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Genome editing has the potential to treat genetic diseases in a variety of tissues, including the lung. We have previously developed and validated a dual adeno-associated virus (AAV) CRISPR platform that supports effective editing in the airways of mice. To validate this delivery vehicle in a large animal model, we have shown that intratracheal instillation of CRISPR/Cas9 in AAV5 can edit a housekeeping gene or a disease-related gene in the lungs of young rhesus monkeys. We observed up to 8% editing of angiotensin-converting enzyme 2 (ACE2) in lung lobes after single-dose administration. Single-nuclear RNA sequencing revealed that AAV5 transduces multiple cell types in the caudal lung lobes, including alveolar cells, macrophages, fibroblasts, endothelial cells, and B cells. These results demonstrate that AAV5 is efficient in the delivery of CRISPR/Cas9 in the lung lobes of young rhesus monkeys. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV)-based vectors are used clinically for gene transfer and persist as extrachromosomal episomes. A small fraction of vector genomes integrate into the host genome, but the theoretical risk of tumorigenesis depends on vector regulatory features. A mouse model was used to investigate integration profiles of an AAV serotype 5 (AAV5) vector produced using Sf and HEK293 cells that mimic key features of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), a gene therapy for severe hemophilia A. The majority (95%) of vector genome reads were derived from episomes, and mean (± standard deviation) integration frequency was 2.70 ± 1.26 and 1.79 ± 0.86 integrations per 1,000 cells for Sf- and HEK293-produced vector. Longitudinal integration analysis suggested integrations occur primarily within 1 week, at low frequency, and their abundance was stable over time. Integration profiles were polyclonal and randomly distributed. No major differences in integration profiles were observed for either vector production platform, and no integrations were associated with clonal expansion. Integrations were enriched near transcription start sites of genes highly expressed in the liver (p = 1 × 10−4) and less enriched for genes of lower expression. We found no evidence of tumorigenesis or fibrosis caused by the vector integrations.

Adeno-associated virus (AAV) vectors are promising gene therapy candidates, but pre-existing anti-AAV neutralizing antibodies (NAbs) pose a significant challenge to successful gene delivery. Knowledge of NAb seroprevalence remains limited and inconsistent. We measured activity of NAbs against six clinically relevant AAV serotypes across 10 countries in adults (n = 502) and children (n = 50) using a highly sensitive transduction inhibition assay. NAb prevalence was generally highest for AAV1 and lowest for AAV5. There was considerable variability across countries and geographical regions. NAb prevalence increased with age and was higher in females, participants of Asian ethnicity, and participants in cancer trials. Co-prevalence was most frequently observed between AAV1 and AAV6 and less frequently between AAV5 and other AAVs. Machine learning analyses revealed a unique clustering of AAVs that differed from previous phylogenetic classifications. These results offer insights into the biological relationships between the immunogenicity of AAVs in humans beyond that observed previously using standard clades, which are based on linear capsid sequences. Our findings may inform improved vector design and facilitate the development of AAV vector-mediated clinical gene therapies.

Agmatine, a decarboxylated form of L-arginine, prevents opioid analgesic tolerance, dependence, and self-administration when given by both central and systemic routes of administration. Endogenous agmatine has been previously detected in the central nervous system. The presence of a biochemical pathway for agmatine synthesis offers the opportunity for sitespecific overexpression of the presumptive synthetic enzyme for local therapeutic effects. In the present study, we evaluated the development of opioid analgesic tolerance in ICR-CD1 mice pre-treated with either vehicle control or intrathecally delivered adeno-associated viral vectors (AAV) carrying the gene for human arginine decarboxylase (hADC). Vehicle-treated or AAVhADC-treated mice were each further divided into two groups which received repeated delivery over three days of either saline or systemically-delivered morphine intended to induce opioid analgesic tolerance. Morphine analgesic dose-response curves were constructed in all subjects on day four using the warm water tail flick assay as the dependent measure. We observed that pretreatment with AAV-hADC prevented the development of analgesic tolerance to morphine. Peripheral and central nervous system tissues were collected and analyzed for presence of hADC mRNA. In a similar experiment, AAV-hADC pre-treatment prevented the development of analgesic tolerance to a high dose of the opioid neuropeptide endomorphin-2. Intrathecal delivery of antiagmatine IgG (but not normal IgG) reversed the inhibition of endomorphin-2 analgesic tolerance in AAV-hADC-treated mice. To summarize, we report here the effects of AAV-mediated gene transfer of human ADC (hADC) in models of opioid-induced analgesic tolerance. This study suggests that gene therapy may contribute to reducing opioid analgesic tolerance. [ABSTRACT FROM AUTHOR]

Agmatine, a decarboxylated form of L-arginine, prevents opioid analgesic tolerance, dependence, and self-administration when given by both central and systemic routes of administration. Endogenous agmatine has been previously detected in the central nervous system. The presence of a biochemical pathway for agmatine synthesis offers the opportunity for site-specific overexpression of the presumptive synthetic enzyme for local therapeutic effects. In the present study, we evaluated the development of opioid analgesic tolerance in ICR-CD1 mice pre-treated with either vehicle control or intrathecally delivered adeno-associated viral vectors (AAV) carrying the gene for human arginine decarboxylase (hADC). Vehicle-treated or AAV-hADC-treated mice were each further divided into two groups which received repeated delivery over three days of either saline or systemically-delivered morphine intended to induce opioid analgesic tolerance. Morphine analgesic dose-response curves were constructed in all subjects on day four using the warm water tail flick assay as the dependent measure. We observed that pre-treatment with AAV-hADC prevented the development of analgesic tolerance to morphine. Peripheral and central nervous system tissues were collected and analyzed for presence of hADC mRNA. In a similar experiment, AAV-hADC pre-treatment prevented the development of analgesic tolerance to a high dose of the opioid neuropeptide endomorphin-2. Intrathecal delivery of anti-agmatine IgG (but not normal IgG) reversed the inhibition of endomorphin-2 analgesic tolerance in AAV-hADC-treated mice. To summarize, we report here the effects of AAV-mediated gene transfer of human ADC (hADC) in models of opioid-induced analgesic tolerance. This study suggests that gene therapy may contribute to reducing opioid analgesic tolerance.

