Adeno-associated Viral Vector Serotype Research Articles

Development and characterization of a non-human primate model of disseminated synucleinopathy.

The presence of a widespread cortical synucleinopathy is the main neuropathological hallmark underlying clinical entities such as Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB). There currently is a pressing need for the development of non-human primate (NHPs) models of PDD and DLB to further overcome existing limitations in drug discovery. Here we took advantage of a retrogradely-spreading adeno-associated viral vector serotype 9 coding for the alpha-synuclein A53T mutated gene (AAV9-SynA53T) to induce a widespread synucleinopathy of cortical and subcortical territories innervating the putamen. Four weeks post-AAV deliveries animals were sacrificed and a comprehensive biodistribution study was conducted, comprising the quantification of neurons expressing alpha-synuclein, rostrocaudal distribution and their specific location. Intraputaminal deliveries of AAV9-SynA53T lead to a disseminated synucleinopathy throughout ipsi- and contralateral cerebral cortices, together with transduced neurons located in the ipsilateral caudal intralaminar nuclei and in the substantia nigra pars compacta (leading to thalamostriatal and nigrostriatal projections, respectively). Cortical afferent systems were found to be the main contributors to putaminal afferents (superior frontal and precentral gyri in particular). Obtained data extends current models of synucleinopathies in NHPs, providing a reproducible platform enabling the adequate implementation of end-stage preclinical screening of new drugs targeting alpha-synuclein.

Retention Rates of Genetic Therapies Based on AAV Serotypes 2 and 8 Using Different Drug-Delivery Materials.

The purpose of this study was to compare the retention rate of Adeno-associated viral vector (AAV) gene therapy agents within different subretinal injection systems. The retention of AAV serotype 2-based voretigene neparvovec (VN) and a clinical-grade AAV serotype 8 vector within four different subretinal cannulas from two different manufacturers was quantified. A standardized qPCR using the universal inverted terminal repeats as a target sequence was developed. The instruments compared were the PolyTip® cannula 25 g/38 g by MedOne Surgical, Inc., Sarasota, FL, USA, and three different subretinal injection needles by DORC, Zuidland, The Netherlands (1270.EXT Extendible 41G subretinal injection needle (23G), DORC 1270.06 23G Dual bore injection cannula, DORC 27G Subretinal injection cannula). The retention rate of VN and within the DORC products (10-28%) was comparable to the retention rate (32%) found for the PolyTip® cannula that is mentioned in the FDA-approved prescribing information for VN. For the AAV8 vector, the PolyTip® cannula showed a retention rate of 14%, and a similar retention rate of 3-16% was found for the DORC products (test-retest variability: mean 4.5%, range 2.5-20.2%). As all the instruments tested showed comparable retention rates, they seem to be equally compatible with AAV2- and AAV8-based gene therapy agents.

Cas13b-mediated RNA targeted therapy alleviates genetic dilated cardiomyopathy in mice

BackgroundRecent advances in gene editing technology have opened up new avenues for in vivo gene therapy, which holds great promise as a potential treatment method for dilated cardiomyopathy (DCM). The CRISPR-Cas13 system has been shown to be an effective tool for knocking down RNA expression in mammalian cells. PspCas13b, a type VI-B effector that can be packed into adeno-associated viruses and improve RNA knockdown efficiency, is a potential treatment for diseases characterized by abnormal gene expression.ResultsUsing PspCas13b, we were able to efficiently and specifically knockdown the mutant transcripts in the AC16 cell line carrying the heterozygous human TNNT2R141W (hTNNT2R141W) mutation. We used adeno-associated virus vector serotype 9 to deliver PspCas13b with specific single guide RNA into the hTNNT2R141W transgenic DCM mouse model, effectively knocking down hTNNT2R141W transcript expression. PspCas13b-mediated knockdown significantly increased myofilament sensitivity to Ca2+, improved cardiac function, and reduced myocardial fibrosis in hTNNT2R141W DCM mice.ConclusionsThese findings suggest that targeting genes through Cas13b is a promising approach for in vivo gene therapy for genetic diseases caused by aberrant gene expression. Our study provides further evidence of Cas13b’s application in genetic disease therapy and paves the way for future applicability of genetic therapies for cardiomyopathy.

The Efficacy of a Human-Ready miniMECP2 Gene Therapy in a Pre-Clinical Model of Rett Syndrome.

