The longitudinal kinetics of AAV5 vector integration profiles and evaluation of clonal expansion in mice.

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.

Exon 1-targeting miRNA reduces the pathogenic exon 1 HTT protein in Huntington disease models.

Huntington disease (HD) is a fatal neurodegenerative disease caused by a trinucleotide repeat expansion in exon 1 of the huntingtin gene (HTT) resulting in toxic gain-of-function and cell death. Despite its monogenic cause, the pathogenesis of HD is highly complex and increasing evidence indicates that, in addition to the full-length (FL) mutant HTT protein, the expanded exon 1 HTT (HTTexon1) protein that is translated from the HTT1a transcript generated by aberrant splicing is prone to aggregate and may contribute to HD pathology. This finding suggests that reducing the expression of HTT1a may achieve a greater therapeutic benefit than targeting only FL mutant HTT. Conversely, strategies that exclusively target FL HTT may not fully prevent the pathogenesis of HD. We have developed an engineered microRNA targeting the HTT exon 1 sequence (miHTT), delivered via adeno-associated virus serotype 5 (AAV5). The target sequence of miHTT is present in both FL HTT and HTT1a transcripts. Preclinical studies with AAV5-miHTT have demonstrated efficacy in several rodent and large animal models by reducing FL HTT mRNA and protein and rescuing HD-like phenotypes, and have been the rationale for phase I/II clinical studies now ongoing in the US and Europe. In the present study, we evaluated the ability of AAV5-miHTT to reduce the levels of aberrantly spliced HTT1a mRNA and the HTTexon1 protein in the brain of two mouse models of HD (heterozygous zQ175 knock-in mice and humanized Hu128/21 mice). Polyadenylated HTT1a mRNA and HTTexon1 protein were detected in the striatum and cortex of heterozygous zQ175 knock-in mice, but not in wild-type, littermate control mice. Intrastriatal administration of AAV5-miHTT resulted in dose-dependent expression of mature miHTT microRNA in cortical brain regions, accompanied by significant lowering of both FL HTT and HTT1a mRNA expression at two months post-injection. Mutant HTT and HTTexon1 protein levels were also significantly reduced in the striatum and cortex of heterozygous zQ175 knock-in at 2 months after AAV5-miHTT treatment and in humanized Hu128/21 mice 7 months post-treatment. The effects were confirmed in primary Hu128/21 neuronal cultures. These results demonstrate that AAV5-miHTT gene therapy is an effective approach to lower both FL HTT and the pathogenic HTTexon1 levels, which could potentially have an additive therapeutic benefit compared to other HTT-targeting modalities.

Global seroprevalence of neutralizing antibodies against adeno-associated virus serotypes used for human gene therapies.

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.

AAV5 Delivery of CRISPR/Cas9 Mediates Genome Editing in the Lungs of Young Rhesus Monkeys.

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.

Tissue-targeted and localized AAV5-DCN and AAV5-PEDF combination gene therapy abrogates corneal fibrosis and concurrent neovascularization in rabbit eyes in vivo.

PURPOSE: Corneal fibrosis and neovascularization (CNV) after ocular trauma impairs vision. This study tested therapeutic potential of tissue-targeted adeno-associated virus5 (AAV5) mediated decorin (DCN) and pigment epithelium-derived factor (PEDF) combination genes in vivo. METHODS: Corneal fibrosis and CNV were induced in New Zealand White rabbits via chemical trauma. Gene therapy in stroma was delivered 30-min after chemical-trauma via topical AAV5-DCN and AAV5-PEDF application using a cloning cylinder. Clinical eye examinations and multimodal imaging in live rabbits were performed periodically and corneal tissues were collected 9-day and 15-day post euthanasia. Histological, cellular, and molecular and apoptosis assays were used for efficacy, tolerability, and mechanistic studies. RESULTS: The AAV5-DCN and AAV5-PEDF combination gene therapy significantly reduced corneal fibrosis (p < 0.01 or p < 0.001) and CNV (p < 0.001) in therapy-given (chemical-trauma and AAV5-DCN + AAV5-PEDF) rabbit eyes compared to the no-therapy given eyes (chemical-trauma and AAV5-naked vector). Histopathological analyses demonstrated significantly reduced fibrotic α-smooth muscle actin and endothelial lectin expression in therapy-given corneas compared to no-therapy corneas on day-9 (p < 0.001) and day-15 (p < 0.001). Further, therapy-given corneas showed significantly increased Fas-ligand mRNA levels (p < 0.001) and apoptotic cell death in neovessels (p < 0.001) compared to no-therapy corneas. AAV5 delivered 2.69 × 107 copies of DCN and 2.31 × 107 copies of PEDF genes per µg of DNA. AAV5 vector and delivered DCN and PEDF genes found tolerable to the rabbit eyes and caused no significant toxicity to the cornea. CONCLUSION: The combination AAV5-DCN and AAV5-PEDF topical gene therapy effectively reduces corneal fibrosis and CNV with high tolerability in vivo in rabbits. Additional studies are warranted.