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) gene transfer provided reduced bleeding for adult clinical trial participants with severe hemophilia A. However, pediatric outcomes are unknown. Using a mouse model of hemophilia A, we investigated the effect of vector dose and age at treatment on transgene production and persistence. We dosed AAV5-hFVIII-SQ to neonatal and adult mice based on body weight or at a fixed dose and assessed human factor VIII-SQ variant (hFVIII-SQ) expression through 16 weeks. AAV5-hFVIII-SQ dosed per body weight in neonatal mice did not result in meaningful plasma hFVIII-SQ protein levels in adulthood. When treated with the same total vector genomes per mouse as adult mice, neonates maintained hFVIII-SQ expression into adulthood, although plasma levels were 3- to 4-fold lower versus mice dosed as adults. Mice

Adeno-associated viruses (AAVs) are one of the most promising tools for gene therapy applications. These vectors are purified using affinity and ion exchange chromatography, typically using packed beds of resin adsorbents. This leads to diffusion and pressure drop limitations that affect process productivity. Due to their high surface area and porosity, electrospun nanofiber adsorbents offer mass transfer and flow rate advantages over conventional chromatographic media. The present work investigated the use of affinity cellulose-based nanofiber adsorbents for adeno-associated virus serotype 5 (AAV5) capture, evaluating dynamic binding capacity, pressure drop, and AAV5 recovery at residence times (RT) less than 5 s. The dynamic binding capacity was found to be residence time-dependent, but nevertheless higher than 1.0 × 1014 TP mL−1 (RT = 1.6 s), with a pressure drop variation of 0.14 MPa obtained after loading more than 2,000 column volumes of clarified AAV5 feedstock. The single affinity chromatography purification step using these new affinity adsorbents resulted in 80% virus recovery, with the removal of impurities comparable to that of bead-based affinity adsorbents. The high binding capacity, virus recovery and reduced pressure drop observed at residence times in the sub-minute range can potentially eliminate the need for prior concentration steps, thereby reducing the overall number of unit operations, process time and costs.

Impressive achievements in clinical trials to treat hemophilia establish a milestone in the development of gene therapy. It highlights the significance of AAV-mediated gene delivery to liver. AAV5 is a unique serotype featured by low neutralizing antibody prevalence. Nevertheless, its liver infectivity is relatively weak. Consequently, it is vital to exploit novel AAV5 capsid mutants with robust liver tropism. To this aim, we performed AAV5-NNK library and barcode screening in mice, from which we identified one capsid variant, called AAVzk2. AAVzk2 displayed a similar yield but divergent post-translational modification sites compared with wild-type serotypes. Mice intravenously injected with AAVzk2 demonstrated a stronger liver transduction than AAV5, roughly comparable with AAV8 and AAV9, with undetectable transduction of other tissues or organs such as heart, lung, spleen, kidney, brain, and skeletal muscle, indicating a liver-specific tropism. Further studies showed a superior human hepatocellular transduction of AAVzk2 to AAV5, AAV8 and AAV9, whereas the seroreactivity of AAVzk2 was as low as AAV5. Overall, we provide a novel AAV serotype that facilitates a robust and specific liver gene delivery to a large population, especially those unable to be treated by AAV8 and AAV9.

[Display omitted] • Impaired motor thalamus and cortex activity causes parkinsonian movement deficits. • Examined utility of optogenetic stimulation with the red-shifted opsin, Chrimson. • Stimulated Chrimson in motor thalamus glutamatergic neurons in parkinsonian rats. • Akinesia was improved by low frequency stimulation with complex and tonic patterns. • Viability of optogenetic stimulation in large brains is improved with Chrimson. Parkinson's disease (PD) is a motor disorder charactertised by altered neural activity throughout the basal ganglia-thalamocortical circuit. Electrical deep brain stimulation (DBS) is efficacious in alleviating motor symptoms, but has several notable side-effects, most likely reflecting the non-specific nature of electrical stimulation and/or the brain regions targeted. We determined whether specific optogenetic activation of glutamatergic motor thalamus (Mthal) neurons alleviated forelimb akinesia in a chronic rat model of PD. Parkinsonian rats (unilateral 6-hydroxydopamine injection) were injected with an adeno-associated viral vector (AAV5-CaMKII-Chrimson-GFP) to transduce glutamatergic Mthal neurons with the red-shifted Chrimson opsin. Optogenetic stimulation with orange light at 15 Hz tonic and a physiological pattern, previously recorded from a Mthal neuron in a control rat, significantly increased forelimb use in the reaching test (p < 0.01). Orange light theta burst stimulation, 15 Hz and control reaching patterns significantly reduced akinesia (p < 0.0001) assessed by the step test. In contrast, forelimb use in the cylinder test was unaffected by orange light stimulation with any pattern. Blue light (control) stimulation failed to alter behaviours. Activation of Chrimson using complex patterns in the Mthal may be an alternative treatment to recover movement in PD. These vector and opsin changes are important steps towards translating optogenetic stimulation to humans. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) is a small ssDNA satellite virus of high interest (in recombinant form) as a safe and effective gene therapy vector. AAV's human cell entry receptor (AAVR) contains polycystic kidney disease (PKD) domains bound by AAV. Seeking understanding of the spectrum of interactions, goat AAVGo.1 is investigated, because its host is the species most distant from human with reciprocal cross-species cell susceptibility. The structure of AAVGo.1, solved by cryo-EM to 2.9 Å resolution, is most similar to AAV5. Through ELISA (enzyme-linked immunosorbent assay) studies, it is shown that AAVGo.1 binds to human AAVR more strongly than do AAV2 or AAV5, and that it joins AAV5 in a class that binds exclusively to PKD domain 1 (PKD1), in contrast to other AAVs that interact primarily with PKD2. The AAVGo.1 cryo-EM structure of a complex with a PKD12 fragment of AAVR at 2.4 Å resolution shows PKD1 bound with minimal change in virus structure. There are only minor conformational adaptations in AAVR, but there is a near-rigid rotation of PKD1 with maximal displacement of the receptor domain by ;1 Å compared to PKD1 bound to AAV5. AAVGo.1 joins AAV5 as the second member of an emerging class of AAVs whose mode of receptor-binding is completely different from other AAVs, typified by AAV2. [ABSTRACT FROM AUTHOR]

In this study, we built upon our previous work to demonstrate the distribution and transport of AAV5-green fluorescent protein (GFP) following a single convection-enhanced delivery infusion into the nonhuman primate cerebellum, with no untoward side effects noted. Dosing under magnetic resonance imaging guidance revealed a sixfold larger volume of distribution compared with the volume of infusion, with no evidence of reflux underscoring the convective properties of the cerebellum and step design of the cannula. Postmortem tissue analysis, 4 weeks post-adeno-associated viral (AAV) delivery, revealed the robust presence of the transgene in situ, with GFP detection in secondary regions not directly targeted by the infusion, denoting distal transport of the vector. Irrespective of tropism, a twofold larger area of transgene expression was found and was corroborated against the presence of contrast on T1-weighted images. Different levels of transduction were detected between animals, which were negatively correlated with the level of antibody titer against the GFP construct, whereby the higher the antibody titer, the lower the level of transgene expression. These findings support the use of the posterior fossa as a potential target site for direct delivery of gene-based therapeutics for cerebellar diseases. [ABSTRACT FROM AUTHOR]