Inactivating mutations and the duplication of methyl-CpG binding protein 2 (MeCP2), respectively, mediate Rett syndrome (RTT) and MECP2 duplication syndrome. These disorders underscore the conceptual dose-dependent risk posed by MECP2 gene therapy for mosaic RTT patients. Recently, a miRNA-Responsive Autoregulatory Element (miRARE) mitigated the dose-dependent toxicity posed by self-complementary adeno-associated viral vector serotype 9 (AAV9) miniMECP2 gene therapy (scAAV9/miniMECP2-myc) in mice. Here, we report an efficacy assessment for the human-ready version of this regulated gene therapy (TSHA-102) in male Mecp2-/y knockout (KO) mice after intracerebroventricular (ICV) administration at postnatal day 2 (P2) and after intrathecal (IT) administration at P7, P14 (±immunosuppression), and P28 (±immunosuppression). We also report qPCR studies on KO mice treated at P7-P35; protein analyses in KO mice treated at P38; and a survival safety study in female adult Mecp2-/+ mice. In KO mice, TSHA-102 improved respiration, weight, and survival across multiple doses and treatment ages. TSHA-102 significantly improved the front average stance and swing times relative to the front average stride time after P14 administration of the highest dose for that treatment age. Viral genomic DNA and miniMECP2 mRNA were present in the CNS. MiniMeCP2 protein expression was higher in the KO spinal cord compared to the brain. In female mice, TSHA-102 permitted survivals that were similar to those of vehicle-treated controls. In all, these pivotal data helped to support the regulatory approval to initiate a clinical trial for TSHA-102 in RTT patients (clinical trial identifier number NCT05606614).

Intraarticular Delivery of AAV5-Mir-125a-5p Regulates Key Molecular Mediators of Hemophilic Arthropathy

Background: Hemophilic arthropathy (HA) is a major comorbidity in patients with hemophilia characterized by chronic inflammation of the joints, stiffness and loss of function. The expression of microRNAs are known to be altered in various joint diseases such as rheumatoid arthritis and osteoarthritis which share clinical attributes with HA. One such microRNA is miR-125a-5p, known to be involved in regulating inflammatory mediators in the arthritic joints. The current study was designed to investigate the role of miR125a-5p in HA and examine the phenotypic effect of its targeted reconstitution in the joints. Methods: We utilized a chronic arthropathy model in hemophilia A mice to study the impact of miR125a-5p in disease progression. First, we performed a targeted screen of miR125a-5p and its target genes (STAT1 and TRAF6) using digital PCR (dPCR). Further, to validate if reconstitution of miR125a-5p aids in modulating the molecular mediators of HA, we performed the Adeno-associated virus vector (AAV) serotype 5 targeted delivery of miR125a (AAV5-miR125a) either alone (2.13x10 11vgs/joint) or in combination with capsid-modified AAV8-human Factor 8 (F8)(AAV8-F8) gene therapy at a low dose (1x10 11vgs/animal) intravenously in F8 tm1kaz/Jmice. Briefly, the animals were administered with vectors (alone or in combination), and seven days post vector administration, the animals were subjected to multiple bleeding episodes in the right knee (Day 0, 14, and 30) to mimic chronic hemarthrosis. On day 45, blood samples were collected from experimental animals for FVIII specific clotting assay, and joint tissues were isolated to perform further molecular and histochemical analysis. Results: Our dPCR analysis revealed a significant reduction (2-fold) of miR125a-5p (3518 copies/µl vs 7242 copies/µl, p≤0.0001) in injured joints compared to control joints. Absolute quantification of target genes, namely STAT1 and TRAF6 showed significantly higher expression in injured joints (7.6 to 10.6 fold, respectively) when compared to control joints. Further immunohistochemical examination of joint sections from the experimental mice confirmed the elevated expression of these immunological mediators (STAT1, TRAF6). Upon administration of AAV vectors containing miR125a and/or F8, we observed a significantly increased expression of miR125a-5p (~12 fold) in only miR125a vector-administered animals while the combination vector treated group had, ~44-fold higher levels of miR125a-5p. The coagulant assay revealed an increased FVIII activity (~17%) in only F8 vector-treated animals and approximately 20% FVIII levels in mice that received both miR125a and F8 vectors at low doses. Surprisingly, the animals that received only F8 vectors also showed significantly upregulated miR125a-5p expression (~20 fold) signifying the role of miR125a-5p in HA pathology. In line with the above findings, immunohistochemical analysis of joint tissues revealed decreased expression of immunological markers STAT1, TRAF6 (direct targets of miR125a-5p), and the chondro-degenerative matrix metalloproteinases 3, 9, 13 in vector-treated groups when compared to injured joints with no treatment. Hematoxylin and Eosin (H&E) staining of joint tissues from experimental animals was performed which revealed that animals that received both miR125a and F8 vectors showed decreased synovial hyperplasia, villi formation, cartilage erosion, and reduced hemosiderin deposition signifying moderate phenotype rescue. Conclusions: Our data confirms that miR125a-5p is a major molecular target that regulates inflammatory response in chronic HA. Targeted delivery of miR125a in tandem with F8 gene augmentation had an impact on several molecular mediators like STAT1, TRAF6, MMP3,9, and 13. The reconstitution of miR125a and a higher dose of F8 vectors might be beneficial to reverse chronic HA.

