Death after High-Dose rAAV9 Gene Therapy in a Patient with Duchenne's Muscular Dystrophy.

We treated a 27-year-old patient with Duchenne's muscular dystrophy (DMD) with recombinant adeno-associated virus (rAAV) serotype 9 containing dSaCas9 (i.e., "dead" Staphylococcus aureus Cas9, in which the Cas9 nuclease activity has been inactivated) fused to VP64; this transgene was designed to up-regulate cortical dystrophin as a custom CRISPR-transactivator therapy. The dose of rAAV used was 1×1014 vector genomes per kilogram of body weight. Mild cardiac dysfunction and pericardial effusion developed, followed by acute respiratory distress syndrome (ARDS) and cardiac arrest 6 days after transgene treatment; the patient died 2 days later. A postmortem examination showed severe diffuse alveolar damage. Expression of transgene in the liver was minimal, and there was no evidence of AAV serotype 9 antibodies or effector T-cell reactivity in the organs. These findings indicate that an innate immune reaction caused ARDS in a patient with advanced DMD treated with high-dose rAAV gene therapy. (Funded by Cure Rare Disease.).

SOD1 Suppression with Adeno-Associated Virus and MicroRNA in Familial ALS.

Two patients with familial amyotrophic lateral sclerosis (ALS) and mutations in the gene encoding superoxide dismutase 1 (SOD1) were treated with a single intrathecal infusion of adeno-associated virus encoding a microRNA targeting SOD1. In Patient 1, SOD1 levels in spinal cord tissue as analyzed on autopsy were lower than corresponding levels in untreated patients with SOD1-mediated ALS and in healthy controls. Levels of SOD1 in cerebrospinal fluid were transiently and only slightly lower in Patient 1 but were not affected in Patient 2. In Patient 1, meningoradiculitis developed after the infusion; Patient 2 was pretreated with immunosuppressive drugs and did not have this complication. Patient 1 had transient improvement in the strength of his right leg, a measure that had been relatively stable throughout his disease course, but there was no change in his vital capacity. Patient 2 had stable scores on a composite measure of ALS function and a stable vital capacity during a 12-month period. This study showed that intrathecal microRNA can be used as a potential treatment for SOD1-mediated ALS.

A Translational Model for Venous Thromboembolism: MicroRNA Expression in Hibernating Black Bears.

BACKGROUND: Hibernating American black bears have significantly different clotting parameters than their summer active counterparts, affording them protection against venous thromboembolism during prolonged periods of immobility. We sought to evaluate if significant differences exist between the expression of microRNAs in the plasma of hibernating black bears compared with their summer active counterparts, potentially contributing to differences in hemostasis during hibernation. MATERIALS AND METHODS: MicroRNA sequencing was assessed in plasma from 21 American black bears in summer active (n = 11) and hibernating states (n = 10), and microRNA signatures during hibernating and active state were established using both bear and human genome. MicroRNA targets were predicted using messenger RNA (mRNA) transcripts from black bear kidney cells. In vitro studies were performed to confirm the relationship between identified microRNAs and mRNA expression, using artificial microRNA and human liver cells. RESULTS: Using the bear genome, we identified 15 microRNAs differentially expressed in the plasma of hibernating black bears. Of these microRNAs, three were significantly downregulated (miR-141-3p, miR-200a-3p, and miR-200c-3p), were predicted to target SERPINC1, the gene for antithrombin, and demonstrated regulatory control of the gene mRNA expression in cell studies. CONCLUSIONS: Our findings suggest that the hibernating black bears' ability to maintain hemostasis and achieve protection from venous thromboembolism during prolonged periods of immobility may be due to changes in microRNA signatures and possible upregulation of antithrombin expression.

Muscle-Directed Delivery of an AAV1 Vector Leads to Capsid-Specific T Cell Exhaustion in Nonhuman Primates and Humans.

With the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) approvals for Zolgensma, Luxturna, and Glybera, recombinant adeno-associated viruses (rAAVs) are considered efficient tools for gene transfer. However, studies in animals and humans demonstrate that intramuscular (IM) AAV delivery can trigger immune responses to AAV capsids and/or transgenes. IM delivery of rAAV1 in humans has also been described to induce tolerance to rAAV characterized by the presence of capsid-specific regulatory T cells (Tregs) in periphery. To understand mechanisms responsible for tolerance and parameters involved, we tested 3 muscle-directed administration routes in rhesus monkeys: IM delivery, venous limb perfusion, and the intra-arterial push and dwell method. These 3 methods were well tolerated and led to transgene expression. Interestingly, gene transfer in muscle led to Tregs and exhausted T cell infiltrates in situ at both day 21 and day 60 post-injection. In human samples, an in-depth analysis of the functionality of these cells demonstrates that capsid-specific exhausted T cells are detected after at least 5 years post-vector delivery and that the exhaustion can be reversed by blocking the checkpoint pathway. Overall, our study shows that persisting transgene expression after gene transfer in muscle is mediated by Tregs and exhausted T cells.

