Structural characterization of antibody-responses from Zolgensma treatment provides the blueprint for the engineering of an AAV capsid suitable for redosing.

Monoclonal antibodies (mAbs) are useful tools to dissect the neutralizing antibody response against the adeno-associated virus (AAV) capsids used as gene therapy delivery vectors. This study structurally characterizes the interactions of 21 human-derived antibodies from patients treated with the AAV9 vector, Zolgensma ® , utilizing high-resolution cryo-electron microscopy. The majority of the bound antibodies do not conform to the icosahedral symmetry of the capsid, thus requiring localized reconstructions. These complex structures provide unprecedented details of the mAbs binding interfaces, with some antibodies inducing structural perturbations of the capsid upon binding. Key surface capsid amino acid residues were identified facilitating the design of capsid variants with an antibody escape phenotype, with the potential to expand the patient cohort treatable with AAV9 vectors to include those that were previously excluded due to their pre-existing neutralizing antibodies, and possibly also to those requiring redosing.

Structural Characterization of Canine Minute Virus, Rat and Porcine Bocavirus.

Bocaparvovirus is an expansive genus of the Parvovirinae, with a wide range of vertebrate hosts. This study investigates Canine minute virus (CnMV), Rat bocavirus (RBoV), and Porcine bocavirus 1 (PBoV1). Both CnMV and PBoV1 have been found in gastrointestinal infections in their respective hosts, with CnMV responsible for spontaneous abortions in dogs, while PBoV has been associated with encephalomyelitis in piglets. The pathogenicity of the recently identified RBoV is currently unknown. To initiate the characterization of these viruses, their capsids structures were determined by cryo-electron microscopy at resolutions ranging from 2.3 to 2.7 Å. Compared to other parvoviruses, the CnMV, PBoV1, and RBoV capsids showed conserved features, such as the channel at the fivefold symmetry axis. However, major differences were observed at the two- and threefold axes. While CnMV displays prominent threefold protrusions, the same region is more recessed in PBoV1 and RBoV. Furthermore, the typical twofold axis depression of parvoviral capsids is absent in CnMV or very small in PBoV and RBoV. These capsid structures extend the structural portfolio for the Bocaparvovirus genus and will allow future characterization of these pathogens on a molecular level. This is important, as no antivirals or vaccines exist for these viruses.

Structural and antigenic characterization of the avian adeno-associated virus capsid.

IMPORTANCE: AAVs are extensively studied as promising therapeutic gene delivery vectors. In order to circumvent pre-existing antibodies targeting primate-based AAV capsids, the AAAV capsid was evaluated as an alternative to primate-based therapeutic vectors. Despite the high sequence diversity, the AAAV capsid was found to bind to a common glycan receptor, terminal galactose, which is also utilized by other AAVs already being utilized in gene therapy trials. However, contrary to the initial hypothesis, AAAV was recognized by approximately 30% of human sera tested. Structural and sequence comparisons point to conserved epitopes in the fivefold region of the capsid as the reason determinant for the observed cross-reactivity.

Structural and Biophysical Analysis of Adeno-Associated Virus Serotype 2 Capsid Assembly Variants.