Safety and efficacy of gene replacement therapy for X-linked myotubular myopathy (ASPIRO): a multinational, open-label, dose-escalation trial

X-linked myotubular myopathy is a rare, life-threatening, congenital muscle disease observed mostly in males, which is caused by mutations in MTM1. No therapies are approved for this disease. We aimed to assess the safety and efficacy of resamirigene bilparvovec, which is an adeno-associated viral vector serotype 8 delivering human MTM1. ASPIRO is an open-label, dose-escalation trial at seven academic medical centres in Canada, France, Germany, and the USA. We included boys younger than 5 years with X-linked myotubular myopathy who required mechanical ventilator support. The trial was initially in two parts. Part 1 was planned as a safety and dose-escalation phase in which participants were randomly allocated (2:1) to either the first dose level (1·3 × 1014 vector genomes [vg]/kg bodyweight) of resamirigene bilparvovec or delayed treatment, then, for later participants, to either a higher dose (3·5 × 1014 vg/kg bodyweight) of resamirigene bilparvovec or delayed treatment. Part 2 was intended to confirm the dose selected in part 1. Resamirigene bilparvovec was administered as a single intravenous infusion. An untreated control group comprised boys who participated in a run-in study (INCEPTUS; NCT02704273) or those in the delayed treatment cohort who did not receive any dose. The primary efficacy outcome was the change from baseline to week 24 in hours of daily ventilator support. After three unexpected deaths, dosing at the higher dose was stopped and the two-part feature of the study design was eliminated. Because of changes to the study design during its implementation, analyses were done on an as-treated basis and are deemed exploratory. All treated and control participants were included in the safety analysis. The trial is registered with ClinicalTrials.gov, NCT03199469. Outcomes are reported as of Feb 28, 2022. ASPIRO is currently paused while deaths in dosed participants are investigated. Between Aug 3, 2017 and June 1, 2021, 30 participants were screened for eligibility, of whom 26 were enrolled; six were allocated to the lower dose, 13 to the higher dose, and seven to delayed treatment. Of the seven children whose treatment was delayed, four later received the higher dose (n=17 total in the higher dose cohort), one received the lower dose (n=7 total in the lower dose cohort), and two received no dose and joined the control group (n=14 total, including 12 children from INCEPTUS). Median age at dosing or enrolment was 12·1 months (IQR 10·0-30·9; range 9·5-49·7) in the lower dose cohort, 31·1 months (16·0-64·7; 6·8-72·7) in the higher dose cohort, and 18·7 months (10·1-31·5; 5·9-39·3) in the control cohort. Median follow-up was 46·1 months (IQR 41·0-49·5; range 2·1-54·7) for lower dose participants, 27·6 months (24·6-29·1; 3·4-41·0) for higher dose participants, and 28·3 months (9·7-46·9; 5·7-32·7) for control participants. At week 24, lower dose participants had an estimated 77·7 percentage point (95% CI 40·22 to 115·24) greater reduction in least squares mean hours per day of ventilator support from baseline versus controls (p=0·0002), and higher dose participants had a 22·8 percentage point (6·15 to 39·37) greater reduction from baseline versus controls (p=0·0077). One participant in the lower dose cohort and three in the higher dose cohort died; at the time of death, all children had cholestatic liver failure following gene therapy (immediate causes of death were sepsis; hepatopathy, severe immune dysfunction, and pseudomonal sepsis; gastrointestinal haemorrhage; and septic shock). Three individuals in the control group died (haemorrhage presumed related to hepatic peliosis; aspiration pneumonia; and cardiopulmonary failure). Most children with X-linked myotubular myopathy who received MTM1 gene replacement therapy had important improvements in ventilator dependence and motor function, with more than half of dosed participants achieving ventilator independence and some attaining the ability to walk independently. Investigations into the risk for underlying hepatobiliary disease in X-linked myotubular myopathy, and the need for monitoring of liver function before gene replacement therapy, are ongoing. Astellas Gene Therapies.

