characterizes the structural and biophysical properties of the SAAV capsid and its receptor interactions and antigenicity. Single particle cryo-electron microscopy (cryo-EM) and thermal stability studies were conducted to characterize the SAAV capsid structure at pH 7.4, 6.0, 5.5, and 4.0, conditions experienced during cellular trafficking. Cell binding assays using Chinese hamster ovary (CHO) cell lines identified terminal sialic acid as the primary attachment receptor for SAAV similar to AAV1, 4, 5, and 6. The binding site of sialic acid to the SAAV capsid was mapped near the 2-fold axis toward the 2/5-fold wall, in a different location than AAV1, 4, 5, and 6. Towards determining the SAAV capsid antigenicity native immunodot blots showed that SAAV evades AAV serotype-specific mouse monoclonal antibodies. However, despite its reptilian origin, it was recognized by ~25% of 50 human sera tested, likely due to the presence of cross-reactive antibodies. These findings will inform future gene delivery applications using SAAV-based vectors and further aid the structural characterization and annotation of the repertoire of available AAV capsids. IMPORTANCE AAVs are widely studied therapeutic gene delivery vectors. However, preexisting antibodies and their detrimental effect on therapeutic efficacy are a primary challenge encountered during clinical trials. In order to circumvent preexisting neutralizing antibodies targeting mammalian AAV capsids, serpentine AAV (SAAV) was evaluated as a potential alternative to existing mammalian therapeutic vectors. The SAAV capsid was found to be thermostable at a wide range of environmental pH conditions, and its structure showed conservation of the core capsid topology but displays high structural variability on the surface. At the same time, it binds to a common receptor, sialic acid, that is also utilized by other AAVs already being utilized in gene therapy trials. Contrary to the initial hypothesis, SAAV capsids were recognized by one in four human sera tested, pointing to conserved amino acids around the 5-fold region as epitopes for cross-reacting antibodies.

Novel AAV44.9-Based Vectors Display Exceptional Characteristics for Retinal Gene Therapy.

The majority of inherited retinal diseases (IRDs) are caused by mutations in genes expressed in photoreceptors (PRs). The ideal vector to address these conditions is one that transduces PRs in large areas of retina with the smallest volume/lowest titer possible, and efficiently transduces foveal cones, the cells responsible for acute, daylight vision that are often the only remaining area of functional retina in IRDs. The purpose of our study was to evaluate the retinal tropism and potency of a novel capsid, AAV44.9, and rationally designed derivatives thereof. We found that AAV44.9 and AAV44.9(E531D) transduced retinas of subretinally injected (SRI) mice with higher efficiency than did benchmark AAV5- and AAV8-based vectors. In macaques, highly efficient cone and rod transduction was observed following submacular and peripheral SRI. AAV44.9- and AAV44.9(E531D)-mediated GFP fluorescence extended laterally well beyond SRI bleb margins. Notably, extrafoveal injection (i.e., fovea not detached during surgery) led to transduction of up to 98% of foveal cones. AAV44.9(E531D) efficiently transduced parafoveal and perifoveal cones, whereas AAV44.9 did not. AAV44.9(E531D) was also capable of restoring retinal function to a mouse model of IRD. These novel capsids will be useful for addressing IRDs that would benefit from an expansive treatment area.

Structure comparison of the chimeric AAV2.7m8 vector with parental AAV2.

The AAV2.7m8 vector is an engineered capsid with a 10-amino acid insertion in adeno-associated virus (AAV) surface variable region VIII (VR-VIII) resulting in the alteration of an antigenic region of AAV2 and the ability to efficiently transduce retina cells following intravitreal administration. Directed evolution and in vivo screening in the mouse retina isolated this vector. In the present study, we sought to identify the structural differences between a recombinant AAV2.7m8 (rAAV2.7m8) vector packaging a GFP genome and its parental serotype, AAV2, by cryo-electron microscopy (cryo-EM) and image reconstruction. The structures of rAAV2.7m8 and AAV2 were determined to 2.91 and 3.02 Å resolution, respectively. The rAAV2.7m8 amino acid side-chains for residues 219-745 (the last C-terminal residue) were interpretable in the density map with the exception of the 10 inserted amino acids. While observable in a low sigma threshold density, side-chains were only resolved at the base of the insertion, likely due to flexibility at the top of the loop. A comparison to parental AAV2 (ordered from residues 217-735) showed the structures to be similar, except at some side-chains that had different orientations and, in VR-VIII containing the 10 amino acid insertion. VR-VIII is part of an AAV2 antigenic epitope, and the difference is consistent with rAAV2.7m8's escape from a known AAV2 monoclonal antibody, C37-B. The observations provide valuable insight into the configuration of inserted surface peptides on the AAV capsid and structural differences to be leveraged for future AAV vector rational design, especially for retargeted tropism and antibody escape.