Objective: Inflammatory hand arthritis (IHA) results in impaired function. Local gene therapy with ART-I02, a recombinant adeno-associated virus (AAV) serotype 5 vector expressing interferon (IFN)-β, under the transcriptional control of nuclear factor κ-B responsive promoter, was preclinically shown to have favorable effects. This study aimed to investigate the safety and tolerability of local gene therapy with ART-I02 in patients with IHA.Methods: In this first-in-human, dose-escalating, cohort study, 12 IHA patients were to receive a single intra-articular (IA) injection of ART-I02 ranging 0.3 × 1012-1.2 × 1013 genome copies in an affected hand joint. Adverse events (AEs), routine safety laboratory and the clinical course of disease were periodically evaluated. Baseline- and follow-up contrast enhanced magnetic resonance images (MRIs), shedding of viral vectors in bodily fluids, and AAV5 and IFN-β immune responses were evaluated. A data review committee provided safety recommendations.Results: Four patients were enrolled. Long-lasting local AEs were observed in 3 patients upon IA injection of ART-I02. The AEs were moderate in severity and could be treated conservative. Given the duration of the AEs and their possible or probable relation to ART-I02, no additional patients were enrolled. No systemic treatment emergent AEs were observed. The MRIs reflected the AEs by (peri)arthritis. No T-cell response against AAV5 or IFN-β, nor IFN-β antibodies could be detected. Neutralizing antibody titers against AAV5 raised post-dose.Conclusion: Single IA doses of 0.6 × 1012 or 1.2 × 1012 ART-I02 vector genomes were administered without systemic side effects or serious AEs. However, local tolerability was insufficient for continuation.Trial Registration: NCT02727764. [ABSTRACT FROM AUTHOR]

Adeno-associated virus 5 (AAV5)-human factor VIII-SQ (hFVIII-SQ; valoctocogene roxaparvovec) is an AAV-mediated product under evaluation for treatment of severe hemophilia A, which contains a B-domain-deleted hFVIII (hFVIII-SQ) transgene and a hybrid liver-specific promotor (HLP). To increase FVIII-SQ expression and reduce the vector dose required, a stronger promoter may be considered. However, because FVIII-SQ is a protein known to be difficult to fold and secrete, this could potentially induce endoplasmic reticulum (ER) stress. We evaluated the effect of two AAV5-hFVIII-SQ vectors with different liver-specific promoter strength (HLP ≪ 100ATGB) on hepatic ER stress in mice. Five weeks after receiving vehicle or vector, the percentage of transduced hepatocytes and levels of liver hFVIII-SQ DNA and RNA increased dose dependently for both vectors. At lower doses, plasma hFVIII-SQ protein levels were higher for 100ATGB. This difference was attenuated at the highest dose. For 100ATGB, liver hFVIII-SQ protein accumulated dose dependently, with increased expression of ER stress markers at the highest dose, suggesting hepatocytes reached or exceeded their capacity to fold/secrete hFVIII-SQ. These data suggest that weaker promoters may require relatively higher doses to distribute expression load across a greater number of hepatocytes, whereas relatively stronger promoters may produce comparable levels of FVIII in fewer hepatocytes, with potential for ER stress.

Adeno-associated virus (AAV) has been used extensively as a vector for gene therapy. Despite its widespread use, the mechanisms by which AAV enters the cell and is trafficked to the nucleus are poorly understood. In this study, we performed two pooled, genome-wide screens to identify positive and negative factors modulating AAV2 transduction. Genome-wide libraries directed against all human genes with four designs per gene or eight designs per gene were transduced into U-2 OS cells. These pools were transduced with AAV2 encoding EGFP and sorted based on the intensity of EGFP expression. Analysis of enriched and depleted barcodes in the sorted samples identified several genes that putatively decreased AAV2 transduction. A subset of screen hits was validated in flow cytometry and imaging studies. In addition to KIAA0319L (AAVR), we confirmed the role of two genes, GPR108 and TM9SF2, in mediating viral transduction in eight different AAV serotypes. Interestingly, GPR108 displayed serotype selectivity and was not required for AAV5 transduction. Follow-up studies suggested that GPR108 localized primarily to the Golgi, where it may interact with AAV and play a critical role in mediating virus escape or trafficking. Cumulatively, these results expand our understanding of the process of AAV transduction in different cell types and serotypes.

Adeno-associated virus (AAV)-based vectors are used clinically for gene transfer and persist as extrachromosomal episomes. A small fraction of vector genomes integrate into the host genome, but the theoretical risk of tumorigenesis depends on vector regulatory features. A mouse model was used to investigate integration profiles of an AAV serotype 5 (AAV5) vector produced using Sf and HEK293 cells that mimic key features of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), a gene therapy for severe hemophilia A. The majority (95%) of vector genome reads were derived from episomes, and mean (± standard deviation) integration frequency was 2.70 ± 1.26 and 1.79 ± 0.86 integrations per 1,000 cells for Sf - and HEK293-produced vector. Longitudinal integration analysis suggested integrations occur primarily within 1 week, at low frequency, and their abundance was stable over time. Integration profiles were polyclonal and randomly distributed. No major differences in integration profiles were observed for either vector production platform, and no integrations were associated with clonal expansion. Integrations were enriched near transcription start sites of genes highly expressed in the liver ( p  = 1 × 10 -4 ) and less enriched for genes of lower expression. We found no evidence of tumorigenesis or fibrosis caused by the vector integrations., Competing Interests: A.M.I., E.W., and B.Y. are employees and stockholders of BioMarin Pharmaceutical Inc. T.B.,W.C., and S.F. are former employees and potential stockholders of BioMarin Pharmaceutical Inc. M.F. is an employee of ProtaGene US Inc., (© 2024 The Authors.)