Prophylactic Prednisolone Promotes AAV5 Hepatocyte Transduction Through the Novel Mechanism of AAV5 Coreceptor Platelet-Derived Growth Factor Receptor Alpha Upregulation and Innate Immune Suppression.

Adeno-associated virus (AAV) vectors are used to deliver therapeutic transgenes, but host immune responses may interfere with transduction and transgene expression. We evaluated prophylactic corticosteroid treatment on AAV5-mediated expression in liver tissue. Wild-type C57BL/6 mice received 6 × 1013 vg/kg AAV5-HLP-hA1AT, an AAV5 vector carrying a human α1-antitrypsin (hA1AT) gene with a hepatocyte-specific promoter. Mice received 4 weeks of daily 2 mg/kg prednisolone or water starting day -1 or 0 before vector dosing. Mice that received prophylactic corticosteroids had significantly higher serum hA1AT protein than mice that did not, starting at 6 weeks and persisting to the study end at 12 weeks, potentially through a decrease in the number of low responders. RNAseq and proteomic analyses investigating mechanisms mediating the improvement of transgene expression found that prophylactic corticosteroid treatment upregulated the AAV5 coreceptor platelet-derived growth factor receptor alpha (PDGFRα) on hepatocytes and downregulated its competitive ligand PDGFα, thus increasing the uptake of AAV5 vectors. Evidently, prophylactic corticosteroid treatment also suppressed acute immune responses to AAV. Together, these mechanisms resulted in increased uptake and preservation of the transgene, allowing more vector genomes to be available to assemble into stable, full-length structures mediating long-term transgene expression. Prophylactic corticosteroids represent a potential actionable strategy to improve AAV5-mediated transgene expression and decrease intersubject variability.

Efficient adeno-associated virus serotype 5 capture with affinity functionalized nanofiber adsorbents.

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.

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients.

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or "empty" capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.

Adeno-associated virus-mediated gene transfer of arginine decarboxylase to the central nervous system prevents opioid analgesic tolerance.

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.

Cross-Species Permissivity: Structure of a Goat Adeno-Associated Virus and Its Complex with the Human Receptor AAVR.

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. IMPORTANCE Adeno-associated virus (AAV) is a small ssDNA satellite parvovirus. As a recombinant vector with a protein shell encapsidating a transgene, recombinant AAV (rAAV) is a leading delivery vehicle for gene therapy, with two FDA-approved treatments and 150 clinical trials for 30 diseases. The human entry receptor AAVR has five PKD domains. To date, all serotypes, except AAV5, have interacted primarily with the second PKD domain, PKD2. Goat is the AAV host most distant from human with cross-species cell infectivity. AAVGo.1 is similar in structure to AAV5, the two forming a class with a distinct mode of receptor-binding. Within the two classes, binding interactions are mostly conserved, giving an indication of the latitude available in modulating delivery vectors.

Patterned Stimulation of the Chrimson Opsin in Glutamatergic Motor Thalamus Neurons Improves Forelimb Akinesia in Parkinsonian Rats.

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.

Structures and implications of the nuclease domain of human parvovirus B19 NS1 protein.

Infection of human parvovirus B19 (B19V) can cause a variety of diseases, such as hydrops fetalis, erythema infectiosum in children and acute arthropathy in women. Although B19V infection mainly occurs during childhood, about 50 % of adults are still susceptible to B19V infection. As the major replication protein of B19V, deletion of NS1 completely abolishes the infectivity of the virus. The nuclease domain of NS1 (NS1_Nuc) is responsible for DNA Ori binding and nicking that is critical for B19V viral DNA replication. NS1 has various variants, the structure and function for the majority of the variants are poorly studied. Here, we report two high-resolution crystal structures of NS1_Nuc, revealed the detailed conformations of many key residues. Structural comparison indicates that these residues are important for ssDNA or dsDNA binding by NS1. NS1 belongs to the HUH-endonuclease superfamily and it shares conserved ssDNA cleavage mechanism with other HUH-endonuclease members. However, our structural analyses, mutagenesis and in vitro assay results all suggested that NS1_Nuc utilizes one unique model in ssDNA binding.

Corneal fibrosis abrogation by a localized AAV-mediated inhibitor of differentiation 3 (Id3) gene therapy in rabbit eyes in vivo.

