Evaluation of Aav Capsids and Delivery Approaches for Hereditary Hemorrhagic Telangiectasia Gene Therapy.

Nosebleeds and intracranial hemorrhage from brain arteriovenous malformations (bAVMs) are among the most devastating symptoms of patients with hereditary hemorrhagic telangiectasis (HHT). All available managements have limitations. We showed that intravenous delivery of soluble FMS-related tyrosine kinase 1 using an adeno-associated viral vector (AAV9-sFLT1) reduced bAVM severity of endoglin deficient mice. However, minor liver inflammation and growth arrest in young mice were observed. To identify AAV variants and delivery methods that can best transduce brain and nasal tissue with an optimal transduction profile, we compared 3 engineered AAV capsids (AAV.cc47, AAV.cc84 and AAV1RX) with AAV9. A single-stranded CBA promoter driven tdTomato transgene was packaged in these capsids and delivered intravenously (i.v.) or intranasally (i.n.) to wild-type mice. A CMV promoter driven Alk1 transgene was packaged into AAV.cc84 and delivered to PdgfbiCre;Alk1 f/f mice through i.v. injection followed by brain AVM induction. Transduced cells in different organs, vessel density and abnormal vessels in the bAVMs, and liver inflammation were analyzed histologically. Liver and kidney function were measured enzymatically. Compared to other viral vectors, AAV.cc84, after i.v. delivery, transduced a high percentage of brain ECs and few hepatocytes; whereas after i.n. delivery, AAV.cc84 transduced ECs and perivascular cells in the brain, and ECs, epithelial cells, and skeletal muscles in the nose with minimum hepatocyte transduction. No changes to liver or kidney function were detected. Delivery of AAV.cc84-Alk1 through i.v. to PdgfbiCre;Alk1 f/f mice reduced bAVM severity. In summary, we propose that AAV.cc84-Alk1 is a promising candidate for developing gene therapy in HHT patients.

2024 update: European consensus statement on gene therapy for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is one of the most common genetic diseases and was, until recently, a leading genetic cause of infant mortality. Three disease-modifying treatments have dramatically changed the disease trajectories and outcome for severely affected infants (SMA type 1), especially when initiated in the presymptomatic phase. One of these treatments is the adeno-associated viral vector 9 (AAV9) based gene therapy onasemnogene abeparvovec (Zolgensma®), which is delivered systemically and has been approved by the European Medicine Agency for SMA patients with up to three copies of the SMN2 gene or with the clinical presentation of SMA type 1. While this broad indication provides flexibility in patient selection, it also raises concerns about the risk-benefit ratio for patients with limited or no evidence supporting treatment. In 2020, we convened a European neuromuscular expert working group to support the rational use of onasemnogene abeparvovec, employing a modified Delphi methodology. After three years, we have assembled a similar yet larger group of European experts who assessed the emerging evidence of onasemnogene abeparvovec's role in treating older and heavier SMA patients, integrating insights from recent clinical trials and real-world evidence. This effort resulted in 12 consensus statements, with strong consensus achieved on 9 and consensus on the remaining 3, reflecting the evolving role of onasemnogene abeparvovec in treating SMA.

Production and Purification of Adeno-Associated Viral Vectors (AAVs) Using Orbitally Shaken HEK293 Cells.

Here, we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium using orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI)-mediated transfection of a three-plasmid system and is specified for production in milliliter-to-liter scales. After PEI and plasmid DNA (pDNA) complex formation, the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 7-day batch process, cell cultures are further processed using a set of methods for cell lysis and vector recovery. Methods for the purification of viral particles are described, including immunoaffinity and anion-exchange chromatography, ultrafiltration, as well as digital PCR to quantify the concentration of vector particles.

Preclinical Milestones in MECP2 Gene Transfer for Treating Rett Syndrome.

BACKGROUND: Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2). After gene transfer in mice, exogenous MeCP2 expression must be regulated to avoid dose-dependent toxicity. SUMMARY: The preclinical gene therapy literature for treating RTT illustrates a duly diligent progression that begins with proof-of-concept studies and advances toward the development of safer, regulated MECP2 viral genome designs. This design progression was partly achieved through international collaborative studies. In 2023, clinicians administered investigational gene therapies for RTT to patients a decade after the first preclinical gene therapy publications for RTT (clinical trial numbers NCT05606614 and NCT05898620). As clinicians take on a more prominent role in MECP2 gene therapy research, preclinical researchers may continue to test more nuanced hypotheses regarding the safety, efficacy, and mechanism of MECP2 gene transfer. KEY MESSAGE: This review summarizes the history of preclinical MECP2 gene transfer for treating RTT and acknowledges major contributions among colleagues in the field. The first clinical injections are a shared milestone.