Serum Western Blot for the Detection of a c-Myc Protein Tag in Non-human Primates and Mice.

This protocol allows for the detection of a c-Myc tag on alpha-1 antitrypsin (AAT) delivered to species that already have endogenous AAT such as non-human primates allowing reliable and repeatable semi-quantitation of serum levels of AAT.

Limb Perfusion Delivery of a rAAV1 Alpha-1 Antitrypsin Vector in Non-Human Primates Is Safe but Insufficient for Therapy.

BACKGROUND/OBJECTIVES: α-1 antitrypsin (AAT) deficiency is an inherited, genetic condition characterized by reduced serum levels of AAT and increased risk of developing emphysema and liver disease. AAT is normally synthesized primarily in the liver, but muscle-targeting with a recombinant adeno-associated virus (rAAV) vector for α-1 antitrypsin (AAT) gene therapy has been used to minimize liver exposure to the virus and hepatotoxicity. Clinical trials of direct intramuscular (IM) administration of rAAV1-hAAT have demonstrated its overall safety and transgene expression for 5 years. However, the failure to reach the therapeutic target level after 100 large-volume (1.5 mL) IM injections of maximally concentrated vector led us to pursue a muscle-targeting approach using isolated limb perfusion. This targets the rAAV to a greater muscle mass and allows for a higher total volume (and thereby a higher dose) than is tolerable by multiple direct IM injections. Limb perfusion has been shown to be feasible in non-human primates using the rAAV1 serotype and a ubiquitous promoter expressing an epitope-tagged AAT matched to the host species. METHODS: In this study, we performed a biodistribution and preclinical safety study in non-human primates with a clinical candidate rAAV1-human AAT (hAAT) vector at doses ranging from 3.0 × 1012 to 1.3 × 1013 vg/kg, bracketing those used in our clinical trials. RESULTS: We found that limb perfusion delivery of rAAV1-hAAT was safe and showed a biodistribution pattern similar to previous studies. However, serum levels of AAT obtained with high-dose limb perfusion still reached only ~50% of the target serum levels. CONCLUSIONS: Our results suggest that clinically effective AAT gene therapy may ultimately require delivery at doses between 3.5 × 1013-1 × 1014 vg/kg, which is within the dose range used for approved rAAV gene therapies. Muscle-targeting strategies could be incorporated when delivering systemic administration of high-dose rAAV gene therapies to increase transduction of muscle tissues and reduce the burden on the liver, especially in diseases that can present with hepatotoxicity such as AAT deficiency.

Biodistribution and safety of a single rAAV3B-AAT vector for silencing and replacement of alpha-1 antitrypsin in Cynomolgus macaques.

Alpha-1 antitrypsin deficiency (AATD) is characterized by both chronic lung disease due to loss of wild-type AAT (M-AAT) antiprotease function and liver disease due to toxicity from delayed secretion, polymerization, and aggregation of misfolded mutant AAT (Z-AAT). The ideal gene therapy for AATD should therefore comprise both endogenous Z-AAT suppression and M-AAT overexpression. We designed a dual-function rAAV3B (df-rAAV3B) construct, which was effective at transducing hepatocytes, resulting in a considerable decrease of Z-AAT levels and safe M-AAT augmentation in mice. We optimized df-rAAV3B and created two variants, AAV3B-E12 and AAV3B-G3, to simultaneously enhance the concentration of M-AAT in the bloodstream to therapeutic levels and silence endogenous AAT liver expression in cynomolgus monkeys. Our results demonstrate that AAV3b-WT, AAV3B-E12, and AAV3B-G3 were able to transduce the monkey livers and achieve high M-AAT serum levels efficiently and safely. In this nondeficient model, we did not find downregulation of endogenous AAT. However, the dual-function vector did serve as a potentially "liver-sparing" alternative for high-dose liver-mediated AAT gene replacement in the context of underlying liver disease.

Liver-directed SERPINA1 gene therapy attenuates progression of spontaneous and tobacco smoke-induced emphysema in α1-antitrypsin null mice.