Adeno-associated virus (AAV) vectors are promising gene therapy candidates, but pre-existing anti-AAV neutralizing antibodies (NAbs) pose a significant challenge to successful gene delivery. Knowledge of NAb seroprevalence remains limited and inconsistent. We measured activity of NAbs against six clinically relevant AAV serotypes across 10 countries in adults ( n  = 502) and children ( n  = 50) using a highly sensitive transduction inhibition assay. NAb prevalence was generally highest for AAV1 and lowest for AAV5. There was considerable variability across countries and geographical regions. NAb prevalence increased with age and was higher in females, participants of Asian ethnicity, and participants in cancer trials. Co-prevalence was most frequently observed between AAV1 and AAV6 and less frequently between AAV5 and other AAVs. Machine learning analyses revealed a unique clustering of AAVs that differed from previous phylogenetic classifications. These results offer insights into the biological relationships between the immunogenicity of AAVs in humans beyond that observed previously using standard clades, which are based on linear capsid sequences. Our findings may inform improved vector design and facilitate the development of AAV vector-mediated clinical gene therapies., Competing Interests: J.E.J.R. has received supply of material (Material Transfer Agreement), consultancy, or honoraria from bluebird bio, Cynata, Novartis, Pfizer, RareCyte, Roche, and SPARK Therapeutics and has shareholdings with RareCyte and Woke. Y.P., C.d.F.P., and I.W. are employees of, and own stock/options in, Pfizer. G.B., P.V.H., S.S., and A.C. were employed at Pfizer at the time of this study. C.J.P. and T.W. are employees of, and hold equity in, Labcorp., (© 2024 The Authors.)

Adeno-associated virus serotype 5 (AAV5) is being developed as a gene delivery vector for several diseases, including hemophilia and Huntington's disease, and has a demonstrated efficient transduction in liver, lung, skeletal muscle, and the central nervous system. One limitation of AAV gene delivery is preexisting neutralizing antibodies, which present a significant challenge for vector effectiveness in therapeutic applications. Here, we report the cryo-electron microscopy (cryo-EM) and image-reconstructed structure of AAV5 in complex with a newly generated monoclonal antibody, HL2476, at 3.1-Å resolution. Unlike other available anti-AAV5 capsid antibodies, ADK5a and ADK5b, with epitopes surrounding the 5-fold channel of the capsid, HL2476 binds to the 3-fold protrusions. To elucidate the capsid-antibody interactions, the heavy and light chains were sequenced and their coordinates, along with the AAV5 viral protein, assigned to the density map. The high resolution of the complex enabled the identification of interacting residues at the 3-fold protrusions of the capsid, including R483, which forms two hydrogen bonds with the light chain of HL2476. A panel of AAV5 variants was generated and analyzed by native dot immunoblot and transduction assays. This identified variants with antibody escape phenotypes that maintain infectivity. IMPORTANCE Biologics based on recombinant AAVs (rAAVs) are increasingly becoming attractive human gene delivery vehicles, especially after the approval of Glybera in Europe and Luxturna in the United States. However, preexisting neutralizing antibodies against the AAV capsids in a large percentage of the human population limit widespread utilization of these vectors. To circumvent this problem, stealth vectors must be generated that are undetectable by these antibodies. This study details the highresolution characterization of a new antigenic region on AAV5, a vector being developed for numerous delivery applications. The structure of AAV5 complexed with HL2476, a novel antibody, was determined by cryo-EM to 3.1-Å resolution. The resolution of the density map enabled the identification of interacting residues between capsid and antibody and the determinants of neutralization. Thus, the information obtained from this study can facilitate the generation of host immune escape vectors. [ABSTRACT FROM AUTHOR]

Huntington disease (HD) is a fatal, neurodegenerative genetic disorder with aggregation of mutant Huntingtin protein (mutHTT) in the brain as a key pathological mechanism. There are currently no disease modifying therapies for HD; however, HTT-lowering therapies hold promise. Recombinant adeno-associated virus serotype 5 expressing a microRNA that targets HTT mRNA (AAV5-miHTT) is in development for the treatment of HD with promising results in rodent and minipig HD models. To support a clinical trial, toxicity studies were performed in non-human primates (NHP, Macaca fascicularis) and Sprague-Dawley rats to evaluate the safety of AAV5-miHTT, the neurosurgical administration procedure, vector delivery and expression of the miHTT transgene during a 6-month observation period. For accurate delivery of AAV5-miHTT to the striatum, real-time magnetic resonance imaging (MRI) with convection-enhanced delivery (CED) was used in NHP. Catheters were successfully implanted in 24 NHP, without neurological symptoms, and resulted in tracer signal in the target areas. Widespread vector DNA and miHTT transgene distribution in the brain was found, particularly in areas associated with HD pathology. Intrastriatal administration of AAV5-miHTT was well tolerated with no clinically relevant changes in either species. These studies demonstrate the excellent safety profile of AAV5-miHTT, the reproducibility and tolerability of intrastriatal administration, and the delivery of AAV5-miHTT to the brain, which support the transition of AAV5-miHTT into clinical studies.

Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry.

Achromatopsia causes severely reduced visual acuity, photoaversion, and inability to discern colors due to cone photoreceptor dysfunction. In 2010, we reported on day-blindness in sheep caused by a stop-codon mutation of the ovine CNGA3 gene and began gene augmentation therapy trials in this naturally occurring large animal model of CNGA3 achromatopsia. The purpose of this study was to evaluate long-term efficacy and safety results of treatment, findings that hold great relevance for clinical trials that started recently in CNGA3 achromatopsia patients. Nine day-blind sheep were available for long-term follow up. The right eye of each sheep was treated with a single subretinal injection of an Adeno-Associated Virus Type 5 (AAV5) vector carrying either a mouse (n = 4) or a human (n = 5) CNGA3 transgene under control of the 2.1-Kb red/green opsin promoter. The efficacy of treatment was assessed periodically with photopic maze tests and electroretinographic (ERG) recordings for as long as 74 months postoperatively. Safety was assessed by repeated ophthalmic examinations and scotopic ERG recordings. The retinas of three animals that died of unrelated causes >5 years post-treatment were studied histologically and immunohistochemically using anti-hCNGA3 and anti-red/green cone opsin antibodies. Passage time and number of collisions of treated sheep in the photopic maze test were significantly lower at all follow-up examinations as compared with pretreatment values (p = 0.0025 and p < 0.001, respectively). ERG Critical Flicker Fusion Frequency and flicker amplitudes at 30 and 40 Hz showed significant improvement following treatment (p < 0.0001) throughout the study. Ophthalmic examinations and rod ERG recordings showed no abnormalities in the treated eyes. Immunohistochemistry revealed the presence of CNGA3 protein in red/green opsin-positive cells (cones) of the treated eyes. Our results show significant, long-term improvement in cone function, demonstrating a robust rescue effect up to six years following a single treatment with a viral vector that provides episomal delivery of the transgene. This unique follow-up duration confirms the safe and stable nature of AAV5 gene therapy in the ovine achromatopsia model. [ABSTRACT FROM AUTHOR]