Effects of unilateral hippocampal surgical procedures needed for calcium imaging on mouse behavior and adult hippocampal neurogenesis.

Hippocampus is essential for episodic memory formation, lesion studies demonstrating its role especially in processing spatial and temporal information. Further, adult hippocampal neurogenesis (AHN) in the dentate gyrus (DG) has also been linked to learning. To study hippocampal neuronal activity during events like learning, in vivo calcium imaging has become increasingly popular. It relies on the use of adeno-associated viral (AAV) vectors, which seem to lead to a decrease in AHN when applied on the DG. More notably, imaging requires the implantation of a relatively large lens into the tissue. Here, we examined how injection of an AAV vector and implantation of a 1-mm-diameter lens into the dorsal DG routinely used to image calcium activity impact the behavior of adult male C57BL/6 mice. To this aim, we conducted open-field, object-recognition and object-location tasks at baseline, after AAV vector injection, and after lens implantation. Finally, we determined AHN from hippocampal slices using a doublecortin-antibody. According to our results, the operations needed for in vivo imaging of the dorsal DG did not have adverse effects on behavior, although we noticed a decrease in AHN ipsilaterally to the operations. Thus, our results suggest that in vivo imaging can be safely used to, for example, correlate patterns of calcium activity with learned behavior. One should still keep in mind that the defects on the operated side might be functionally compensated by the (hippocampus in the) contralateral hemisphere.

Integration and the risk of liver cancer-Is there a real risk?

Adeno-associated virus (AAV)-based gene therapies are in clinical development for haemophilia and other genetic diseases. Since the recombinant AAV genome primarily remains episomal, it provides the opportunity for long-term expression in tissues that are not proliferating and reduces the safety concerns compared with integrating viral vectors. However, AAV integration events are detected at a low frequency. Preclinical studies in mouse models have reported hepatocellular carcinoma (HCC) after systemic AAV administration in some settings, though this has not been reported in large animal models. The risk of HCC or other cancers after AAV gene therapy in clinical studies thus remains theoretical. Potential risk factors for HCC after gene therapy are beginning to be elucidated through animal studies, but their relevance to human studies remains unknown. Studies to investigate the factors that may influence the risk of oncogenesis as well as detailed investigation of cases of cancer in AAV gene therapy patients will be important to define the potential risk of AAV genotoxicity.

Inhibition of Granule Cell Dispersion and Seizure Development by Astrocyte Elevated Gene-1 in a Mouse Model of Temporal Lobe Epilepsy.

Although granule cell dispersion (GCD) in the hippocampus is known to be an important feature associated with epileptic seizures in temporal lobe epilepsy (TLE), the endogenous molecules that regulate GCD are largely unknown. In the present study, we have examined whether there is any change in AEG-1 expression in the hippocampus of a kainic acid (KA)-induced mouse model of TLE. In addition, we have investigated whether the modulation of astrocyte elevated gene-1 (AEG-1) expression in the dentate gyrus (DG) by intracranial injection of adeno-associated virus 1 (AAV1) influences pathological phenotypes such as GCD formation and seizure susceptibility in a KA-treated mouse. We have identified that the protein expression of AEG-1 is upregulated in the DG of a KA-induced mouse model of TLE. We further demonstrated that AEG-1 upregulation by AAV1 delivery in the DG-induced anticonvulsant activities such as the delay of seizure onset and inhibition of spontaneous recurrent seizures (SRS) through GCD suppression in the mouse model of TLE, while the inhibition of AEG-1 expression increased susceptibility to seizures. The present observations suggest that AEG-1 is a potent regulator of GCD formation and seizure development associated with TLE, and the significant induction of AEG-1 in the DG may have therapeutic potential against epilepsy.

A drug cocktail of rapamycin, acarbose, and phenylbutyrate enhances resilience to features of early-stage Alzheimer's disease in aging mice.

