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Viral vector technologies are commonly used in neuroscience research to understand and manipulate neural circuits, but successful applications of these technologies in non-human primate models have been inconsistent. An essential component to improve these technologies is an impartial and accurate assessment of the effectiveness of different viral constructs in the primate brain. We tested a diverse array of viral vectors delivered to the brain and extraocular muscles of macaques and compared three methods for histological assessment of viral-mediated fluorescent transgene expression: epifluorescence (Epi), immunofluorescence (IF), and immunohistochemistry (IHC). Importantly, IF and IHC identified a greater number of transduced neurons compared to Epi. Furthermore, IF and IHC reliably provided enhanced visualization of transgene in most cellular compartments (i.e., dendritic, axonal, and terminal fields), whereas the degree of labeling provided by Epi was inconsistent and predominantly restricted to somas and apical dendrites. Because Epi signals are unamplified (in contrast to IF and IHC), Epi likely provides a more veridical assessment for the amount of accumulated transgene and, thus, the potential to chemo- or optogenetically manipulate neuronal activity. The comparatively weak Epi signals suggest that the current generations of viral constructs, regardless of delivered transgene, are not optimized for primates. This reinforces an emerging viewpoint that viral vectors tailored for the primate brain are necessary for basic research and human gene therapy.

Glucocorticoids have anti-inflammatory and immunosuppressive functions and have commonly been used for preventing liver toxicity after the systemic application of a high dose of adeno-associated virus (AAV) vector for gene therapy. Clinical studies have reported that glucocorticoids have rescued factor IX (FIX) expression in patients with hemophilia B who showed a reduced FIX expression at 6 to 10 weeks post-AAV vector administration. In this study, we explored whether glucocorticoids could affect transgene expression in AAV targeted livers in animal models. When dexamethasone was applied before AAV9/FIX vector administration in the wild-type C57BL/6 mice, FIX expression was much higher than that of the control mice at any time point. More importantly, FIX expression transiently increased after dexamethasone was administered at week 6 or later post-AAV injection regardless of the various dexamethasone treatments applied. The transient enhancement in transgene expression was observed once there were one to several consecutive dexamethasone treatments completed. A similar result was also achieved in other wild-type BALB/c and hemophilia B mice that were treated with AAV9/FIX and dexamethasone. This mechanism study demonstrated that the administration of dexamethasone did not change either AAV genome copy number or transgene expression at the transcription level but transiently decreased interferon beta (IFN-β) and tumor necrosis factor alpha (TNF-α) expression in the livers of mice at a later time after AAV injection. Next, we studied the effect of dexamethasone on late transgene expression in hemophilia B dogs. Dexamethasone was administered 1 year after AAV9/FIX injection. Inconsistent with the results in mice, no significant change of FIX expression was observed in hemophilia B dogs. In summary, the results from this study indicate that dexamethasone may have various effects on transgene expression in AAV-transduced livers in different species, which provides valuable information about the rational application of dexamethasone in future clinical studies.

AAV virion biology is still lacking a complete understanding of the role that the various structural subunits (VP1, 2, and 3) play in virus assembly, infectivity, and therapeutic delivery for clinical indications. In this study, we focus on the less studied adeno-associated virus AAV3B and generate a collection of AAV plasmid substrates that assemble virion particles deficient specifically in VP1, VP2, or VP1 and 2 structural subunits. Using a collection of biological and structural assays, we observed that virions devoid of VP1, VP2, or VP1 and 2 efficiently assembled virion particles, indistinguishable by cryoelectron microscopy (cryo-EM) from that of wild type (WT), but unique in virion transduction (WTVP2VP1VP1 and 2 mutants). We also observed that the missing structural subunit was mostly compensated by additional VP3 protomers in the formed virion particle. Using cryo-EM analysis, virions fell into three classes, namely full, empty, and partially filled, based on comparison of density values within the capsid. Further, we characterize virions described as "broken" or "disassembled" particles, and provide structural information that supports the particle dissolution occurring through the two-fold symmetry sites. Finally, we highlight the unique value of employing cryo-EM as an essential tool for release criteria with respect to AAV manufacturing.

