Characterization and biodistribution of under-employed gene therapy vector AAV7.

IMPORTANCE: The use of adeno-associated viruses (AAVs) as gene delivery vectors has vast potential for the treatment of many severe human diseases. Over one hundred naturally existing AAV capsid variants have been described and classified into phylogenetic clades based on their sequences. AAV8, AAV9, AAVrh.10, and other intensively studied capsids have been propelled into pre-clinical and clinical use, and more recently, marketed products; however, less-studied capsids may also have desirable properties (e.g., potency differences, tissue tropism, reduced immunogenicity, etc.) that have yet to be thoroughly described. These data will help build a broader structure-function knowledge base in the field, present capsid engineering opportunities, and enable the use of novel capsids with unique properties.

Kinetics and durability of transgene expression after intrastriatal injection of AAV9 vectors.

Understanding the kinetics and durability of AAV-mediated transgene expression in the brain is essential for conducting basic neuroscience studies as well as for developing gene therapy approaches for CNS diseases. Here, we characterize and compare the temporal profile of transgene expression after bilateral injections into the mouse striatum of rAAV9 encoding GFP under the control of either a ubiquitous promoter (CAG), or the neuron-specific human synapsin (hSyn) and CamKII promoters. GFP protein expression with the CAG promoter was highest at 3 weeks, and then decreased to stable levels at 3 and 6 months. Surprisingly, GFP mRNA levels continued to increase from 3 weeks to 3 months, despite GFP protein expression decreasing during this time. GFP protein expression with hSyn increased more slowly, reaching a maximum at 3 months, which was equivalent to protein expression levels from CAG at that time point. Importantly, transgene expression driven by the hSyn promoter at 6 months was not silenced as previously reported, and GFP mRNA was continuing to rise even at the final 6-month time point. Thus, hSyn as a promoter for transgene expression demonstrates long-term durability but may require more time after vector administration to achieve steady-state levels. Because CAG had the highest GFP protein expression in our comparison, which was at 3 weeks post administration, the early kinetics of transgene expression from CAG was examined (1, 2, 5, and 10 days after injection). This analysis showed that GFP protein expression and GFP mRNA increased during the first 3 weeks after administration. Interestingly, vector DNA rapidly decreased 10-fold over the first 3 weeks following injection as it assembled into stable circular episomes and concatemers. Surprisingly, the processing of vector genomes into circular episomes and concatemers was continually dynamic up to 3 months after injection. These results provide novel insight into the dynamic processing of vector genomes and promoter-specific temporal patterns of transgene expression in the brain.

Isolating Human Monoclonal Antibodies Against Adeno-Associated Virus From Donors With Pre-existing Immunity.

With the advent of single B-cell cloning technology, we can isolate antibodies against virtually any antigen to study the interaction of a given pathogen with the immune system and develop novel therapeutic strategies. Antibodies directed against the capsid of adeno-associated viruses (AAV) are a significant obstacle to effectively leveraging AAV as a gene-delivery vector in seropositive individuals. In order to design next-generation vectors that can evade neutralization by these antibodies, studies have mapped the epitopes of mouse monoclonal antibodies generated by immunization with AAV. Although these studies provide critical information regarding capsid immunogenicity, they cannot address (1) differences in the antibody repertoire generated in humans following AAV natural infection; or (2) how reactions can vary when generated in response to vector administration. Here, we isolated and evaluated a panel of novel, fully human anti-AAV antibodies by cloning single memory B cells from a seropositive normal donor. We have validated the utility of this approach to study AAV immunology. Our goal is to leverage this knowledge to design novel AAV variants that can effectively transduce target tissues in individuals with AAV-neutralizing antibodies.

TLR9 signaling mediates adaptive immunity following systemic AAV gene therapy.

An ongoing concern of in vivo gene therapy is adaptive immune responses against the protein product of a transgene, particularly for recessive diseases in which antigens are not presented to lymphocytes during central tolerance induction. Here we show that Toll-like receptor 9 (TLR9) signaling activates T cells against an epitope tagged mitochondria-targeted ornithine transcarbamylase (OTC) following the administration of a systemic adeno-associated virus (AAV) vector. Using a transgenic mouse model system, we demonstrate that TLR9 signaling extrinsic to T cells induces a robust cytotoxic T-cell response against the transgene and results in transgene expression loss. Overall, our results suggest that inflammation mediated by TLR9 signaling and the presence of high affinity transgene-specific T cells is important for the development of adaptive immune responses to transgene products following AAV gene therapy.

Deamidation of Amino Acids on the Surface of Adeno-Associated Virus Capsids Leads to Charge Heterogeneity and Altered Vector Function.