The process of aging is defined by the breakdown of critical maintenance pathways leading to an accumulation of damage and its associated phenotypes. Aging affects many systems and is considered the greatest risk factor for a number of diseases. Therefore, interventions aimed at establishing resilience to aging should delay or prevent the onset of age-related diseases. Recent studies have shown a three-drug cocktail consisting of rapamycin, acarbose, and phenylbutyrate delayed the onset of physical, cognitive, and biological aging phenotypes in old mice. To test the ability of this drug cocktail to impact Alzheimer's disease (AD), an adeno-associated-viral vector model of AD was created. Mice were fed the drug cocktail 2 months prior to injection and allowed 3 months for phenotypic development. Cognitive phenotypes were evaluated through a spatial navigation learning task. To quantify neuropathology, immunohistochemistry was performed for AD proteins and pathways of aging. Results suggested the drug cocktail was able to increase resilience to cognitive impairment, inflammation, and AD protein aggregation while enhancing autophagy and synaptic integrity, preferentially in female cohorts. In conclusion, female mice were more susceptible to the development of early stage AD neuropathology and learning impairment, and more responsive to treatment with the drug cocktail in comparison to male mice. Translationally, a model of AD where females are more susceptible would have greater value as women have a greater burden and incidence of disease compared to men. These findings validate past results and provide the rationale for further investigations into enhancing resilience to early-stage AD by enhancing resilience to aging.

AAV8 gene therapy reverses cardiac pathology and prevents early mortality in a mouse model of Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an autosomal-recessive disorder primarily attributed to biallelic GAA repeat expansions that reduce expression of the mitochondrial protein frataxin (FXN). FRDA is characterized by progressive neurodegeneration, with many patients developing cardiomyopathy that progresses to heart failure and death. The potential to reverse or prevent progression of the cardiac phenotype of FRDA was investigated in a mouse model of FRDA, using an adeno-associated viral vector (AAV8) containing the coding sequence of the FXN gene. The Fxnflox/null::MCK-Cre conditional knockout mouse (FXN-MCK) has an FXN gene ablation that prevents FXN expression in cardiac and skeletal muscle, leading to cardiac insufficiency, weight loss, and morbidity. FXN-MCK mice received a single intravenous injection of an AAV8 vector containing human (hFXN) or mouse (mFXN) FXN genes under the control of a phosphoglycerate kinase promoter. Compared to vehicle-treated FXN-MCK control mice, AAV-treated FXN-MCK mice displayed increases in body weight, reversal of cardiac deficits, and increases in survival without apparent toxicity in the heart or liver for up to 12 weeks postdose. FXN protein expression in heart tissue was detected in a dose-dependent manner, exhibiting wide distribution throughout the heart similar to wild type, but more speckled. These results support an AAV8-based approach to treat FRDA-associated cardiomyopathy.

The Road towards Gene Therapy for X-Linked Juvenile Retinoschisis: A Systematic Review of Preclinical Gene Therapy in Cell-Based and Rodent Models of XLRS.

X-linked juvenile retinoschisis (XLRS) is an early-onset progressive inherited retinopathy affecting males. It is characterized by abnormalities in the macula, with formation of cystoid retinal cavities, frequently accompanied by splitting of the retinal layers, impaired synaptic transmission of visual signals, and associated loss of visual acuity. XLRS is caused by loss-of-function mutations in the retinoschisin gene located on the X chromosome (RS1, MIM 30083). While proof-of-concept studies for gene augmentation therapy have been promising in in vitro and rodent models, clinical trials in XLRS patients have not been successful thus far. We performed a systematic literature investigation using search strings related to XLRS and gene therapy in in vivo and in vitro models. Three rounds of screening (title/abstract, full text and qualitative) were performed by two independent reviewers until consensus was reached. Characteristics related to study design and intervention were extracted from all studies. Results were divided into studies using (1) viral and (2) non-viral therapies. All in vivo rodent studies that used viral vectors were assessed for quality and risk of bias using the SYRCLE's risk-of-bias tool. Studies using alternative and non-viral delivery techniques, either in vivo or in vitro, were extracted and reviewed qualitatively, given the diverse and dispersed nature of the information. For in-depth analysis of in vivo studies using viral vectors, outcome data for optical coherence tomography (OCT), immunohistopathology and electroretinography (ERG) were extracted. Meta-analyses were performed on the effect of recombinant adeno-associated viral vector (AAV)-mediated gene augmentation therapies on a- and b-wave amplitude as well as the ratio between b- and a-wave amplitudes (b/a-ratio) extracted from ERG data. Subgroup analyses and meta-regression were performed for model, dose, age at injection, follow-up time point and delivery method. Between-study heterogeneity was assessed with a Chi-square test of homogeneity (I2). We identified 25 studies that target RS1 and met our search string. A total of 19 of these studies reported rodent viral methods in vivo. Six of the 25 studies used non-viral or alternative delivery methods, either in vitro or in vivo. Of these, five studies described non-viral methods and one study described an alternative delivery method. The 19 aforementioned in vivo studies were assessed for risk of bias and quality assessments and showed inconsistency in reporting. This resulted in an unclear risk of bias in most included studies. All 19 studies used AAVs to deliver intact human or murine RS1 in rodent models for XLRS. Meta-analyses of a-wave amplitude, b-wave amplitude, and b/a-ratio showed that, overall, AAV-mediated gene augmentation therapy significantly ameliorated the disease phenotype on these parameters. Subgroup analyses and meta-regression showed significant correlations between b-wave amplitude effect size and dose, although between-study heterogeneity was high. This systematic review reiterates the high potential for gene therapy in XLRS, while highlighting the importance of careful preclinical study design and reporting. The establishment of a systematic approach in these studies is essential to effectively translate this knowledge into novel and improved treatment alternatives.