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-ß 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.

Application of in-vitro-cultured primary hepatocytes to evaluate species translatability and AAV transduction mechanisms of action.

Recombinant adeno-associated virus (AAV) is an effective platform for therapeutic gene transfer; however, tissue-tropism differences between species are a challenge for successful translation of preclinical results to humans. We evaluated the use of in vitro primary hepatocyte cultures to predict in vivo liver-directed AAV expression in different species. We assessed whether in vitro AAV transduction assays in cultured primary hepatocytes from mice, nonhuman primates (NHPs), and humans could model in vivo liver-directed AAV expression of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), an experimental gene therapy for hemophilia A with a hepatocyte-selective promoter. Relative levels of DNA and RNA in hepatocytes grown in vitro correlated with in vivo liver transduction across species. Expression in NHP hepatocytes more closely reflected expression in human hepatocytes than in mouse hepatocytes. We used this hepatocyte culture model to assess transduction efficacy of a novel liver-directed AAV capsid across species and identified which of 3 different canine factor VIII vectors produced the most transgene expression. Results were confirmed in vivo. Further, we determined mechanisms mediating inhibition of AAV5-hFVIII-SQ expression by concomitant isotretinoin using primary human hepatocytes. These studies support using in vitro primary hepatocyte models to predict species translatability of liver-directed AAV gene therapy and improve mechanistic understanding of drug-drug interactions.

Directed evolution of adeno-associated virus 5 capsid enables specific liver tropism.

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.

Molecular analysis of AAV5-hFVIII-SQ vector-genome-processing kinetics in transduced mouse and nonhuman primate livers.

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adeno-associated virus serotype 5 (AAV5)-based gene therapy vector containing a B-domain-deleted human coagulation factor VIII (hFVIII) gene controlled by a liver-selective promoter. AAV5-hFVIII-SQ is currently under clinical investigation as a treatment for severe hemophilia A. The full-length AAV5-hFVIII-SQ is >4.9 kb, which is over the optimal packaging limit of AAV5. Following administration, the vector must undergo a number of genome-processing, assembly, and repair steps to form full-length circularized episomes that mediate long-term FVIII expression in target tissues. To understand the processing kinetics of the oversized AAV5-hFVIII-SQ vector genome into circular episomes, we characterized the various molecular forms of the AAV5-hFVIII-SQ genome at multiple time points up to 6 months postdose in the liver of murine and non-human primate models. Full-length circular episomes were detected in liver tissue beginning 1 week postdosing. Over 6 months, quantities of circular episomes (in a predominantly head-to-tail configuration) increased, while DNA species lacking inverted terminal repeats were preferentially degraded. Levels of duplex, circular, full-length genomes significantly correlated with levels of hFVIII-SQ RNA transcripts in mice and non-human primates dosed with AAV5-hFVIII-SQ. Altogether, we show that formation of full-length circular episomes in the liver following AAV5-hFVIII-SQ transduction was associated with long-term FVIII expression.

Feasibility of Targeted Delivery of AAV5-GFP into the Cerebellum of Nonhuman Primates Following a Single Convection-Enhanced Delivery Infusion.

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.

Administration of an adeno-associated viral vector expressing interferon-ß in patients with inflammatory hand arthritis, results of a phase I/II study.

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)-ß, 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-ß 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-ß, nor IFN-ß 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.

AAV5 delivery of CRISPR-Cas9 supports effective genome editing in mouse lung airway.

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.

Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method.

Adeno-associated virus (AAV)-based gene therapies have recently shown promise as a novel treatment for hereditary diseases. Due to the viral origin of the vector capsid, however, cellular immune response may be elicited that could eliminate transduced target cells. To monitor cellular immune responses in clinical trials, we optimized and bioanalytically validated a sensitive, robust, and reliable interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assay. For method performance validation, human peripheral blood mononuclear cells (PBMCs) were stimulated with peptides derived from AAV5 capsid proteins and the encoded transgene product, human blood clotting factor VIII (FVIII), in addition to positive controls, such as peptides from the 65-kDa phosphoprotein of cytomegalovirus. We statistically assessed the limit of detection and confirmatory cutpoint, evaluated precision and linearity, and confirmed specificity using HIV peptides. Robustness parameter ranges and sample stability periods were established. The validated IFN-γ ELISpot assay was then implemented in an AAV5-FVIII gene therapy clinical trial. Cellular immune responses against the AAV5 capsid were observed in most participants as soon as 2 weeks following dose administration; only limited responses against the transgene product were detected. These data underscore the value of using validated methods for monitoring cellular immunity in AAV gene therapy trials.