Grafting of cell-seeded alginate capillary hydrogels into a spinal cord lesion site provides an axonal bridge while physically directing regenerating axonal growth in a linear pattern. However, without an additional growth stimulus, bridging axons fail to extend into the distal host spinal cord. Here we examined whether a combinatory strategy would support regeneration of descending axons across a cervical (C5) lateral hemisection lesion in the rat spinal cord. Following spinal cord transections, Schwann cell (SC)-seeded alginate hydrogels were grafted to the lesion site and AAV5 expressing brain-derived neurotrophic factor (BDNF) under control of a tetracycline-regulated promoter was injected caudally. In addition, we examined whether SC injection into the caudal spinal parenchyma would further enhance regeneration of descending axons to re-enter the host spinal cord. Our data show that both serotonergic and descending axons traced by biotinylated dextran amine (BDA) extend throughout the scaffolds. The number of regenerating axons is significantly increased when caudal BDNF expression is activated and transient BDNF delivery is able to sustain axons after gene expression is switched off. Descending axons are confined to the caudal graft/host interface even with continuous BDNF expression for 8 weeks. Only with a caudal injection of SCs, a pathway facilitating axonal regeneration through the host/graft interface is generated allowing axons to successfully re-enter the caudal spinal cord. Statement of Significance Recovery from spinal cord injury is poor due to the limited regeneration observed in the adult mammalian central nervous system. Biomaterials, cell transplantation and growth factors that can guide axons across a lesion site, provide a cellular substrate, stimulate axon growth and have shown some promise in increasing the growth distance of regenerating axons. In the present study, we combined an alginate biomaterial with linear channels with transplantation of Schwann cells within and beyond the lesion site and injection of a regulatable vector for the transient expression of brain-derived neurotrophic factor (BDNF). Our data show that only with the full combination axons extend across the lesion site and that expression of BDNF beyond 4 weeks does not further increase the number of regenerating axons. [ABSTRACT FROM AUTHOR]

Adeno-associated viruses (AAVs) are one of the most promising tools for gene therapy applications. These vectors are purified using affinity and ion exchange chromatography, typically using packed beds of resin adsorbents. This leads to diffusion and pressure drop limitations that affect process productivity. Due to their high surface area and porosity, electrospun nanofiber adsorbents offer mass transfer and flow rate advantages over conventional chromatographic media. The present work investigated the use of affinity cellulose-based nanofiber adsorbents for adeno-associated virus serotype 5 (AAV5) capture, evaluating dynamic binding capacity, pressure drop, and AAV5 recovery at residence times (RT) less than 5 s. The dynamic binding capacity was found to be residence time-dependent, but nevertheless higher than 1.0 × 10 14  TP mL -1 (RT = 1.6 s), with a pressure drop variation of 0.14 MPa obtained after loading more than 2,000 column volumes of clarified AAV5 feedstock. The single affinity chromatography purification step using these new affinity adsorbents resulted in 80% virus recovery, with the removal of impurities comparable to that of bead-based affinity adsorbents. The high binding capacity, virus recovery and reduced pressure drop observed at residence times in the sub-minute range can potentially eliminate the need for prior concentration steps, thereby reducing the overall number of unit operations, process time and costs., Competing Interests: Author SS was employed by the company Cytiva and Author AS was employed by the company Cytiva. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Neto, Mendes, Santos, Solbrand, Carrondo, Peixoto and Silva.)

We report the pattern of transgene expression across brain regions after intrathecal delivery of adeno-associated virus serotype 5 (AAV5). Labeling in hindbrain appeared to be primarily neuronal, and was detected in sensory nuclei of medulla, pontine nuclei, and all layers of cerebellar cortex. Expression in midbrain was minimal, and generally limited to isolated neurons and astrocytes in the cerebral peduncles. GFP immunoreactivity (-ir) in thalamus was most prominent in medial geniculate nucleus, and otherwise limited to posterior nuclei of the dorsal and lateral margins. Labeling was also observed in neurons and astrocytes of the hippocampal formation and amygdaloid complex. In the hippocampal formation, GFP-ir was found in neuronal cell bodies of the rostral ventral portion, but was largely restricted to fiber-like staining in the molecular layer of dentate gyrus and stratum lacunosum-moleculare of the rostral dorsal region. GFP-ir was seen in neurons and astroglia throughout caudal cortex, whereas in rostral regions of neocortex it was limited to isolated astrocytes and neurons. Labeling was also present in olfactory bulb. These results demonstrate that intrathecal delivery of AAV5 vector leads to transgene expression in discrete CNS regions throughout the rostro-caudal extent of the neuraxis. A caudal-to-rostral gradient of decreasing GFP-ir was present in choroid plexus and Purkinje cells, suggesting that spread of virus through cerebrospinal fluid plays a role in the resulting transduction pattern. Other factors contributing to the observed expression pattern likely include variations in cell-surface receptors and inter-parenchymal space.

We report the pattern of transgene expression across brain regions after intrathecal delivery of adeno-associated virus serotype 5 (AAV5). Labeling in hindbrain appeared to be primarily neuronal, and was detected in sensory nuclei of medulla, pontine nuclei, and all layers of cerebellar cortex. Expression in midbrain was minimal, and generally limited to isolated neurons and astrocytes in the cerebral peduncles. GFP immunoreactivity (-ir) in thalamus was most prominent in medial geniculate nucleus, and otherwise limited to posterior nuclei of the dorsal and lateral margins. Labeling was also observed in neurons and astrocytes of the hippocampal formation and amygdaloid complex. In the hippocampal formation, GFP-ir was found in neuronal cell bodies of the rostral ventral portion, but was largely restricted to fiber-like staining in the molecular layer of dentate gyrus and stratum lacunosum-moleculare of the rostral dorsal region. GFP-ir was seen in neurons and astroglia throughout caudal cortex, whereas in rostral regions of neocortex it was limited to isolated neurons and non-neuronal cells. Labeling was also present in olfactory bulb. These results demonstrate that intrathecal delivery of AAV5 vector leads to transgene expression in discrete CNS regions throughout the rostro-caudal extent of the neuraxis. A caudal-to-rostral gradient of decreasing GFP-ir was present in choroid plexus and Purkinje cells, suggesting that spread of virus through cerebrospinal fluid plays a role in the resulting transduction pattern. Other factors contributing to the observed expression pattern likely include variations in cell-surface receptors and inter-parenchymal space. [ABSTRACT FROM AUTHOR]