Effects of gene replacement therapy with resamirigene bilparvovec (AT132) on skeletal muscle pathology in X-linked myotubular myopathy: results from a substudy of the ASPIRO open-label clinical trial.

BACKGROUND: X-linked myotubular myopathy (XLMTM) is a rare, life-threatening congenital muscle disease caused by mutations in the MTM1 gene that result in profound muscle weakness, significant respiratory insufficiency, and high infant mortality. There is no approved disease-modifying therapy for XLMTM. Resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) is an investigational adeno-associated virus (AAV8)-mediated gene replacement therapy designed to deliver MTM1 to skeletal muscle cells and achieve long-term correction of XLMTM-related muscle pathology. The clinical trial ASPIRO (NCT03199469) investigating resamirigene bilparvovec in XLMTM is currently paused while the risk:benefit balance associated with this gene therapy is further investigated. METHODS: Muscle biopsies were taken before treatment and 24 and 48 weeks after treatment from ten boys with XLMTM in a clinical trial of resamirigene bilparvovec (ASPIRO; NCT03199469). Comprehensive histopathological analysis was performed. FINDINGS: Baseline biopsies uniformly showed findings characteristic of XLMTM, including small myofibres, increased internal or central nucleation, and central aggregates of organelles. Biopsies taken at 24 weeks post-treatment showed marked improvement of organelle localisation, without apparent increases in myofibre size in most participants. Biopsies taken at 48 weeks, however, did show statistically significant increases in myofibre size in all nine biopsies evaluated at this timepoint. Histopathological endpoints that did not demonstrate statistically significant changes with treatment included the degree of internal/central nucleation, numbers of triad structures, fibre type distributions, and numbers of satellite cells. Limited (predominantly mild) treatment-associated inflammatory changes were seen in biopsy specimens from five participants. INTERPRETATION: Muscle biopsies from individuals with XLMTM treated with resamirigene bilparvovec display statistically significant improvement in organelle localisation and myofibre size during a period of substantial improvements in muscle strength and respiratory function. This study identifies valuable histological endpoints for tracking treatment-related gains with resamirigene bilparvovec, as well as endpoints that did not show strong correlation with clinical improvement in this human study. FUNDING: Astellas Gene Therapies (formerly Audentes Therapeutics, Inc.).

Enhancement of adeno-associated virus serotype 6 transduction into T cells with cell-penetrating peptides.