Development of capsid- and genome-modified optimized AAVrh74 vectors for muscle gene therapy

The first generation of adeno-associated virus (AAV) vectors composed of the naturally occurring capsids and genomes, although effective in some instances, are unlikely to be optimal for gene therapy in humans. The use of the first generation of two different AAV serotype vectors (AAV9 and AAVrh74) in four separate clinical trials failed to be effective in patients with Duchenne muscular dystrophy, although some efficacy was observed in a subset of patients with AAVrh74 vectors leading to US Food and Drug Administration approval (Elevidys). In two trials with the first generation of AAV9 vectors, several serious adverse events were observed, including the death of a patient in one trial, and more recently, in the death of a second patient in an N-of-1 clinical trial. In a fourth trial with the first generation of AAVrh74 vectors, myositis and myocarditis were also observed. Here, we report that capsid- and genome-modified optimized AAVrh74 vectors are significantly more efficient in transducing primary human skeletal muscle cells invitro and in all major muscle tissues invivo following systemic administration in a murine model. The availability of optimized AAVrh74 vectors promises to be safe and effective in the potential gene therapy of muscle diseases in humans.

Production of adeno-associated viral vector serotype 6 by triple transfection of suspension HEK293 cells at higher cell densities.

In recent years, the use of adeno-associated viruses (AAV) as vectors for gene and cell therapy has increased, leading to a rise in the amount of AAV vectors required during pre-clinical and clinical trials. AAV serotype 6 (AAV6) has been found to be efficient in transducing different cell types and has been successfully used in gene and cell therapy protocols. However, the number of vectors required to effectively deliver the transgene to one single cell has been estimated at 106 viral genomes (VG), making large-scale production of AAV6 necessary. Suspension cell-based platforms are currently limited to low cell density productions due to the widely reported cell density effect (CDE), which results in diminished production at high cell densities and decreased cell-specific productivity. This limitation hinders the potential of the suspension cell-based production process to increase yields. In this study, we investigated the improvement of the production of AAV6 at higher cell densities by transiently transfecting HEK293SF cells. The results showed that when the plasmid DNA was provided on a cell basis, the production could be carried out at medium cell density (MCD, 4 × 106 cells/mL) resulting in titers above 1010 VG/mL. No detrimental effects on cell-specific virus yield or cell-specific functional titer were observed at MCD production. Furthermore, while medium supplementation alleviated the CDE in terms of VG/cell at high cell density (HCD, 10 × 106 cells/mL) productions, the cell-specific functional titer was not maintained, and further studies are necessary to understand the observed limitations for AAV production in HCD processes. The MCD production method reported here lays the foundation for large-scale process operations, potentially solving the current vector shortage in AAV manufacturing. This article is protected by copyright. All rights reserved.

Immunobiology of a rationally-designed AAV2 capsid following intravitreal delivery in mice.

Adeno-associated virus serotype 2 (AAV2) is a viral vector that can be used to deliver therapeutic genes to diseased cells in the retina. One strategy for altering AAV2 vectors involves the mutation of phosphodegron residues, which are thought to be phosphorylated/ubiquitinated in the cytosol, facilitating degradation of the vector and the inhibition of transduction. As such, mutation of phosphodegron residues have been correlated with increased transduction of target cells, however, an assessment of the immunobiology of wild-type and phosphodegron mutant AAV2 vectors following intravitreal (IVT) delivery to immunocompetent animals is lacking in the current literature. In this study, we show that IVT of a triple phosphodegron mutant AAV2 capsid is associated with higher levels of humoral immune activation, infiltration of CD4 and CD8 T-cells into the retina, generation of splenic germinal centre reactions, activation of conventional dendritic cell subsets, and elevated retinal gliosis compared to wild-type AAV2 capsids. However, we did not detect significant changes in electroretinography arising after vector administration. We also demonstrate that the triple AAV2 mutant capsid is less susceptible to neutralisation by soluble heparan sulphate and anti-AAV2 neutralising antibodies, highlighting a possible utility for the vector in terms of circumventing pre-existing humoral immunity. In summary, the present study highlights novel aspects of rationally-designed vector immunobiology, which may be relevant to their application in preclinical and clinical settings.