Previously we found that inhibitor of differentiation 3 (Id3) gene, a transcriptional repressor, efficiently inhibits corneal keratocyte differentiation to myofibroblasts in vitro. This study evaluated the potential of adeno-associated virus 5 (AAV5)-mediated Id3 gene therapy to treat corneal scarring using an established rabbit in vivo disease model. Corneal scarring/fibrosis in rabbit eyes was induced by alkali trauma, and 24 h thereafter corneas were administered with either balanced salt solution AAV5-naked vector, or AAV5-Id3 vector (n = 6/group) via an optimized reported method. Therapeutic effects of AAV5-Id3 gene therapy on corneal pathology and ocular health were evaluated with clinical, histological, and molecular techniques. Localized AAV5-Id3 gene therapy significantly inhibited corneal fibrosis/haze clinically from 2.7 to 0.7 on the Fantes scale in live animals (AAV5-naked versus AAV5-Id3; p < 0.001). Furthermore, AAV5-Id3 treatment significantly reduced profibrotic gene mRNA levels: α-smooth muscle actin (α-SMA) (2.8-fold; p < 0.001), fibronectin (3.2-fold; p < 0.001), collagen I (0.8-fold; p < 0.001), and collagen III (1.4-fold; p < 0.001), as well as protein levels of α-SMA (23.8%; p < 0.001) and collagens (1.8-fold; p < 0.001). The anti-fibrotic activity of AAV5-Id3 is attributed to reduced myofibroblast formation by disrupting the binding of E-box proteins to the promoter of α-SMA, a transforming growth factor-β signaling downstream target gene. In conclusion, these results indicate that localized AAV5-Id3 delivery in stroma caused no clinically relevant ocular symptoms or corneal cellular toxicity in the rabbit eyes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) gene transfer provided reduced bleeding for adult clinical trial participants with severe hemophilia A. However, pediatric outcomes are unknown. Using a mouse model of hemophilia A, we investigated the effect of vector dose and age at treatment on transgene production and persistence. We dosed AAV5-hFVIII-SQ to neonatal and adult mice based on body weight or at a fixed dose and assessed human factor VIII-SQ variant (hFVIII-SQ) expression through 16 weeks. AAV5-hFVIII-SQ dosed per body weight in neonatal mice did not result in meaningful plasma hFVIII-SQ protein levels in adulthood. When treated with the same total vector genomes per mouse as adult mice, neonates maintained hFVIII-SQ expression into adulthood, although plasma levels were 3- to 4-fold lower versus mice dosed as adults. Mice <1 week old initially exhibited high hFVIII-SQ plasma levels and maintained meaningful levels into adulthood, despite a partial decline potentially due to age-related body mass and blood volume increases. Spatial transduction patterns differed between mice dosed as neonates versus adults. No features of hepatotoxicity or endoplasmic reticulum stress were observed with dosing at any age. These data suggest that young mice require the same total vector genomes as adult mice to sustain hFVIII-SQ plasma levels., Competing Interests: L.Z., B.Y., B.H., C-R.S., S.F., S. Bunting, S. Bullens, R.M., L.X., J.A., J.H., A.V.T., T.B., J.H., C.V., D.T., C.S., and C.O. are employees and stockholders of BioMarin Pharmaceutical Inc. W.Z., S.S., N.G., A.C., S.L., C.F., S.C., and J.S. are former employees of BioMarin Pharmaceutical Inc. and may hold stock., (© 2022 BioMarin Pharmaceutical Inc.)

Abstract: Due to its non-pathogenic lifecycle, little is known about the cellular determinants of infection by adeno-associated virus (AAV). To identify these critical cellular factors, we took advantage of the gene transfer abilities of AAV in combination with a forward genetic selection to identify proteins critical for transduction by this virus. AAV serotype 5 (AAV5) vectors encoding the furin gene were used to transduce furin-deficient cells followed by selection with furin-dependent toxins. A population of cells specifically resistant to AAV5 transduction was identified and sequence analysis suggested all had a single amino acid mutation in the leader sequence of the platelet-derived growth factor receptor alpha (PDGFRα) gene. Characterization of this mutation suggested it inhibited PDGFRα trafficking resulting in limited expression on the plasma membrane. Mutagenesis and transfection experiments confirmed the effect of this mutation on PDGFRα trafficking, and the AAV5 resistant phenotype could be rescued by transfection with wild type PDGFRα. [Copyright &y& Elsevier]

Abstract: Recombinant adeno-associated virus type 2 (AAV2) vectors have shown great promise in current ophthalmology clinical trials targeting gene delivery to the retinal pigment epithelium (RPE). To treat the majority of retinal diseases, however, gene delivery would need to be targeted to photoreceptor neurons of the outer retina. AAV2 pseudotyped with the AAV5 capsid (AAV2/5) has shown far greater transduction efficiency in photoreceptors compared to standard AAV2 vectors. For clinical trial applications using gene therapy, it is helpful to generate pre-clinical data in human cells wherever possible. There is however very little data, indeed some controversy, as to whether AAV2/5 can be used effectively in differentiated neurons in culture. In this study we show that transduction of the human neuroblastoma cell line SH-SY5Y with recombinant AAV2/5 expressing GFP is well tolerated. Furthermore, we explore the mechanism whereby exposure to retinoic acid (RA) and the phorbol ester 12-O-Tetradecanoylphorbol-13- acetate (TPA) can induce this cell line to differentiate into a stable population of human neurons, with significantly increased levels of AAV2/5 transduction. These observations may be helpful for assessing AAV2/5 vectors in vitro, particularly where it is necessary to generate pre-clinical data for clinical trials of gene therapy to the human central nervous system. [Copyright &y& Elsevier]

Abstract: Since transgenes were first cloned into recombinant adenoviruses almost 30 years ago, a variety of viral vectors have become important tools in genetic research. Viruses adeptly transport genetic material into eukaryotic cells, and replacing all or part of the viral genome with genes of interest or silencing sequences creates a method of gene expression modulation in which the timing and location of manipulations can be specific. The hypothalamo-neurohypophyseal system (HNS), consisting of the paraventricular (PVN) and supraoptic (SON) nuclei in the hypothalamus, regulates fluid balance homeostasis and is highly plastic, yet tightly regulated by extracellular fluid (ECF) osmolality and volume. Its reversible plasticity and physiological relevance make it a good system for studying interactions between gene expression and physiology. Here, four viral vectors were compared for their ability to transduce magnocellular neurosecretory neurons (MNCs) of the SON in adult rats. The vectors included an adenovirus, a lentivirus (HIV) and two serotypes of adeno-associated viruses (AAV5 and AAV2). Though adenovirus and AAV2 vectors have previously been used to transduce SON neurons, HIV and AAV5 have not. All four vectors transduced MNCs, but the AAV vectors were the most effective, transducing large numbers of MNCs, with minimal or no glial transduction. The AAV vectors were injected using a convection enhanced delivery protocol to maximize dispersal through the tissue, resulting in the transduction of neurons throughout the anterior to posterior length of the SON (∼1.5mm). AAV5, but not AAV2, showed some selectivity for SON neurons relative to those in the surrounding hypothalamus. [Copyright &y& Elsevier]