Adeno-associated virus (AAV) is a nonenveloped single-stranded DNA (ssDNA) icosahedral T=1 virus being developed as a vector for clinical gene delivery systems. Currently, there are approximately 160 AAV clinical trials, with AAV2 being the most widely studied serotype. To further understand the AAV gene delivery system, this study investigates the role of viral protein (VP) symmetry interactions on capsid assembly, genome packaging, stability, and infectivity. A total of 25 (seven 2-fold, nine 3-fold, and nine 5-fold symmetry interface) AAV2 VP variants were studied. Six 2-fold and two 5-fold variants did not assemble capsids based on native immunoblots and anti-AAV2 enzyme-linked immunosorbent assays (ELISAs). Seven of the 3-fold and seven of the 5-fold variants that assembled capsids were less stable, while the only 2-fold variant that assembled had ~2°C higher thermal stability (Tm) than recombinant wild-type AAV2 (wtAAV2). Three of the 3-fold variants (AAV2-R432A, AAV2-L510A, and N511R) had an approximately 3-log defect in genome packaging. Consistent with previous reports of the 5-fold axes, the region of the capsid is important for VP1u externalization and genome ejection, and one 5-fold variant (R404A) had a significant defect in viral infectivity. The structures of wtAAV2 packaged with a transgene (AAV2-full) and without a transgene (AAV2-empty) and one 5-fold variant (AAV2-R404A) were determined by cryo-electron microscopy and three dimensional (3D)-image reconstruction to 2.8, 2.9, and 3.6 Å resolution, respectively. These structures revealed the role of stabilizing interactions on the assembly, stability, packaging, and infectivity of the virus capsid. This study provides insight into the structural characterization and functional implications of the rational design of AAV vectors. IMPORTANCE Adeno-associated viruses (AAVs) have been shown to be useful vectors for gene therapy applications. Consequently, AAV has been approved as a biologic for the treatment of several monogenic disorders, and many additional clinical trials are ongoing. These successes have generated significant interest in all aspects of the basic biology of AAV. However, to date, there are limited data available on the importance of the capsid viral protein (VP) symmetry-related interactions required to assemble and maintain the stability of the AAV capsids and the infectivity of the AAV capsids. Characterizing the residue type and interactions at these symmetry-driven assembly interfaces of AAV2 has provided the foundation for understanding their role in AAV vectors (serotypes and engineered chimeras) and has determined the residues or regions of the capsid that can or cannot tolerate alterations.

Structural and functional characterization of capsid binding by anti-AAV9 monoclonal antibodies from infants after SMA gene therapy.

Success in the treatment of infants with spinal muscular atrophy (SMA) underscores the potential of vectors based on adeno-associated virus (AAV). However, a major obstacle to the full realization of this potential is pre-existing natural and therapy-induced anti-capsid humoral immunity. Structure-guided capsid engineering is one possible approach to surmounting this challenge but necessitates an understanding of capsid-antibody interactions at high molecular resolution. Currently, only mouse-derived monoclonal antibodies (mAbs) are available to structurally map these interactions, which presupposes that mouse and human-derived antibodies are functionally equivalent. In this study, we have characterized the polyclonal antibody responses of infants following AAV9-mediated gene therapy for SMA and recovered 35 anti-capsid mAbs from the abundance of switched-memory B (smB) cells present in these infants. For 21 of these mAbs, seven from each of three infants, we have undertaken functional and structural analysis measuring neutralization, affinities, and binding patterns by cryoelectron microscopy (cryo-EM). Four distinct patterns were observed akin to those reported for mouse-derived mAbs, but with early evidence of differing binding pattern preference and underlying molecular interactions. This is the first human and largest series of anti-capsid mAbs to have been comprehensively characterized and will prove to be powerful tools for basic discovery and applied purposes.

Structurally Mapping Antigenic Epitopes of Adeno-associated Virus 9: Development of Antibody Escape Variants.

Adeno-associated viruses (AAV) serve as vectors for therapeutic gene delivery. AAV9 vectors have been FDA approved, as Zolgensma, for the treatment of spinal muscular atrophy and are being evaluated in clinical trials for the treatment of neurotropic and musculotropic diseases. A major hurdle for AAV-mediated gene delivery is the presence of preexisting neutralizing antibodies in 40 to 80% of the general population. These preexisting antibodies can reduce therapeutic efficacy through viral neutralization and the size of the patient cohort eligible for treatment. In this study, cryo-electron microscopy and image reconstruction were used to define the epitopes of five anti-AAV9 monoclonal antibodies (MAbs), ADK9, HL2368, HL2370, HL2372, and HL2374, on the capsid surface. Three of these, ADK9, HL2370, and HL2374, bound to or near the icosahedral 3-fold axes, HL2368 bound to the 2/5-fold wall, and HL2372 bound to the region surrounding the 5-fold axes. Pseudoatomic modeling enabled the mapping and identification of antibody contact amino acids on the capsid, including S454 and P659. These epitopes overlap previously defined parvovirus antigenic sites. Capsid amino acids critical for the interactions were confirmed by mutagenesis, followed by biochemical assays testing recombinant AAV9 (rAAV9) variants capable of escaping recognition and neutralization by the parental MAbs. These variants retained parental tropism and had similar or improved transduction efficiency compared to AAV9. These engineered rAAV9 variants could expand the patient cohort eligible for AAV9-mediated gene delivery by avoiding preexisting circulating neutralizing antibodies. IMPORTANCE The use of recombinant adeno-associated viruses (rAAVs) as delivery vectors for therapeutic genes is becoming increasingly popular, especially following the FDA approval of Luxturna and Zolgensma, based on serotypes AAV2 and AAV9, respectively. However, high-titer anti-AAV neutralizing antibodies in the general population exempt patients from treatment. The goal of this study is to circumvent this issue by creating AAV variant vectors not recognized by preexisting neutralizing antibodies. The mapping of the antigenic epitopes of five different monoclonal antibodies (MAbs) on AAV9, to recapitulate a polyclonal response, enabled the rational design of escape variants with minimal disruption to cell tropism and gene expression. This study, which included four newly developed and now commercially available MAbs, provides a platform for the engineering of rAAV9 vectors that can be used to deliver genes to patients with preexisting AAV antibodies.