Functional Exploration of Conserved Sequences in the Distal Face of Angiotensinogen-Brief Report.

Angiotensinogen (AGT) is an essential component in the renin-angiotensin system. AGT has highly conserved sequences in the loop and β-sheet regions among species; however, their functions have not been studied. Adeno-associated viral vector (AAV) serotype 2/8 encoding mouse AGT with mutations of conserved sequences in the loop (AAV.loop-Mut), β-sheet (AAV.βsheet-Mut), or both regions (AAV.loop/βsheet-Mut) was injected into male hepatocyte-specific AGT-deficient (hepAGT-/-) mice in an LDL (low-density lipoprotein) receptor-deficient background. AAV containing mouse wild-type AGT (AAV.mAGT) or a null vector (AAV.null) were used as controls. Two weeks after AAV administration, all mice were fed a western diet for 12 weeks. To determine how AGT secretion is regulated in hepatocytes, AAVs containing the above mutations were transducted into HepG2 cells. In hepAGT-/- mice infected with AAV.loop-Mut or βsheet-Mut, plasma AGT concentrations, systolic blood pressure, and atherosclerosis were comparable to those in AAV.mAGT-infected mice. Interestingly, plasma AGT concentrations, systolic blood pressure, and atherosclerotic lesion size in hepAGT-/- mice infected with AAV.loop/βsheet-Mut were not different from mice infected with AAV.null. In contrast, hepatic Agt mRNA abundance was elevated to a comparable magnitude as AAV.mAGT-infected mice. Immunostaining showed that AGT protein was accumulated in hepatocytes of mice infected with AAV.loop/βsheet-Mut or HepG2 cells transducted with AAV.loop/βsheet-Mut. Accumulated AGT was not located in the endoplasmic reticulum. The conserved sequences in either the loop or β-sheet region individually have no effect on AGT regulation, but the conserved sequences in both regions synergistically contribute to the secretion of AGT from hepatocytes.

Efficacy and muscle safety assessment of fukutin-related protein gene therapy

Limb-girdle muscular dystrophy type R9 (LGMDR9) is a muscle-wasting disease that begins in the hip and shoulder regions of the body. This disease is caused by mutations in fukutin-related protein (FKRP), a glycosyltransferase critical for maintaining muscle cell integrity. Here we investigated potential gene therapies for LGMDR9 containing an FKRP expression construct with untranslated region (UTR) modifications. Initial studies treated an aged dystrophic mouse model (FKRPP448L) with adeno-associated virus vector serotype 6 (AAV6). Grip strength improved in a dose- and time-dependent manner, injected mice exhibited fewer central nuclei and serum creatine kinase levels were 3- and 5-fold lower compared to those in non-injected FKRPP448L mice. Treatment also partially stabilized the respiratory pattern during exercise and improved treadmill running, partially protecting muscle from exercise-induced damage. Western blotting of C2C12 myotubes using a novel rabbit antibody confirmed heightened translation with the UTR modifications. We further explored the question of FKRP toxicity in wild-type mice using high doses of two additional muscle-tropic capsids: AAV9 and AAVMYO1. No toxic effects were detected with either therapeutic agent. These data further support the feasibility of gene therapy to treat LGMDR9.

Genetic modulation of the HTR2A gene reduces anxiety-related behavior in mice.

The expanding field of precision gene editing using CRISPR/Cas9 has demonstrated its potential as a transformative technology in the treatment of various diseases. However, whether this genome-editing tool could be used to modify neural circuits in the central nervous system (CNS), which are implicated in complex behavioral traits, remains uncertain. In this study, we demonstrate the feasibility of noninvasive, intranasal delivery of adeno-associated virus serotype 9 (AAV9) vectors containing CRISPR/Cas9 cargo within the CNS resulting in modification of the HTR2A receptor gene. In vitro, exposure to primary mouse cortical neurons to AAV9 vectors targeting the HT2RA gene led to a concentration-dependent decrease in spontaneous electrical activity following multielectrode array (MEA) analysis. In vivo, at 5 weeks postintranasal delivery in mice, analysis of brain samples revealed single base pair deletions and nonsense mutations, leading to an 8.46-fold reduction in mRNA expression and a corresponding 68% decrease in the 5HT-2A receptor staining. Our findings also demonstrate a significant decrease in anxiety-like behavior in treated mice. This study constitutes the first successful demonstration of a noninvasive CRISPR/Cas9 delivery platform, capable of bypassing the blood-brain barrier and enabling modulation of neuronal 5HT-2A receptor pathways. The results of this study targeting the HTR2A gene provide a foundation for the development of innovative therapeutic strategies for a broad range of neurological disorders, including anxiety, depression, attentional deficits, and cognitive dysfunction.