Recent advances in adeno-associated viral (AAV) capsid variants with novel oligotropism require validation in models of disease in order to be viable candidates for white matter disease gene therapy. We present here an assessment of the biodistribution, tropism, and efficacy of a novel AAV capsid variant (AAV/ Olig001) in a model of Canavan disease. We first define a combination of dose and route of administration of an AAV/Olig001-GFP reporter conducive to widespread CNS oligodendrocyte transduction in acutely symptomatic animals that model the Canavan brain at time of diagnosis. Administration of AAV/Olig001-GFP resulted in >70% oligotropism in all regions of interest except the cerebellum without the need for lineage-specific expression elements. Intracerebroventricular infusion into the cerebrospinal fluid (CSF) was identified as the most appropriate route of administration and employed for delivery of an AAV/Olig001 vector to reconstitute oligodendroglial aspartoacylase (ASPA) in adult Canavan mice, which resulted in a dose-dependent rescue of ASPA activity, motor function, and a near-total reduction in vacuolation. A head-to-head efficacy comparison with astrogliotropic AAV9 highlighted a significant advantage conferred by oligotropic AAV/Olig001 that was independent of overall transduction efficiency. These results support the continued development of AAV/Olig001 for advancement to clinical application to white matter disease., Graphical Abstract, This study documents the unique target engagement features of a novel viral vector (AAV/Olig001) that promotes phenotypic rescue in a preclinical model of a white matter disease caused by an inherited defect in a white matter cell enzyme.

Gene therapy has the potential to maintain therapeutic blood clotting factor IX (FIX) levels in patients with hemophilia B by delivering a functional human F9 gene into liver cells. This phase 1/2, open-label dose-escalation study investigated BAX 335 (AskBio009, AAV8.sc-TTR-FIXR338Lopt), an adeno-associated virus serotype 8 (AAV8)–based FIX Padua gene therapy, in patients with hemophilia B. This report focuses on 12-month interim analyses of safety, pharmacokinetic variables, effects on FIX activity, and immune responses for dosed participants. Eight adult male participants (aged 20-69 years; range FIX activity, 0.5% to 2.0%) received 1 of 3 BAX 335 IV doses: 2.0 × 1011; 1.0 × 1012; or 3.0 × 1012 vector genomes/kg. Three (37.5%) participants had 4 serious adverse events, all considered unrelated to BAX 335. No serious adverse event led to death. No clinical thrombosis, inhibitors, or other FIX Padua–directed immunity was reported. FIX expression was measurable in 7 of 8 participants; peak FIX activity displayed dose dependence (32.0% to 58.5% in cohort 3). One participant achieved sustained therapeutic FIX activity of ∼20%, without bleeding or replacement therapy, for 4 years; in others, FIX activity was not sustained beyond 5 to 11 weeks. In contrast to some previous studies, corticosteroid treatment did not stabilize FIX activity loss. We hypothesize that the loss of transgene expression could have been caused by stimulation of innate immune responses, including CpG oligodeoxynucleotides introduced into the BAX 335 coding sequence by codon optimization. This trial was registered at www.clinicaltrials.gov as #NCT01687608.

Recombinant adeno-associated virus (rAAV) vectors have become one of the most promising and efficacious delivery vehicles for human gene therapy; however, low infectivity remains a major ongoing obstacle in the clinical application of rAAV vectors. Multiple strategies, including rAAV capsid modification and the application of pharmacological reagents, have been explored to enhance rAAV vector gene delivery. Recently, a new strategy using native proteins or various peptides has shown promise for increasing rAAV transduction locally or globally. This review summarizes the current status of protein- and peptide-based strategies and mechanisms to modulate rAAV transduction. We also provide a potential insight regarding the design of effective approaches for rAAV transduction enhancement in future clinical studies.

Recently, we established an adeno-associated virus (AAV9) capsid-promoter interaction that directly determined cell-specific gene expression across two synthetic promoters, Cbh and CBA, in the rat striatum. These studies not only expand this capsid-promoter interaction to include another promoter in the rat striatum but also establish AAV capsid-promoter interactions in the nonhuman primate brain. When AAV serotype 9 (AAV9) vectors were injected into the rat striatum, the minimal synthetic promoter JetI drove green fluorescent protein (GFP) gene expression predominantly in oligodendrocytes. However, similar to our previous findings, the insertion of six alanines into VP1/VP2 of the AAV9 capsid (AAV9AU) significantly shifted JetI-driven GFP gene expression to neurons. In addition, previous retrograde tracing studies in the nonhuman primate brain also revealed the existence of a capsid-promoter interaction. When rAAV2-Retro vectors were infused into the frontal eye field (FEF) of rhesus macaques, local gene expression was prominent using either the hybrid chicken beta actin (CAG) or human synapsin (hSyn) promoters. However, only the CAG promoter, not the hSyn promoter, led to gene expression in the ipsilateral claustrum and contralateral FEF. Conversely, infusion of rAAV2-retro-hSyn vectors, but not rAAV2-retro-CAG, into the macaque superior colliculus led to differential and selective retrograde gene expression in cerebellotectal afferent cells. Clearly, this differential promoter/capsid expression profile could not be attributed to promoter inactivation from retrograde transport of the rAAV2-Retro vector. In summary, we document the potential for AAV capsid/promoter interactions to impact cell-specific gene expression across species, experimental manipulations, and engineered capsids, independent of capsid permissivity.