Post-translational modification of the adeno-associated virus capsids is a poorly understood factor in the development of these viral vectors into pharmaceutical products. Here we report the extensive capsid deamidation of adeno-associated virus serotype 8 and seven other diverse adeno-associated virus serotypes, with supporting evidence from structural, biochemical, and mass spectrometry approaches. The extent of deamidation at each site depended on the vector's age and multiple primary-sequence and three-dimensional structural factors. However, the extent of deamidation was largely independent of the vector recovery and purification conditions. We demonstrate the potential for deamidation to impact transduction activity and, moreover, correlate an early time point loss in vector activity to rapidly progressing spontaneous deamidation at several adeno-associated virus 8 asparagines. We explore mutational strategies that stabilize side-chain amides, improving vector transduction and reducing the lot-to-lot molecular variability that presents a key concern in biologics manufacturing. This study illuminates a previously unknown aspect of adeno-associated virus capsid heterogeneity and highlights its importance in the development of these vectors for gene therapy.

Mapping an Adeno-associated Virus 9-Specific Neutralizing Epitope To Develop Next-Generation Gene Delivery Vectors.

Recent clinical trials have demonstrated the potential of adeno-associated virus (AAV)-based vectors for treating rare diseases. However, significant barriers remain for the translation of these vectors into widely available therapies. In particular, exposure to the AAV capsid can generate an immune response of neutralizing antibodies. One approach to overcome this response is to map the AAV-specific neutralizing epitopes and rationally design an AAV capsid able to evade neutralization. To accomplish this, we isolated a monoclonal antibody against AAV9 following immunization of BALB/c mice and hybridoma screening. This antibody, PAV9.1, is specific for intact AAV9 capsids and has a high neutralizing titer of >1:160,000. We used cryo-electron microscopy to reconstruct PAV9.1 in complex with AAV9. We then mapped its epitope to the 3-fold axis of symmetry on the capsid, specifically to residues 496-NNN-498 and 588-QAQAQT-592. Capsid mutagenesis demonstrated that even a single amino acid substitution within this epitope markedly reduced binding and neutralization by PAV9.1. In addition, in vivo studies showed that mutations in the PAV9.1 epitope conferred a "liver-detargeting" phenotype to the mutant vectors, unlike AAV9, indicating that the residues involved in PAV9.1 interactions are also responsible for AAV9 tropism. However, we observed minimal changes in binding and neutralizing titer when we tested these mutant vectors for evasion of polyclonal sera from mice, macaques, or humans previously exposed to AAV. Taken together, these studies demonstrate the complexity of incorporating mapped neutralizing epitopes and previously identified functional motifs into the design of novel capsids able to evade immune response.IMPORTANCE Gene therapy utilizing viral vectors has experienced recent success, culminating in U.S. Food and Drug Administration approval of the first adeno-associated virus vector gene therapy product in the United States: Luxturna for inherited retinal dystrophy. However, application of this approach to other tissues faces significant barriers. One challenge is the immune response to viral infection or vector administration, precluding patients from receiving an initial or readministered dose of vector, respectively. Here, we mapped the epitope of a novel neutralizing antibody generated in response to this viral vector to design a next-generation capsid to evade immune responses. Epitope-based mutations in the capsid interfered with the binding and neutralizing ability of the antibody but not when tested against polyclonal samples from various sources. Our results suggest that targeted mutation of a greater breadth of neutralizing epitopes will be required to evade the repertoire of neutralizing antibodies responsible for blocking viral vector transduction.

Adeno-associated virus serotypes 1, 8, and 9 share conserved mechanisms for anterograde and retrograde axonal transport.

Adeno-associated virus (AAV) vectors often undergo long-distance axonal transport after brain injection. This leads to transduction of brain regions distal to the injection site, although the extent of axonal transport and distal transduction varies widely among AAV serotypes. The mechanisms driving this variability are poorly understood. This is a critical problem for applications that require focal gene expression within a specific brain region, and also impedes the utilization of vector transport for applications requiring widespread delivery of transgene to the brain. Here, we compared AAV serotypes 1 and 9, which frequently demonstrate distal transduction, with serotype 8, which rarely spreads beyond the injection site. To examine directional AAV transport in vitro, we used a microfluidic chamber to apply dye-labeled AAV to the axon termini or to the cell bodies of primary rat embryonic cortical neurons. All three serotypes were actively transported along axons, with transport characterized by high velocities and prolonged runs in both the anterograde and retrograde directions. Coinfection with pairs of serotypes indicated that AAV1, 8, and 9 share the same intracellular compartments for axonal transport. In vivo, both AAV8 and 9 demonstrated anterograde and retrograde transport within a nonreciprocal circuit after injection into adult mouse brain, with highly similar distributions of distal transduction. However, in mass-cultured neurons, we found that AAV1 was more frequently transported than AAV8 or 9, and that the frequency of AAV9 transport could be enhanced by increasing receptor availability. Thus, while these serotypes share conserved mechanisms for axonal transport both in vitro and in vivo, the frequency of transport can vary among serotypes, and axonal transport can be markedly increased by enhancing vector uptake. This suggests that variability in distal transduction in vivo likely results from differential uptake at the plasma membrane, rather than fundamental differences in transport mechanisms among AAV serotypes.