Eladocagene Exuparvovec Gene Therapy Improves Motor Development in Patients With Aromatic L-Amino Acid Decarboxylase Deficiency (S34.004)

<h3>Objective:</h3> Investigate the efficacy and safety of eladocagene exuparvovec in patients with aromatic l-amino acid decarboxylase deficiency (AADCd). <h3>Background:</h3> AADCd results from mutations in the dopa decarboxylase gene leading to reduced AADC enzyme activity; it is characterized by motor impairments and inability to attain developmental milestones. Eladocagene exuparvovec is a recombinant adeno-associated viral vector serotype 2 carrying the coding sequence for human AADC. <h3>Design/Methods:</h3> Eladocagene exuparvovec was infused bilaterally in the putamina of 30 children with AADC deficiency in 3 studies (AADC-CU/1601 [n=8], AADC-010 [n=10], and AADC-011 [n=12] in patients aged 18–102 months. Data were extracted on January 4, 2022. Patients receiving a total of 1.8 × 10¹¹ vg (n=21) or 2.4 × 10¹¹ vg (n=9; AADC-011) were followed for up to 120 months and assessed for motor milestone attainment using the Peabody Developmental Motor Scale, 2nd edition (PDMS-2). Specific motor skill items of the PDMS-2 were used to assess motor milestones including head control (partial or full), sitting (supported or independently), standing (with/away from support; up from cross-legged position), and walking (with/without assistance; 10 feet; taped line) Motor milestones and development were measured every 3 months for 1 year following gene therapy, then every 6–12 months for ≤120 months. <h3>Results:</h3> At baseline, no patients mastered head control or more advanced milestones. At year 1 of follow-up, patients were gaining the following skills (n): partial head control (26); full head control (15), sitting unassisted (7), supported standing (2). Progression of development was noted at years 5 and 10. By year 5, more advanced milestones were achieved (n): full head control (24), sitting unassisted (21) assisted walking (5), walking 10 feet (3), or walking up stairs (3). These abilities were maintained for as long as 10 years <h3>Conclusions:</h3> These data indicate eladocagene exuparvovec can provide a durable, positive impact on motor development in patients with AADCd. <b>Disclosure:</b> Mrs. Russell has received personal compensation for serving as an employee of PTC Therapeutics. Mrs. Russell has stock in PTC Therapeutics. Prof. ROUBERTIE has received personal compensation in the range of $500-$4,999 for serving as a Consultant for PTC. Prof. ROUBERTIE has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for PTC. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Sanofi Genzyme. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Biogen. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Novartis. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Amicus. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Sanofi. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Sanofi Genzyme. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Avexis/Norvatis. Yin-Hsiu Chien has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Biogen. The institution of Yin-Hsiu Chien has received research support from Sanofi Genzyme. Ni-Chung Lee has nothing to disclose. Dr. Sierra has received personal compensation for serving as an employee of PTC Therapeutics Inc. Dr. Sierra has stock in PTC Therapeutics Inc. Dr. Turna has received personal compensation for serving as an employee of PTC Therapeutics. Dr. Turna has stock in PTC Therapeutics. Dr. Wang has received personal compensation for serving as an employee of PTC Therapeutics. Dr. Wang has received stock or an ownership interest from PTC Therapeutics, Inc.. The institution of Chun-Hwei Tai has received research support from National Taiwan University Hospital. Paul Wuh-Liang Hwu has received personal compensation in the range of $500-$4,999 for serving as a Consultant for PTC Therapeutics Inc.. Paul Wuh-Liang Hwu has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for PTC therapeutics. The institution of Paul Wuh-Liang Hwu has received research support from PTC therapeutics.