Abstract: To clarify whether transduction efficiency and cell type specificity of self-complementary (sc) AAV5 vectors are similar to those of standard, single-stranded AAV5 vectors in normal retina, one micro liter of scAAV5-smCBA-GFP vector (1×1012 genome-containing particles/ml) and AAV5-smCBA-GFP vector (1×1012 genome-containing particles/ml) were subretinally or intravitreally (in both cases through the cornea) injected into the right and left eyes of adult C57BL/6J mice, respectively. On post-injection day (PID) 1, 2, 5, 7, 10, 14, 21, 28 and 35, eyes were enucleated; retinal pigment epithelium (RPE) wholemounts, neuroretinal wholemounts and eyecup sections were prepared to evaluate green fluorescent protein (GFP) expression by fluorescent microscopy. GFP expression following trans-cornea subretinal injection of scAAV5-smCBA-GFP vector was first detected in RPE wholemounts around PID 1 and in neuroretinal wholemounts between PID 2 and 5; GFP expression peaked and stabilized between PID 10–14 in RPE wholemounts and between P14 and P21 in neuroretinal wholemounts with strong, homogeneous green fluorescence covering the entire wholemounts. The frozen sections supported the following findings from the wholemounts: GFP expression appeared first in RPE around PID 1–2 and soon spread to photoreceptors (PR) cells; by PID 7, moderate GFP expression was found mainly in PR and RPE layers; between PID 14 and 21, strong and homogenous GFP expression was observed in RPE and PR cells. GFP expression following subretinal injection of AAV5-smCBA-GFP was first detected in RPE wholemounts around PID 5–7 and in neuroretinal wholemounts around PID 7–10; ssAAV5-mediated GFP expression peaked at PID 21 in RPE wholemounts and around PID 28 in neuroretinal wholemounts; sections from AAV5 treated eyes also supported findings obtained from wholemounts: GFP expression was first detected in RPE and then spread to the PR cells. Peak GFP expression in RPE mediated by scAAV5 was similar to that mediated by AAV5. However, peak GFP expression mediated by scAAV5 in PR cells was stronger than that mediated by AAV5. No GFP fluorescence was detected in any retinal cells (RPE wholemounts, neuroretinal wholemounts and retinal sections) after trans-cornea intravitreal delivery of either scAAV5-GFP or AAV5-GFP. Neither scAAV5 nor AAV5 can transduce retinal cells following trans-cornea intravitreal injection. The scAAV5 vector used in this study directs an earlier onset of transgene expression than the matched AAV5 vector, and has stronger transgene expression in PR cells following subretinal injection. Our data confirm the previous reports that scAAV vectors have an earlier onset than the standard, single strand AAV vectors (). scAAV5 vectors may be more useful than standard, single-stranded AAV vector when addressing certain RPE and/or PR cell-related models of retinal dystrophy, particularly for mouse models of human retinitis pigmentosa that require rapid and robust transgene expression to prevent early degeneration in PR cells. [Copyright &y& Elsevier]

Abstract: The parvoviruses bovine adeno-associated virus (B-AAV) and adeno-associated virus type 5 (AAV5) have similar transcription maps. However, while the AAV5 capsid gene promoter P41 possesses a high basal level in 293 cells, and is further activated only poorly by Rep during adenovirus type 5 (Ad5) infection, the B-AAV P41 promoter has a low basal activity within RepCap constructs in these cells and can be strongly activated by its Rep protein in the presence of Ad5 when a Rep-binding element (RBE) is included in cis at either end of the molecule. These differences are not due to differences in the intrinsic activating capability of the individual Rep proteins. Both viral promoters contain AP1 and CRE elements that contribute to their basal activity; however, the nature of the B-AAV P41 promoter itself and the surrounding sequences contribute to its relatively lower basal activity. In addition, the B-AAV upstream transcription units themselves also are activated in the presence of Ad5 and Rep. Thus, although the transcription map of B-AAV is much more closely related to AAV5, activation of its promoters is functionally more like the prototype AAV2. [Copyright &y& Elsevier]

Recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) have been used to deliver transgenes to the airways in a variety of pre-clinical and clinical studies. Gene transfer in these studies has been severely restricted by the basolateral localization of rAAV2 receptors. Here, we studied vectors constructed from the AAV5 genome and capsid, which utilize N-linked sialic acid-containing receptors found on the apical surface of airway epithelial cells. We investigated gene transfer efficacy and duration of transgene expression following delivery of rAAV5/5 vectors to the mouse respiratory tract. Robust, dose-dependent transgene expression was observed in the epithelium lining the nose for at least 32 weeks, and for at least 52 weeks in the lung. Importantly, in the lung, transgene expression mediated by rAAV5/5 was 40-fold greater than by rAAV2/2 vectors. A distinct cellular preference for rAAV5/5-mediated transduction was observed, with transgene expression being predominantly restricted to sustentacular cells of the olfactory epithelium in the nose and alveolar type II cells in the lung. Administration of rAAV5/5 vectors to both the nose and lungs led to the rapid development of rAAV5/5-neutralizing antibodies, suggesting that repeated administration may be severely hampered by host immune responses.Gene Therapy (2006) 13, 1703–1713. doi:10.1038/sj.gt.3302815; published online 20 July 2006 [ABSTRACT FROM AUTHOR]

Impressive achievements in clinical trials to treat hemophilia establish a milestone in the development of gene therapy. It highlights the significance of AAV-mediated gene delivery to liver. AAV5 is a unique serotype featured by low neutralizing antibody prevalence. Nevertheless, its liver infectivity is relatively weak. Consequently, it is vital to exploit novel AAV5 capsid mutants with robust liver tropism. To this aim, we performed AAV5-NNK library and barcode screening in mice, from which we identified one capsid variant, called AAVzk2. AAVzk2 displayed a similar yield but divergent post-translational modification sites compared with wild-type serotypes. Mice intravenously injected with AAVzk2 demonstrated a stronger liver transduction than AAV5, roughly comparable with AAV8 and AAV9, with undetectable transduction of other tissues or organs such as heart, lung, spleen, kidney, brain, and skeletal muscle, indicating a liver-specific tropism. Further studies showed a superior human hepatocellular transduction of AAVzk2 to AAV5, AAV8 and AAV9, whereas the seroreactivity of AAVzk2 was as low as AAV5. Overall, we provide a novel AAV serotype that facilitates a robust and specific liver gene delivery to a large population, especially those unable to be treated by AAV8 and AAV9., Competing Interests: X.X., K.Z., C.Y., H.H., and Z.D. are co-inventors of a patent about the novel AAV serotype generated in this paper., (© 2022 The Author(s).)