BACKGROUND: Adeno-associated viruses (AAVs) are gaining interest in the development of cellular immunotherapy. Compared to other viral vectors, AAVs can reduce the risk of insertional oncogenesis. AAV serotype 6 (AAV6) shows the highest efficiency for transducing T cells. Nevertheless, a multiplicity of infection (MOI) of up to one million viral genomes per cell is required to transduce the target cells effectively. Cell-penetrating peptides (CPPs) are short, positively charged peptides that easily translocate the plasma membranes and can facilitate the cellular uptake of a wide variety of cargoes, including small molecules, nucleic acids, drugs, proteins and viral vectors. METHODS: The present study evaluated five CPPs (Antp, TAT-HA2, LAH4, TAT1 and TAT2) on their effects on enhancing transduction of AAV6 packaging a green fluorescent protein transgene into Jurkat T cell line. RESULTS: Vector incubation with peptides TAT-HA2 and LAH4 at a final concentration of 0.2 mm resulted in an approximately two-fold increase in transduced cells. At the lowest MOI tested (1.25 × 104 ), using LAH4 resulted in a 10-fold increase in transduction efficiency. The peptide LAH4 increased the uptake of AAV6 viral particles in both Jurkat cells and mouse primary T cells. Regardless of the large size of the AAV6-LAH4 complexes, their internalization does not appear to depend on macropinocytosis. CONCLUSIONS: Overall, the present study reports an approach to significantly improve the delivery of transgenes into T cells using AAV6 vectors. Notably, the peptides TAT-HA2 and LAH4 contribute to improving the use of AAV6 as a gene delivery vector for the engineering of T cells.

Local delivery of adeno-associated viral vectors with electrospun gelatin nanofiber mats.

Adeno-associated viral (AAV) vectors play a significant role in gene therapy, yet the typical delivery methods, like systemic and local AAV injections, often lead to unintended off-target distribution and tissue damage due to injection. In this study, we propose a localized delivery approach for AAV vectors utilizing electrospun gelatin nanofiber mats, which are cross-linked with glutaraldehyde. The AAV vectors, which encoded a green fluorescent protein (GFP), were loaded onto the mats by immersing them in a solution containing the vectors. The amount of AAV vector loaded onto the mats increased as the vector concentration in the solution increased. The loaded AAV vector was steadily released into the cell culture medium over 3 days. The mats incubated for 3 days also showed the ability to transduce into the cells cultured on them. We evaluated the effectiveness of this delivery system by attaching the mats to mouse livers. GFP expression was visible on the surface of the liver beneath the attached mats, but not in areas in direct contact with the mats. These findings suggest that the attachment of AAV vector-loaded electrospun gelatin nanofiber mats to a target site present a promising solution for localized gene delivery while reducing off-target distribution.

CRISPR-Cas9-mediated somatic correction of a one-base deletion in the Ugt1a gene ameliorates hyperbilirubinemia in Crigler-Najjar syndrome mice.

(AAV)-mediated episomal gene replacement therapy for monogenic liver disorders is currently limited in pediatric settings due to the loss of vector DNA, associated with hepatocyte duplication during liver growth. Genome editing is a promising strategy leading to a permanent and specific genome modification that is transmitted to daughter cells upon proliferation. Using genome targeting, we previously rescued neonatal lethality in mice with Crigler-Najjar syndrome. This rare monogenic disease is characterized by severe neonatal unconjugated hyperbilirubinemia, neurological damage, and death. Here, using the CRISPR-Staphylococcus aureus Cas9 (SaCas9) platform, we edited the disease-causing mutation present in the Ugt1a locus of these mice. Newborn mice were treated with two AAV8 vectors: one expressing the SaCas9 and single guide RNA, and the other carrying the Ugt1a homology regions with the corrected sequence, while maintained in a temporary phototherapy setting rescuing mortality. We observed a 50% plasma bilirubin reduction that remained stable for up to 6 months. We then tested different Cas9:donor vector ratios, with a 1:5 ratio showing the greatest efficacy in lowering plasma bilirubin, with partial lethality rescue when more severe, lethal conditions were applied. In conclusion, we reduced plasma bilirubin to safe levels and partially rescued neonatal lethality by correcting the mutant Ugt1a1 gene of a Crigler-Najjar mouse model.

A C1qTNF3 collagen domain fusion chaperones diverse secreted proteins and anti-Aß scFvs: Applications for gene therapies.

Enhancing production of protein cargoes delivered by gene therapies can improve efficacy by reducing the amount of vector or simply increasing transgene expression levels. We explored the utility of a 126-amino acid collagen domain (CD) derived from the C1qTNF3 protein as a fusion partner to chaperone secreted proteins, extracellular "decoy receptor" domains, and single-chain variable fragments (scFvs). Fusions to the CD domain result in multimerization and enhanced levels of secretion of numerous fusion proteins while maintaining functionality. Efficient creation of bifunctional proteins using the CD domain is also demonstrated. Recombinant adeno-associated viral vector delivery of the CD with a signal peptide resulted in high-level expression with minimal biological impact as assessed by whole-brain transcriptomics. As a proof-of-concept in vivo study, we evaluated three different anti-amyloid Aß scFvs (anti-Aß scFvs), alone or expressed as CD fusions, following viral delivery to neonatal CRND8 mice. The CD fusion increased half-life, expression levels, and improved efficacy for amyloid lowering of a weaker binding anti-Aß scFv. These studies validate the potential utility of this small CD as a fusion partner for secretory cargoes delivered by gene therapy and demonstrate that it is feasible to use this CD fusion to create biotherapeutic molecules with enhanced avidity or bifunctionality.