Recombinant adeno-associated viral (rAAV) vector mobilization is a largely theoretical process in which intact AAV vectors spread or “mobilize” from transduced cells and infect additional cells within, or external of, the initial host. This process can be helper virus-independent (vector alone) or helper virus-dependent (de novo rAAV production facilitated by superinfection of both wild-type AAV [wtAAV] and Adenovirus 5 [Ad] helper virus). Herein, rAAV production and mobilization with and without wtAAV were analyzed following plasmid transfection or viral transduction utilizing well-established in vitro conditions and analytical measurements. During in vitro production, wtAAV produced the highest titer with rAAV-luc (4.1 kb), rAAV-IDUA (3.7 kb), and rAAV-Nano-dysferlin (4.9 kb) generating 2.5-, 5.9-, or 10.7-fold lower amounts, respectively. Surprisingly, cotransfection of a wtAAV and an rAAV plasmid resulted in a uniform decrease in production of wtAAV in all instances with a concomitant increase of rAAV such that wtAAV:rAAV titers were at a ratio of 1:1 for all constructs investigated. These results were shown to be independent of the rAAV transgenic sequence, size, transgene, or promoter choice and point to novel aspects of wtAAV complementation that enhance current vector production systems yet to be defined. In a mobilization assay, a sizeable amount of rAAV recovered from infected 293 cell lysate remained intact and competent for a secondary round of infection (termed Ad-independent mobilization). In rAAV-infected cells coinfected with Ad and wtAAV, rAAV particle production was increased >50-fold compared with no Ad conditions. In addition, Ad-dependent rAAV vectors mobilized and resulted in >1,000-fold transduction upon a subsequent second-round infection, highlighting the reality of these theoretical safety concerns that can be manifested under various conditions. Overall, these studies document and signify the need for mobilization-resistant vectors and the opportunity to derive better vector production systems.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disease characterized by severe phenotypes, including corneal clouding. MPS I is caused by mutations in alpha-l-iduronidase (IDUA), a ubiquitous enzyme that catalyzes the hydrolysis of glycosaminoglycans. Currently, no treatment exists to address MPS I corneal clouding other than corneal transplantation, which is complicated by a high risk for rejection. Investigation of an adeno-associated virus (AAV) IDUA gene addition strategy targeting the corneal stroma addresses this deficiency. In MPS I canines with early or advanced corneal disease, a single intrastromal AAV8G9-IDUA injection was well tolerated at all administered doses. The eyes with advanced disease demonstrated resolution of corneal clouding as early as 1 week post-injection, followed by sustained corneal transparency until the experimental endpoint of 25 weeks. AAV8G9-IDUA injection in the MPS I canine eye with early corneal disease prevented the development of advanced corneal changes while restoring clarity. Biodistribution studies demonstrated vector genomes in ocular compartments other than the cornea and in some systemic organs; however, a capsid antibody response was detected in only the highest dosed subject. Collectively, the results suggest that intrastromal AAV8G9-IDUA therapy prevents and reverses visual impairment associated with MPS I corneal clouding.

Cell-selective gene expression comprises a critical element of many adeno-associated virus (AAV) vector-based gene therapies, and to date achieving this goal has focused on AAV capsid engineering, cell-specific promoters, or cell-specific enhancers. Recently, we discovered that the capsid of AAV9 exerts a differential influence on constitutive promoters of sufficient magnitude to alter cell type gene expression in the rat CNS. For AAV9 vectors chicken β-actin (CBA) promoter-driven gene expression exhibited a dominant neuronal gene expression in the rat striatum. Surprisingly, for otherwise identical AAV9 vectors, the truncated CBA hybrid (CBh) promoter shifted gene expression toward striatal oligodendrocytes. In contrast, AAV2 vector gene expression was restricted to striatal neurons, regardless of the constitutive promoter used. Furthermore, a six-glutamate residue insertion immediately after the VP2 start residue shifted CBA-driven cellular gene expression from neurons to oligodendrocytes. Conversely, a six-alanine insertion in the same AAV9 capsid region reversed the CBh-mediated oligodendrocyte expression back to neurons without changing AAV9 capsid access to oligodendrocytes. Given the preponderance of AAV9 in ongoing clinical trials and AAV capsid engineering, this AAV9 capsid-promoter interaction reveals a previously unknown novel contribution to cell-selective AAV-mediated gene expression in the CNS.

Adeno-associated virus (AAV) vector-mediated gene delivery was recently approved for the treatment of inherited blindness and spinal muscular atrophy, and long-term therapeutic effects have been achieved for other rare diseases, including haemophilia and Duchenne muscular dystrophy. However, current research indicates that the genetic modification of AAV vectors may further facilitate the success of AAV gene therapy. Vector engineering can increase AAV transduction efficiency (by optimizing the transgene cassette), vector tropism (using capsid engineering) and the ability of the capsid and transgene to avoid the host immune response (by genetically modifying these components), as well as optimize the large-scale production of AAV. Adeno-associated virus (AAV) vector-mediated gene delivery has had long-term therapeutic effects for several diseases, including haemophilia and Duchenne muscular dystrophy. Genetically modifying AAV vectors to increase their transduction efficiency, vector tropism and ability to avoid the host immune response may further increase the success of AAV gene therapy.