Structural Study of Aavrh.10 Receptor and Antibody Interactions.

Recombinant adeno-associated virus (rAAV) vectors are one of the leading tools for the delivery of therapeutic genes in human gene therapy applications. For a successful transfer of their payload, the AAV vectors have to circumvent potential preexisting neutralizing host antibodies and bind to the receptors of the target cells. Both of these aspects have not been structurally analyzed for AAVrh.10. Here, cryo-electron microscopy and three-dimensional image reconstruction were used to map the binding site of sulfated N-acetyllactosamine (LacNAc; previously shown to bind AAVrh.10) and a series of four monoclonal antibodies (MAbs). LacNAc was found to bind to a pocket located on the side of the 3-fold capsid protrusion that is mostly conserved to AAV9 and equivalent to its galactose-binding site. As a result, AAVrh.10 was also shown to be able to bind to cell surface glycans with terminal galactose. For the antigenic characterization, it was observed that several anti-AAV8 MAbs cross-react with AAVrh.10. The binding sites of these antibodies were mapped to the 3-fold capsid protrusions. Based on these observations, the AAVrh.10 capsid surface was engineered to create variant capsids that escape these antibodies while maintaining infectivity. IMPORTANCE Gene therapy vectors based on adeno-associated virus rhesus isolate 10 (AAVrh.10) have been used in several clinical trials to treat monogenetic diseases. However, compared to other AAV serotypes little is known about receptor binding and antigenicity of the AAVrh.10 capsid. Particularly, preexisting neutralizing antibodies against capsids are an important challenge that can hamper treatment efficiency. This study addresses both topics and identifies critical regions of the AAVrh.10 capsid for receptor and antibody binding. The insights gained were utilized to generate AAVrh.10 variants capable of evading known neutralizing antibodies. The findings of this study could further aid the utilization of AAVrh.10 vectors in clinical trials and help the approval of the subsequent biologics.

Improved Genome Packaging Efficiency of Adeno-associated Virus Vectors Using Rep Hybrids.

Recombinant adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last 2 decades, research focused primarily on the characterization and isolation of new cap, genes resulting in hundreds of natural and engineered AAV capsid variants, while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the single-stranded DNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major by-product of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2-rep system. In further experiments the 3' end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids approximately 2- to -4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production. IMPORTANCE A major by-product of all adeno-associated virus (AAV) vector production systems are "empty" capsids, void of the desired therapeutic gene, and thus do not provide any curative benefit for the treatment of the targeted disease. In fact, empty capsids can potentially elicit additional immune responses in vivo gene therapies if not removed by additional purification steps. Thus, there is a need to increase the genome packaging efficiency and reduce the number of empty capsids from AAV biologics. The novel Rep hybrids from different AAV serotypes described in this study are capable of reducing the percentage of empty capsids in all tested AAV serotypes and improve overall yields of genome-containing AAV capsids at the same time. They can likely be integrated easily into existing AAV manufacturing protocols to optimize the production of the generated AAV gene therapy products.