Detrimental role of SIX1 in hepatic lipogenesis and fibrosis of non-alcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. Aberrant lipid metabolism and accumulation of extracellular matrix proteins are hallmarks of the disease, but the underlying mechanisms are largely unknown. This study aims to elucidate the key role of sine oculis homeobox homologue 1 (SIX1) in the development of NAFLD. Alb-Cre mice were administered the AAV9 vector for SIX1 liver-specific overexpression or knockdown. Metabolic disorders, hepatic steatosis, and inflammation were monitored in mice fed with HFHC or MCD diet. High throughput CUT&Tag analysis was employed to investigate the mechanism of SIX1 in diet-induced steatohepatitis. Here, we found increased SIX1 expression in the livers of NAFLD patients and animal models. Liver-specific overexpression of SIX1 using adeno-associated virus serotype 9 (AAV9) provoked more severe inflammation, metabolic disorders, and hepatic steatosis in the HFHC or MCD-induced mice model. Mechanistically, we demonstrated that SIX1 directly activated the expression of liver X receptor α (LXRα) and liver X receptor β (LXRβ), thus inducing de novo lipogenesis (DNL). In addition, our results also illustrated a critical role of SIX1 in regulating the TGF-β pathway by increasing the levels of type I and II TGF-β receptor (TGFβRI/TGFβRII) in hepatic stellate cells (HSCs). Finally, we found that liver-specific SIX1 deficiency could ameliorate diet-induced NAFLD pathogenesis. Our findings suggest a detrimental function of SIX1 in the progression of NAFLD. The direct regulation of LXRα/β and TGF-β signalling by SIX1 provides a new regulatory mechanism in hepatic steatosis and fibrosis.

Successful liver transduction by re-administration of different adeno-associated virus vector serotypes in mice.

Intravenous administration of adeno-associated virus (AAV) vectors is a promising gene therapy approach for monogenic diseases. However, re-administration of the same AAV serotype is impossible because of the induction of anti-AAV neutralizing antibodies (NAbs). Here, we examined the feasibility of re-administrating AAV vector serotypes different from the initial AAV vector serotype. Liver-targeting AAV3B, AAV5, and AAV8 vectors were intravenously injected in C57BL/6 mice, and the emergence of NAbs and the transduction efficacy following re-administration were evaluated. For all serotypes, re-administration of the same serotype was not possible. Although the highest neutralizing activity of NAb was induced by AAV5, anti-AAV5 NAbs did not react with other serotypes, resulting in successful re-administration with the other serotypes. AAV5 re-administration was also successful in all mice treated with AAV3B and AAV8. Effective secondary administration of AAV3B and AAV8 was observed in most mice initially administrated AAV8 and AAV3B, respectively. However, few mice developed NAbs cross-reacting with the other serotypes, especially those with close sequence homology. In summary, AAV vector administration induced NAbs relatively specific to the administrated serotype. Secondary administration of AAVs targeting liver transduction could be successfully achieved by switching AAV serotypes in mice.

Development of an icIEF assay for monitoring AAV capsid proteins and application to gene therapy products

Adeno-associated virus (AAV) gene therapy vectors, which contain a DNA transgene packaged into a protein capsid, have shown tremendous therapeutic potential in recent years. Methods traditionally used in quality control labs, such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), do not provide a complete understanding of capsid viral protein (VP) charge heterogeneity. In the present study, we developed simple, one-step sample preparation and charge-based VP separation using imaged capillary isoelectric focusing (icIEF) for monitoring AAV products. The robustness of the method was confirmed through a design of experiments (DoE) exercise. An orthogonal reverse-phase (RP) HPLC method coupled with mass spectrometry was developed to separate and identify charge species. Additionally, capsid point mutants demonstrate the capability of the method to resolve deamidation at a single site on the viral proteins. Finally, case studies using two different AAV serotype vectors establish the icIEF method as stability indicating and demonstrate that increases in acidic species measured by icIEF correlate with increased deamidation, which, we show, results in decreased transduction efficiency. The addition of a rapid and robust icIEF method to the AAV capsid analytical toolkit enables development and consistent manufacturing of well-characterized gene therapy products.

Tyrosine kinase receptor B attenuates liver fibrosis by inhibiting TGF-β/SMAD signaling.