Adeno-associated virus (AAV) vectors have become an important tool for delivering therapeutic genes for a wide range of neurological diseases. AAV serotypes possess differential cellular tropism in the central nervous system. Although several AAV serotypes or mutants have been reported to transduce the brain efficiently, conflicting data occur across studies with the use of various rodent strains from different genetic backgrounds. Herein, we performed a systematic comparison of the brain transduction properties among five AAV serotypes (AAV2, 5, 7, 8, and 9) in two common rodent strains (C57BL/6J and FVB/N), following local intrastriatal injection of AAV vectors encoding enhanced green fluorescent protein (EGFP) driven by the CBh promoter. Important differences were found regarding overall cellular tropism and transduction efficiency, including contralateral transduction among the AAV serotypes and between the mouse strains. We have further found loss of NeuN-immunoreactivity and microglial activation from AAV transduction in the different mouse strains. The important strain-specific differences from our study suggest that the genetic background of the mouse may affect AAV serotype transduction properties in the brain. These data can provide valuable information about how to choose an effective AAV vector for clinical application and interpret the data obtained from preclinical studies and clinical trials.

Sensorineural hearing loss is one of the most common disabilities worldwide. Such prevalence necessitates effective tools for studying the molecular workings of cochlear cells. One prominent and effective vector for expressing genes of interest in research models is adeno-associated virus (AAV). However, AAV efficacy in transducing cochlear cells can vary for a number of reasons including serotype, species, and methodology, and oftentimes requires high multiplicity of infection which can damage the sensory cells. Reports in other systems suggest multiple approaches can be used to enhance AAV transduction including self-complementary vector design and pharmacological inhibition of degradation. Here we produced AAV to drive green fluorescent protein (GFP) expression in explanted neonatal mouse cochleae. Treatment with eeyarestatin I, tyrphostin 23, or lipofectamine 2000 did not result in increased transduction, however, self-complementary vector design resulted in significantly more GFP positive cells when compared to single-stranded controls. Similarly, self-complementary AAV2 vectors demonstrated enhanced transduction efficiency compared to single stranded AAV2 when injected via the posterior semicircular canal, in vivo. Self-complementary vectors for AAV1, 8, and 9 serotypes also demonstrated robust GFP transduction in cochlear cells in vivo, though these were not directly compared to single stranded vectors. These findings suggest that second-strand synthesis may be a rate limiting step in AAV transduction of cochlear tissues and that self-complementary AAV can be used to effectively target large numbers of cochlear cells in vitro and in vivo.

Recombinant Adeno-associated viral vector (rAAV) mobilization is a largely theoretical process in which intact AAV vectors spread or “mobilize” from transduced cells and infect additional cells within, or external, of the initial host. This process can be replication independent (vector alone), or replication-dependent (de novo rAAV production facilitated by super-infection of both wild-type AAV (wtAAV) and Ad helper virus). Herein, rAAV production and mobilization with and without wtAAV were analyzed following plasmid transfection or viral transduction utilizing well established in vitro conditions and analytical measurements. During in vitro production, wtAAV produced the highest titer with rAAV-luc (4.1 Kb), rAAV-IDUA (3.7 Kb), and rAAV-NanoDysferlin (4.9 Kb) generating 2.5-, 5.9-, or 10.7-fold lower amounts, respectively. Surprisingly, cotransfection of a wtAAV and a rAAV plasmid resulted in a uniform decrease in production of wtAAV in all instances with a concomitant increase of rAAV such that wtAAV:rAAV titers were at a ratio of 1:1 for all constructs investigated. These results were shown to be independent of the rAAV transgenic sequence, size, transgene, or promoter choice and point to novel aspects of wtAAV complementation that enhance current vector production systems yet to be de fined. In a mobilization assay, a sizeable amount of rAAV recovered from infected 293 cell lysate remained intact and competent for a secondary round of infection (termed non-replicative mobilization). In rAAV infected cells co-infected with Ad5 and wtAAV, rAAV particle production was increased > 50-fold compared to non-replicative conditions. In addition, replicative dependent rAAV vectors mobilized and resulted in >1,000 -fold transduction upon a subsequent 2nd round infection, highlighting the reality of these theoretical safety concerns that can be manifested under various conditions. Overall, these studies document and signify the need for mobilization resistant vectors and the opportunity to derive better vector production systems.