Etranacogene dezaparvovec for the treatment of adult patients with severe and moderately severe hemophilia B.

INTRODUCTION: Etranacogene dezaparvovec is the first gene therapy approved for treatment of adults with severe and moderately severe hemophilia B. AREAS COVERED: This review describes the results of the clinical trial program of AMT-060 and etranacogene dezaparvovec, outlining the pharmacokinetic, clinical efficacy and safety data. With the entry of etranacogene dezaparvovec into the market, this review summarizes the treatment landscape in hemophilia B and discusses the current unknowns in the field. EXPERT OPINION: Gene therapy appears to be a feasible option for adults with severe and moderately severe hemophilia B. Etranacogene dezaparvovec enables most patients to reach stable factor IX (FIX) levels after a single intravenous infusion, eliminating the need for regular prophylaxis; thus, drastically reducing treatment burden and avoiding variable bleeding risk owing to fluctuating FIX activity levels. Efficacy of etranacogene dezaparvovec has been demonstrated even in the presence of preexisting neutralizing antibodies (up to a titer of 1:678), with a relative low risk of transaminitis and its associated potential loss of transgene expression. However, long-term data are required to ascertain the durability of FIX levels achieved and safety. The cost-effectiveness and adoption of innovative payment models for reimbursement are key in choosing gene therapy over existing treatments.

Lethality rescue and long-term amelioration of a citrullinemia type I mouse model by neonatal gene-targeting combined to SaCRISPR-Cas9.

Citrullinemia type I is a rare autosomal-recessive disorder caused by deficiency of argininosuccinate synthetase (ASS1). The clinical presentation includes the acute neonatal form, characterized by ammonia and citrulline accumulation in blood, which may lead to encephalopathy, coma, and death, and the milder late-onset form. Current treatments are unsatisfactory, and the only curative treatment is liver transplantation. We permanently modified the hepatocyte genome in lethal citrullinemia mice (Ass1fold/fold) by inserting the ASS1 cDNA into the albumin locus through the delivery of two AAV8 vectors carrying the donor DNA and the CRISPR-Cas9 platform. The neonatal treatment completely rescued mortality ensuring survival up to 5 months of age, with plasma citrulline levels significantly decreased, while plasma ammonia levels remained unchanged. In contrast, neonatal treatment with a liver-directed non-integrative AAV8-AAT-hASS1 vector failed to improve disease parameters. To model late-onset citrullinemia, we dosed postnatal day (P) 30 juvenile animals using the integrative approach, resulting in lifespan improvement and a minor reduction in disease markers. Conversely, treatment with the non-integrative vector completely rescued mortality, reducing plasma ammonia and citrulline to wild-type values. In summary, the integrative approach in neonates is effective, although further improvements are required to fully correct the phenotype. Non-integrative gene therapy application to juvenile mice ensures a stable and very efficient therapeutic effect.

Adeno-Associated Viral Vector-Delivered Pannexin-1 Mimetic Peptide Alleviates Airway Inflammation in an Allergen-Sensitized Mouse Model.