Liver fibrosis is a leading indicator for increased mortality and long-term comorbidity in NASH. Activation of HSCs and excessive extracellular matrix production are the hallmarks of liver fibrogenesis. Tyrosine kinase receptor (TrkB) is a multifunctional receptor that participates in neurodegenerative disorders. However, paucity of literature is available about TrkB function in liver fibrosis. Herein, the regulatory network and therapeutic potential of TrkB were explored in the progression of hepatic fibrosis. The protein level of TrkB was decreased in mouse models of CDAHFD feeding or carbon tetrachloride-induced hepatic fibrosis. TrkB suppressed TGF-β-stimulated proliferation and activation of HSCs in 3-dimensional liver spheroids and significantly repressed TGF-β/SMAD signaling pathway either in HSCs or in hepatocytes. The cytokine, TGF-β, boosted Nedd4 family interacting protein-1 (Ndfip1) expression, promoting the ubiquitination and degradation of TrkB through E3 ligase Nedd4-2. Moreover, carbon tetrachloride intoxication-induced hepatic fibrosis in mouse models was reduced by adeno-associated virus vector serotype 6 (AAV6)-mediated TrkB overexpression in HSCs. In addition, in murine models of CDAHFD feeding and Gubra-Amylin NASH (GAN), fibrogenesis was reduced by adeno-associated virus vector serotype 8 (AAV8)-mediated TrkB overexpression in hepatocytes. TGF-β stimulated TrkB degradation through E3 ligase Nedd4-2 in HSCs. TrkB overexpression inhibited the activation of TGF-β/SMAD signaling and alleviated the hepatic fibrosis both in vitro and in vivo . These findings demonstrate that TrkB could be a significant suppressor of hepatic fibrosis and confer a potential therapeutic target in hepatic fibrosis.

Recombinant Adeno-associated Viral Vectors Serotypes 6 and 9 are Able to Transduce Human Tracheal Epithelial Cells but Not Human Induced Pluripotent Stem Cells.

Recombinant adeno-associated viruses (rAAVs) may be useful for the development of gene therapy for hereditary diseases. Patient-specific human induced pluripotent stem cells (hiPSCs) can be differentiated into a variety of cells which are difficult or impossible to obtain by biopsy. To date, few research on the efficiency of rAAV transduction of hiPSCs has been published, but the obtained data are very contradictory and do not answer the actual question: how effective are rAAVs for the delivery of transgenes into hiPSCs. In this work, we used rAAV serotypes 5, 6, and 9 carrying the GFP transgene. The transduction efficiency of rAAV2/9-GFP and rAAV2/6-GFP for the immortalized tracheal epithelial cell line derived from a patient with cystic fibrosis (CFTE29o-) was relatively high. At the same time, the efficiency of transduction of iPSCs from a healthy donor and a cystic fibrosis (CF) donor was extremely low. Thus, our results show that the efficiency of hiPSC transduction by rAAV serotypes 5, 6, and 9 is not suitable for the delivery of transgenes.

Non-invasive intravenous administration of AAV9 transducing iduronate sulfatase leads to global metabolic correction and prevention of neurologic deficits in a mouse model of Hunter syndrome

Hunter syndrome is a rare x-linked recessive genetic disorder that affects lysosomal metabolism due to deficiency of iduronate-2-sulfatase (IDS), with subsequent accumulation of glycosaminoglycans heparan and dermatan sulfates (GAG). Enzyme replacement therapy is the only FDA-approved remedy and is an expensive life-time treatment that alleviates some symptoms of the disease without neurocognitive benefit. We previously reported successful treatment in a mouse model of mucopolysaccharidosis type II (MPS II) using adeno-associated viral vector serotype 9 encoding human IDS (AAV9.hIDS) via intracerebroventricular injection. As a less invasive and more straightforward procedure, here we report intravenously administered AAV9.hIDS in a mouse model of MPS II. In animals administered 1.5 × 1012 vg of AAV9.hIDS at 2 months of age, we observed supraphysiological levels of IDS enzyme activity in the circulation (up to 9100-fold higher than wild-type), in the tested peripheral organs (up to 560-fold higher than wild-type), but only 4% to 50% of wild type levels in the CNS. GAG levels were normalized on both sides of the blood-brain-barrier (BBB) in most of tissues tested. Despite low levels of the IDS observed in the CNS, this treatment prevented neurocognitive decline as shown by testing in the Barnes maze and by fear conditioning. This study demonstrates that a single dose of IV-administered AAV9.hIDS may be an effective and non-invasive procedure to treat MPS II that benefits both sides of the BBB, with implications for potential use of IV-administered AAV9 for other neuronopathic lysosomal diseases.

Quality control for Adeno-associated viral vector production.

Adeno-associated viral vectors (AAV) are frequently used by neuroscientists to deliver tools, such as biosensors and optogenetic and chemogenetic actuators, in vivo. Despite its widespread use, AAV vector characterization and quality control can vary between labs and viral vector cores leading to variable results and irreproducibility. This protocol describes some of the characterization and quality control assays necessary to confirm an AAV vector's titer, genomic identity, serotype and purity.