Adeno-associated virus (AAV) vectors have been successfully used in patients with bleeding disorders and blindness. For human liver targeting, two major factors restrict effective AAV transduction after systemic administration of AAV vectors: human hepatocyte tropism and neutralizing antibodies (Nabs). In this study, we attempted to isolate AAV variants with the ability to transduce human hepatocytes and escape Nabs using a directed evolution approach in vivo. After four cycles of selection, 14 AAV capsid mutants were identified from a capsid shuffling library selected in the presence of human Intravenous Immunoglobulin (IVIG) and isolated from human hepatocytes xenografted into chimeric mice. AAV neutralization assays using IVIG showed that most of the mutants showed the Nab escape pattern in a manner similar to that of AAV8 or AAV9 and better than that of other AAV serotypes. Different mutants displayed varying capacities to escape Nab activity from individual serum samples collected from healthy subjects or hemophilia patients. The mutant AAV LP2-10 was found in 12 colonies out of 25, which was composed of capsids from AAV serotypes 2, 6, 8, and 9, with VP3 subunits derived from AAV8 swapped with AAV6 from residues 261 to 272. The mutant AAV LP2-10 manifested a higher ability than that of other serotypes to escape Nabs in IVIG and most human serum samples. After injection of AAV vectors encoding a self-complementary GFP cassette into chimeric mice, LP2-10 transduced human hepatocytes with efficiency similar to that of AAV8. In summary, AAV mutants can be isolated in humanized mice with both human hepatocyte tropism and the ability to evade Nab activity., Graphical Abstract, AAV mutant vectors were identified from a capsid shuffling library selected in the presence of human IVIG and isolated from human hepatocytes xenografted into chimeric mice. LP2-10, whose VP3 subunits were derived from AAV8 and AAV6, manifested the highest ability to escape Nabs in IVIG and most human serum samples.

Adeno-associated viral vectors have been successfully used in laboratory and clinical settings for efficient gene delivery. In these vectors, 96% of the adeno-associated virus (AAV) genome is replaced with a gene cassette of interest, leaving only the 145 bp inverted terminal repeat (ITR) sequences. These cis-elements, primarily from AAV serotype 2, are required for genome rescue, replication, packaging, and vector persistence. Previous work from our lab and others have demonstrated that the AAV ITR2 sequence has inherent transcriptional activity, which may confound intended transgene expression in therapeutic applications. Currently, AAV capsids are extensively study for vector contribution; however, a comprehensive analysis of ITR promoter activity of various AAV serotypes has not been described to date. Here, the transcriptional activity of AAV ITRs from different serotypes (1–4, 6, and 7) was compared in numerous cell lines and a mouse model. Under the conditions used here, all ITRs tested were capable of promoting transgene expression both in vitro and in vivo. However, we observed three classes of AAV ITR expression in vitro. Class I ITRs (AAV2 and 3) generated the highest level, whereas class II (AAV 4) had intermediate levels, and class III (AAV1 and 6) had the lowest levels. These expression levels were consistent across multiple cell lines. Only ITR7 demonstrated cell-type dependent transcriptional activity. In vivo, all classes had promoter activity. Next-generation sequencing revealed multiple transcriptional start sites that originated from the ITR sequence, with most arising from within the Rep binding element. The collective results demonstrate that the serotype ITR sequence may have multiple levels of influence on transgene expression cassettes independent of promoter selection.

Recombinant adeno-associated virus (rAAV) vectors have been widely used as favored delivery vehicles for the treatment of inherited diseases in clinical trials, including neurological diseases. However, the noninvasive systemic delivery of rAAV to the central nervous system is severely hampered by the blood-brain barrier (BBB). Several approaches have been exploited to enhance AAV vector brain transduction after systemic administration, including genetic modification of AAV capsids and physical methods. However, these approaches are not always predictive of desirable outcomes in humans and induce complications. It is imperative to explore novel strategies to increase the ability of AAV9 to cross the BBB for enhanced brain transduction. Herein, we have conducted a combinatorial in vivo/in vitro phage display library screening in mouse brains and purified AAV9 virions to identify a customized BBB shuttle peptide, designated as PB5-3. The PB5-3 peptide specifically bound to AAV9 virions and enhanced widespread transduction of AAV9 in mouse brains, especially in neuronal cells, after systemic administration. Further study demonstrated that systemic administration of AAV9 vectors encoding IDUA complexed with PB5-3 increased the phenotypic correction in the brains of MPS I mice. Mechanistic studies revealed that the PB5-3 peptide effectively increased AAV9 trafficking and transcytosis efficiency in the human BBB model hCMEC/D3 cell line but did not interfere with AAV9 binding to the receptor terminal N-linked galactosylated glycans. Additionally, the PB5-3 peptide slowed the clearance of AAV9 from blood without hepatic toxicity. This study highlights, for the first time, the potential of this combinatorial approach for the isolation of peptides that interact with specific AAV vectors for enhanced and targeted AAV transduction. This promising approach will open new combined therapeutic avenues and shed light on the potential applications of peptides for the treatment of human diseases in future clinical trials with AAV vector-mediated gene delivery.