Genome editing in the lung has the potential to provide long-term expression of therapeutic protein to treat lung genetic diseases. Yet efficient delivery of CRISPR to the lung remains a challenge. The NIH Somatic Cell Genome Editing (SCGE) Consortium is developing safe and effective methods for genome editing in disease tissues. Methods developed by consortium members are independently validated by the SCGE small animal testing center to establish rigor and reproducibility. We have developed and validated a dual adeno-associated virus (AAV) CRISPR platform that supports effective editing of a lox-stop-lox-Tomato reporter in mouse lung airway. After intratracheal injection of the AAV serotype 5 (AAV5)-packaged S. pyogenes Cas9 (SpCas9) and single guide RNAs (sgRNAs), we observed ∼19%-26% Tomato-positive cells in both large and small airways, including club and ciliated epithelial cell types. This highly effective AAV delivery platform will facilitate the study of therapeutic genome editing in the lung and other tissue types., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Huntington disease (HD) is a fatal, neurodegenerative genetic disorder with aggregation of mutant Huntingtin protein (mutHTT) in the brain as a key pathological mechanism. There are currently no disease modifying therapies for HD; however, HTT-lowering therapies hold promise. Recombinant adeno-associated virus serotype 5 expressing a microRNA that targets HTT mRNA (AAV5-miHTT) is in development for the treatment of HD with promising results in rodent and minipig HD models. To support a clinical trial, toxicity studies were performed in non-human primates (NHP, Macaca fascicularis) and Sprague-Dawley rats to evaluate the safety of AAV5-miHTT, the neurosurgical administration procedure, vector delivery and expression of the miHTT transgene during a 6-month observation period. For accurate delivery of AAV5-miHTT to the striatum, real-time magnetic resonance imaging (MRI) with convection-enhanced delivery (CED) was used in NHP. Catheters were successfully implanted in 24 NHP, without neurological symptoms, and resulted in tracer signal in the target areas. Widespread vector DNA and miHTT transgene distribution in the brain was found, particularly in areas associated with HD pathology. Intrastriatal administration of AAV5-miHTT was well tolerated with no clinically relevant changes in either species. These studies demonstrate the excellent safety profile of AAV5-miHTT, the reproducibility and tolerability of intrastriatal administration, and the delivery of AAV5-miHTT to the brain, which support the transition of AAV5-miHTT into clinical studies. [ABSTRACT FROM AUTHOR]

Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry. [ABSTRACT FROM AUTHOR]

Adeno-associated virus 5 (AAV5)-human factor VIII-SQ (hFVIII-SQ; valoctocogene roxaparvovec) is an AAV-mediated product under evaluation for treatment of severe hemophilia A, which contains a B-domain-deleted hFVIII (hFVIII-SQ) transgene and a hybrid liver-specific promotor (HLP). To increase FVIII-SQ expression and reduce the vector dose required, a stronger promoter may be considered. However, because FVIII-SQ is a protein known to be difficult to fold and secrete, this could potentially induce endoplasmic reticulum (ER) stress. We evaluated the effect of two AAV5-hFVIII-SQ vectors with different liver-specific promoter strength (HLP << 100ATGB) on hepatic ER stress in mice. Five weeks after receiving vehicle or vector, the percentage of transduced hepatocytes and levels of liver hFVIII-SQ DNA and RNA increased dose dependently for both vectors. At lower doses, plasma hFVIII-SQ protein levels were higher for 100ATGB. This difference was attenuated at the highest dose. For 100ATGB, liver hFVIII-SQ protein accumulated dose dependently, with increased expression of ER stress markers at the highest dose, suggesting hepatocytes reached or exceeded their capacity to fold/secrete hFVIII-SQ. These data suggest that weaker promoters may require relatively higher doses to distribute expression load across a greater number of hepatocytes, whereas relatively stronger promoters may produce comparable levels of FVIII in fewer hepatocytes, with potential for ER stress., (© 2020 The Authors.)

Adeno-associated virus (AAV) has been used extensively as a vector for gene therapy. Despite its widespread use, the mechanisms by which AAV enters the cell and is trafficked to the nucleus are poorly understood. In this study, we performed two pooled, genome-wide screens to identify positive and negative factors modulating AAV2 transduction. Genome-wide libraries directed against all human genes with four designs per gene or eight designs per gene were transduced into U-2 OS cells. These pools were transduced with AAV2 encoding EGFP and sorted based on the intensity of EGFP expression. Analysis of enriched and depleted barcodes in the sorted samples identified several genes that putatively decreased AAV2 transduction. A subset of screen hits was validated in flow cytometry and imaging studies. In addition to KIAA0319L ( AAVR ), we confirmed the role of two genes, GPR108 and TM9SF2 , in mediating viral transduction in eight different AAV serotypes. Interestingly, GPR108 displayed serotype selectivity and was not required for AAV5 transduction. Follow-up studies suggested that GPR108 localized primarily to the Golgi, where it may interact with AAV and play a critical role in mediating virus escape or trafficking. Cumulatively, these results expand our understanding of the process of AAV transduction in different cell types and serotypes., (© 2020 Amgen Inc.)

Huntington disease (HD) is a fatal neurodegenerative genetic disorder, thought to reflect a toxic gain of function in huntingtin (Htt) protein. Adeno-associated viral vector serotype 5 (AAV5)- microRNA targeting huntingtin (miHTT) is a HD gene-therapy candidate that efficiently lowers HTT using RNAi. This study analyzed the efficacy and potential for off-target effects with AAV5-miHTT in neuronal and astrocyte cell cultures differentiated from induced pluripotent stem cells (iPSCs) from two individuals with HD (HD71 and HD180). One-time AAV5-miHTT treatment significantly reduced human HTT mRNA by 57% and Htt protein by 68% in neurons. Small RNA sequencing showed that mature miHTT was processed correctly without off-target passenger strand. No cellular microRNAs were dysregulated, indicating that endogenous RNAi machinery was unaffected by miHTT overexpression. qPCR validation of in silico -predicted off-target transcripts, next-generation sequencing, and pathway analysis confirmed absence of dysregulated genes due to sequence homology or seed-sequence activity of miHTT. Minor effects on gene expression were observed in both AAV5-miHTT and AAV5-GFP-treated samples, suggesting that they were due to viral transduction rather than miHTT. This study confirms the efficacy of AAV5-miHTT in HD patient iPSC-derived neuronal cultures and lack of off-target effects in gene expression and regulation in neuronal cells and astrocytes., (© 2019 The Author(s).)