Asthma is a chronic inflammatory disease around the world. Extracellular adenosine triphosphate works as a dangerous signal in responding to cellular stress, irritation, or inflammation. It has also been reported its association with the pathogenicity in asthma, with increased level in lungs of asthmatics. Pannexin-1 is one of the routes that contributes to the release of adenosine triphosphate form intracellular to extracellular. The aim of this study was to apply pannexin-1 peptide antagonist 10Panx1 into adeno-associated viral (AAV) vectors on ovalbumin (OVA)-induced asthmatic mouse model. The results demonstrated that this treatment was able to reduce the adenosine triphosphate level in bronchoalveolar lavage fluid and downregulate the major relevant to the symptom of asthma attack, airway hyperresponsiveness to methacholine. The histological data also gave a positive support with decreased tissue remodeling and mucus deposition. Other asthmatic related features, including eosinophilic inflammation and OVA-specific T helper type 2 responses, were also decreased by the treatment. Beyond the index of inflammation, the proportion of effector and regulatory T cells was examined to survey the potential mechanism behind. The data provided a slightly downregulated pattern in lung GATA3+ CD4 T cells. However, an upregulated population of CD25+FoxP3+ CD4 T cells was seen in spleens. These data suggested that exogeneous expression of 10Panx1 peptide was potential to alleviated asthmatic airway inflammation, and this therapeutic effect might be from 10Panx1-mediated disruption of T cell activation or differentiation. Collectively, AAV vector-mediated 10Panx1 expression could be a naval therapy option to develop.

Efficient removal of antibodies to adeno-associated viruses by immunoadsorption.

BACKGROUND: Gene therapies based on adeno-associated viruses (AAV) are a therapeutic option to successfully treat monogenetic diseases. However, the influence of pre-existing immunity to AAV can compromise the application of AAV gene therapy, most notably by the presence of neutralizing antibodies (NAb) to AAV. METHODS: In the following study, we investigated to what extent the treatment by immunoadsorption (IA) would reduce the levels of human anti-AAV antibodies to AAV2 and AAV5. To that end, we screened blood sera from 40 patients receiving IA treatment because of underlying autoimmune disease or transplant rejection, with detectable AAV-antibodies in 23 patients (22 by NAb detection, and 1 additionally by anti-AAV5 ELISA analysis). RESULTS: Our results show that IA efficiently depleted anti-AAV2 NAb with a mean reduction of 3.92 ± 1.09 log2 titer steps (93.4%) after three to five single IA treatments, 45% of seropositive subjects had an anti-AAV2 titer below the threshold titer of 1:5 after the IA treatment series. Anti-AAV5 NAb were reduced to below the threshold titer of 1:5 in all but one of five seropositive subjects. Analysis of total anti-AAV5 antibodies by ELISA demonstrated an anti-AAV5 antibody reduction over the IA treatment series of 2.67 ± 1.16 log2 titer steps (84.3%). CONCLUSION: In summary, IA may represent a safe strategy to precondition patients with pre-existing anti-AAV antibodies to make this population eligible for an effective AAV-based gene therapy.

Recapitulation of Skewed X-Inactivation in Female Ornithine Transcarbamylase-Deficient Primary Human Hepatocytes in the FRG Mouse: A Novel System for Developing Epigenetic Therapies.

Realization of the immense therapeutic potential of epigenetic editing requires development of clinically predictive model systems that faithfully recapitulate relevant aspects of the target disease pathophysiology. In female patients with ornithine transcarbamylase (OTC) deficiency, an X-linked condition, skewed inactivation of the X chromosome carrying the wild-type OTC allele is associated with increased disease severity. The majority of affected female patients can be managed medically, but a proportion require liver transplantation. With rapid development of epigenetic editing technology, reactivation of silenced wild-type OTC alleles is becoming an increasingly plausible therapeutic approach. Toward this end, privileged access to explanted diseased livers from two affected female infants provided the opportunity to explore whether engraftment and expansion of dissociated patient-derived hepatocytes in the FRG mouse might produce a relevant model for evaluation of epigenetic interventions. Hepatocytes from both infants were successfully used to generate chimeric mouse-human livers, in which clusters of primary human hepatocytes were either OTC positive or negative by immunohistochemistry (IHC), consistent with clonal expansion from individual hepatocytes in which the mutant or wild-type OTC allele was inactivated, respectively. Enumeration of the proportion of OTC-positive or -negative human hepatocyte clusters was consistent with dramatic skewing in one infant and minimal to modest skewing in the other. Importantly, IHC and fluorescence-activated cell sorting analysis of intact and dissociated liver samples from both infants showed qualitatively similar patterns, confirming that the chimeric mouse-human liver model recapitulated the native state in each infant. Also of importance was the induction of a treatable metabolic phenotype, orotic aciduria, in mice, which correlated with the presence of clonally expanded OTC-negative primary human hepatocytes. We are currently using this unique model to explore CRISPR-dCas9-based epigenetic targeting strategies in combination with efficient adeno-associated virus (AAV) gene delivery to reactivate the silenced functional OTC gene on the inactive X chromosome.