The adeno-associated virus (AAV) vector has been used in preclinical and clinical trials of gene therapy for central nervous system (CNS) diseases. One of the biggest challenges of effectively delivering AAV to the brain is to surmount the blood-brain barrier (BBB). Herein, we identified several potential BBB shuttle peptides that significantly enhanced AAV8 transduction in the brain after a systemic administration, the best of which was the THR peptide. The enhancement of AAV8 brain transduction by THR is dose-dependent, and neurons are the primary THR targets. Mechanism studies revealed that THR directly bound to the AAV8 virion, increasing its ability to cross the endothelial cell barrier. Further experiments showed that binding of THR to the AAV virion did not interfere with AAV8 infection biology, and that THR competitively blocked transferrin from binding to AAV8. Taken together, our results demonstrate, for the first time, that BBB shuttle peptides are able to directly interact with AAV and increase the ability of the AAV vectors to cross the BBB for transduction enhancement in the brain. These results will shed important light on the potential applications of BBB shuttle peptides for enhancing brain transduction with systemic administration of AAV vectors.

Adeno-associated virus (AAV) vectors have been successfully applied in hemophilia clinical trials. However, this approach is limited to patients without AAV-neutralizing antibodies (NAbs). In this study, we explored the feasibility of AAV re-administration in hemophilia A dogs treated initially 8 years ago with AAV8.canine FVIII. After the re-administration in two NAb-negative dogs with AAV8 vectors carrying human factor VIII (hFVIII), along with the proteasome inhibitor bortezomib, we observed a phenotypic improvement in both dogs that persisted in one dog. Phenotypic improvement disappeared at 59 days after re-administration in the other dog, and specific cytotoxic T lymphocytes (CTLs) to the capsid were detected at day 17, but not to hFVIII. hFVIII inhibitors were observed at day 59 and gradually increased. Mechanistic studies demonstrated an increase in pro-inflammatory cytokines, a decrease in immunomodulatory cytokines, as well as lower Tregs after re-administration. These results suggest that hFVIII inhibitor development may contribute to the therapeutic failure via immune response activation. Interestingly, it takes about 30–50 days for AAV NAb titers to decrease by half. Collectively, this study suggests that re-administration of the same AAV serotype after long-term follow-up is feasible and that the study of AAV NAb kinetics will provide important information for predicating the efficacy of re-administration. Keywords: AAV, hemophilia, neutralizing antibodies, NAbs, re-administration, human factor VIII, hFVIII, hFVIII inhibitor

Our previous studies have demonstrated that haploid AAV vectors made from capsids of two different serotypes induced high transduction and prevented serotype-specific antibody binding. In this study, we explored the transduction efficiency of several haploid viruses, which were made from the VP1/VP2 of one serotype and VP3 of another compatible serotype. After systemic injection of 2 &times, 1010 vg of AAV vectors into mice, the haploid AAV vectors, composed of VP1/VP2 from serotypes 8 or 9, and VP3 from AAV2, displayed a two to seven-fold increase in liver transduction compared with those of parental AAV2 vectors. Furthermore, a chimeric AAV2/8 VP1/VP2 with N-terminus of VP1/VP2 from AAV2 and C-terminus (VP3 domain) from AAV8 was constructed, and produced the haploid vector 28m-2VP3 with AAV2 VP3. The haploid 28m-2VP3 vector showed a five-fold higher transduction than that of the vectors composed solely of AAV2 VPs. Remarkably, the 28m-2VP3 vectors also induced a significant increase in transgene expression compared to the vectors composed of AAV8 VP1/VP2 with AAV2 VP3. The results suggest that the difference in the VP1/VP2 N-terminal region between AAV2 and AAV8 may allow better &ldquo, communication&rdquo, between the VP1/VP2 N-terminus of AAV2 with its cognate VP3. Similarly, the haploid vectors, VP1/VP2 from serotypes 8 or 9 and VP3 from AAV3, achieved higher transductions in multiple tissue types beyond typical tropism compared with those of AAV3 vectors. Consistently, higher vector genome copy numbers were detected in these tissues, indicating that an incorporation of non-cognate VP1/VP2 might influence the cellular tropism of the haploid vectors. However, there was no significant difference or even decreased transductions when compared with those of parental AAV8 or AAV9 vectors. In summary, these studies provide insight into current development strategies of AAV vectors that can increase AAV transduction across multiple tissues.

Adeno-associated virus (AAV) vector-mediated gene delivery was recently approved for the treatment of inherited blindness and spinal muscular atrophy, and long-term therapeutic effects have been achieved for other rare diseases, including haemophilia and Duchenne muscular dystrophy. However, current research indicates that the genetic modification of AAV vectors may further facilitate the success of AAV gene therapy. Vector engineering can increase AAV transduction efficiency (by optimizing the transgene cassette), vector tropism (using capsid engineering) and the ability of the capsid and transgene to avoid the host immune response (by genetically modifying these components), as well as optimize the large-scale production of AAV.

Adeno-associated viruses (AAVs) are being developed as vectors for the treatment of genetic disorders. However, pre-existing antibodies present a significant limitation to achieving optimal efficacy for the AAV gene delivery system. Efforts aimed at engineering vectors with the ability to evade the immune response include identification of residues on the virus capsid important for these interactions and changing them. Here K531 is identified as the determinant of monoclonal antibody ADK6 recognition by AAV6, and not the closely related AAV1. The AAV6-ADK6 complex structure was determined by cryo-electron microscopy and the footprint confirmed by cell-based assays. The ADK6 footprint overlaps previously identified AAV antigenic regions and neutralizes by blocking essential cell surface glycan attachment sites. This study thus expands the available repertoire of AAV-antibody information that can guide the design of host immune escaping AAV vectors able to maintain capsid functionality.

Adeno-associated virus (AAV) vectors have been used successfully in clinical trials for patients with hemophilia or blindness, but pre-existing neutralizing antibodies (Nab) are common in the general population and exclude many patients from clinical trials. Exploration of effective strategies to enhance AAV transduction and escape from Nab activity is still imperative. Previous studies have shown the compatibility of capsids from AAV serotypes and homology of recognition sites of AAV Nab located on different capsid subunits from one virion. In this study, we co-transfected AAV2 and AAV8 helper plasmids at different ratios (3:1, 1:1 and 1:3) to assemble haploid capsids and study both their transduction efficiency and Nab escape activity. After muscular injection, all of the haploid viruses induced higher transduction than their parental AAV vectors (2- to 9-fold over AAV2), with the highest of these being the haploid vector AAV2/8 3:1. After systemic administration, a 4-fold higher transduction in the liver was observed with haploid AAV2/8 1:3 than that with AAV8 alone. We then packaged the therapeutic factor IX cassette into haploid AAV2/8 1:3 capsids and injected them into FIX knockout mice via the tail vein. Higher FIX expression and improved phenotypic correction were achieved with the haploid AAV2/8 1:3 virus vector when compared to that of AAV8. Additionally, the haploid virus AAV2/8 1:3 was able to escape AAV2 neutralization and did not increase capsid antigen presentation capacity when compared to AAV8. To improve the Nab evasion ability of the haploid virus, we produced the triploid vector AAV2/8/9 by co-transfecting AAV2, AAV8 and AAV9 helper plasmids at a ratio of 1:1:1. After systemic administration, a 2-fold higher transduction in the liver was observed with the triploid vector AAV2/8/9 than that with AAV8. Nab analysis demonstrated that the triploid AAV2/8/9 vector was able to escape Nab activity from mouse sera immunized with parental serotypes. These results indicate that polyploid viruses might potentially acquire advantages from parental serotypes for enhancement of AAV transduction and evasion of Nab recognition without increasing capsid antigen presentation in target cells. Polyploid AAV vectors can be generated from any AAV serotype, whether natural, rational, library derived or a combination thereof, providing a novel strategy that should be explored in future clinical trials in patients with neutralizing antibodies.

NRF2 is a transcription factor that drives antioxidant gene expression in multiple organ systems. We hypothesized that Nrf2 overexpression could be therapeutically applied toward diseases in which redox homeostasis is disrupted. In this study, adeno-associated virus (AAV)-Nrf2 was tested in a mouse model of acute acetaminophen-induced liver toxicity and successfully conferred protection from hepatotoxicity, validating the vector design and early onset of NRF2-mediated protection. Furthermore, therapeutic potential of AAV-Nrf2 in chronic disease also was tested in a light-induced mouse model of age-related macular degeneration. Adult BALB/c mice were intravitreally injected with AAV-Nrf2 and subject to light damage following injection. Retinal thickness and function were monitored following light damage using optical coherence tomography and electroretinography, respectively. By 3 months post-damage, injected eyes had greater retinal thickness compared to uninjected controls. At 1 month post-damage, AAV-Nrf2 injection facilitated full functional recovery from light damage. Our results suggest a therapeutic potential for Nrf2 overexpression in acute and long-term capacities in multiple organ systems, opening up doors for combination gene therapy where replacement gene therapy requires additional therapeutic support to prevent further degeneration.

Breakdown of neuro-glial N-acetyl-aspartate (NAA) metabolism results in the failure of developmental myelination, manifest in the congenital pediatric leukodystrophy Canavan disease caused by mutations to the sole NAA catabolizing enzyme aspartoacylase. Canavan disease is a major point of focus for efforts to define NAA function, with available evidence suggesting NAA serves as an acetyl donor for fatty acid synthesis during myelination. Elevated NAA is a diagnostic hallmark of Canavan disease, which contrasts with a broad spectrum of alternative neurodegenerative contexts in which levels of NAA are inversely proportional to pathological progression. Recently generated data in the nur7 mouse model of Canavan disease suggests loss of aspartoacylase function results in compromised energetic integrity prior to oligodendrocyte death, abnormalities in myelin content, spongiform degeneration, and motor deficit. The present study utilized a next-generation "oligotropic" adeno-associated virus vector (AAV-Olig001) to quantitatively assess the impact of aspartoacylase reconstitution on developmental myelination. AAV-Olig001-aspartoacylase promoted normalization of NAA, increased bioavailable acetyl-CoA, and restored energetic balance within a window of postnatal development preceding gross histopathology and deteriorating motor function. Long-term effects included increased oligodendrocyte numbers, a global increase in myelination, reversal of vacuolation, and rescue of motor function. Effects on brain energy observed following AAV-Olig001-aspartoacylase gene therapy are shown to be consistent with a metabolic profile observed in mild cases of Canavan disease, implicating NAA in the maintenance of energetic integrity during myelination via oligodendroglial aspartoacylase.

Many adeno-associated virus (AAV) serotypes efficiently transduce the retina when delivered to the subretinal space but show limited success when delivered to the vitreous due to the inner limiting membrane (ILM). Subretinal delivery of AAV serotype 2 (AAV2) and its heparan sulfate (HS)-binding-deficient capsid led to similar expression, indicating transduction of the outer retina occurred by HS-independent mechanisms. However, intravitreal delivery of HS-ablated recombinant AAV2 (rAAV2) led to a 300-fold decrease in transduction compared to AAV2. Fluorescence in situ hybridization of AAV transgenes was used to identify differences in retinal trafficking and revealed that HS binding was responsible for AAV2 accumulation at the ILM. This mechanism was tested on human ex vivo retinas and showed similar accumulation with HS-binding AAV2 capsid only. To evaluate if HS binding could be applied to other AAV serotypes to enhance their transduction, AAV1 and AAV8 were modified to bind HS with a single-amino-acid mutation and tested in mice. Both HS-binding mutants of AAV1 and AAV8 had higher intravitreal transduction than their non-HS-binding parent capsid due to increased retinal accumulation. To understand the influence that HS binding has on tropism, chimeric AAV2 capsids with dual-glycan usage were tested intravitreally in mice. Compared to HS binding alone, these chimeric capsids displayed enhanced transduction that was correlated with a change in tropism. Taken together, these data indicate that HS binding serves to sequester AAV capsids from the vitreous to the ILM but does not influence retinal tropism. The enhanced retinal transduction of HS-binding capsids provides a rational design strategy for engineering capsids for intravitreal delivery. IMPORTANCE Adeno-associated virus (AAV) has become the vector of choice for viral gene transfer and has shown great promise in clinical trials. The need for development of an easy, less invasive injection route for ocular gene therapy is met by intravitreal delivery, but delivery of AAV by this route results in poor transduction outcomes. The inner limiting membrane (ILM) creates a barrier separating the vitreous and the retina. Binding of AAV to heparan sulfate proteoglycan (HSPG) at the ILM may allow the virus to traverse this barrier for better retinal transduction. We show that HSPG binding is correlated with greater accumulation and penetration of AAV in the retina. We demonstrated that this accumulation is conserved across mouse and human retinas and that the addition of HSPG binding to other AAV capsids can increase the number of vectors accumulating at the ILM without dictating tropism.

Adeno-associated virus (AAV) continues to be a promising viral vector for delivery of therapeutic genes in human gene therapy. In 2012, the European Commission approved Glybera (alipogene tiparvovec),1 an AAV vector carrying the human lipoprotein lipase gene for commercial use in treatment of lipoprotein lipase deficiency. In addition, within the past five years there have been numerous successes in preclinical, early, and late-stage clinical trials of AAV for treatment of a variety of diseases, including muscular dystrophy, hemophilia, Leber’s congenital amaurosis retinopathy, age-related macular degeneration, cardiac disorders, cancer, and neurodegenerative disorders, such as Parkinson’s disease and Canavan disease. However, despite this progress, the infectious pathway of AAV vectors is an evolving story. In a study recently reported in Nature, Pillay and colleagues2 employed a powerful genome-wide haploid genetic screen to gain a better understanding of the AAV infectious entry pathway. Among other factors consistent with the known pathways of entry and infection, the authors identified a new cellular factor that is required for infection of multiple AAV serotypes, which they term AAV receptor (AAVR).