α1-antitrypsin deficiency is a rare genetic condition that can cause liver and/or lung disease. There is currently no cure for this disorder, although repeated infusions of plasma-purified protein may slow down emphysema progression. Gene therapy in which a single recombinant adeno-associated viral vector (rAAV) administration would lead to sustained protein expression could therefore similarly affect disease progression, and provide the added benefits of reducing treatment burden and thereby improving the patient's quality of life. The study presented here tests whether treating the Serpina1a-e knockout mouse model of α1-antitrypsin-deficiency lung disease with gene therapy would have an impact on the disease course, either on spontaneous disease caused by aging or on accelerated disease caused by exposure to cigarette smoke. Liver-directed gene therapy led to dose-dependent levels of biologically active human α1-antitrypsin protein. Furthermore, decreased lung compliance and increased elastic recoil indicate that treated mice had largely preserved lung tissue elasticity and alveolar wall integrity compared with untreated mice. rAAV-mediated gene augmentation is therefore able to compensate for the loss of function and restore a beneficial lung protease-antiprotease balance. This work constitutes a preclinical study report of a disease-modifying treatment in the Serpina1a-e knockout mouse model using a liver-specific rAAV serotype 8 capsid.

CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro.

A GGGGCC24+ hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), fatal neurodegenerative diseases with no cure or approved treatments that substantially slow disease progression or extend survival. Mechanistic underpinnings of neuronal death include C9ORF72 haploinsufficiency, sequestration of RNA-binding proteins in the nucleus, and production of dipeptide repeat proteins. Here, we used an adeno-associated viral vector system to deliver CRISPR/Cas9 gene-editing machineries to effectuate the removal of the HRE from the C9ORF72 genomic locus. We demonstrate successful excision of the HRE in primary cortical neurons and brains of three mouse models containing the expansion (500-600 repeats) as well as in patient-derived iPSC motor neurons and brain organoids (450 repeats). This resulted in a reduction of RNA foci, poly-dipeptides and haploinsufficiency, major hallmarks of C9-ALS/FTD, making this a promising therapeutic approach to these diseases.

AAV gene therapy for Tay-Sachs disease.

Tay-Sachs disease (TSD) is an inherited neurological disorder caused by deficiency of hexosaminidase A (HexA). Here, we describe an adeno-associated virus (AAV) gene therapy expanded-access trial in two patients with infantile TSD (IND 18225) with safety as the primary endpoint and no secondary endpoints. Patient TSD-001 was treated at 30 months with an equimolar mix of AAVrh8-HEXA and AAVrh8-HEXB administered intrathecally (i.t.), with 75% of the total dose (1 × 1014 vector genomes (vg)) in the cisterna magna and 25% at the thoracolumbar junction. Patient TSD-002 was treated at 7 months by combined bilateral thalamic (1.5 × 1012 vg per thalamus) and i.t. infusion (3.9 × 1013 vg). Both patients were immunosuppressed. Injection procedures were well tolerated, with no vector-related adverse events (AEs) to date. Cerebrospinal fluid (CSF) HexA activity increased from baseline and remained stable in both patients. TSD-002 showed disease stabilization by 3 months after injection with ongoing myelination, a temporary deviation from the natural history of infantile TSD, but disease progression was evident at 6 months after treatment. TSD-001 remains seizure-free at 5 years of age on the same anticonvulsant therapy as before therapy. TSD-002 developed anticonvulsant-responsive seizures at 2 years of age. This study provides early safety and proof-of-concept data in humans for treatment of patients with TSD by AAV gene therapy.

Modulating immune responses to AAV by expanded polyclonal T-regs and capsid specific chimeric antigen receptor T-regulatory cells.

Immune responses to adeno-associated virus (AAV) capsids limit the therapeutic potential of AAV gene therapy. Herein, we model clinical immune responses by generating AAV capsid-specific chimeric antigen receptor (AAV-CAR) T cells. We then modulate immune responses to AAV capsid with AAV-CAR regulatory T cells (Tregs). AAV-CAR Tregs in vitro display phenotypical Treg surface marker expression, and functional suppression of effector T cell proliferation and cytotoxicity. In mouse models, AAV-CAR Tregs mediated continued transgene expression from an immunogenic capsid, despite antibody responses, produced immunosuppressive cytokines, and decreased tissue inflammation. AAV-CAR Tregs are also able to bystander suppress immune responses to immunogenic transgenes similarly mediating continued transgene expression, producing immunosuppressive cytokines, and reducing tissue infiltration. Taken together, AAV-CAR T cells and AAV-CAR Tregs are directed and powerful immunosuppressive tools to model and modulate immune responses to AAV capsids and transgenes in the local environment.

Safe and effective superoxide dismutase 1 silencing using artificial microRNA in macaques.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease caused by degeneration of motor neurons leading to rapidly progressive paralysis. About 10% of cases are caused by gain-of-function mutations that are transmitted as dominant traits. A potential therapy for these cases is to suppress the expression of the mutant gene. Here, we investigated silencing of SOD1, a gene commonly mutated in familial ALS, using an adeno-associated virus (AAV) encoding an artificial microRNA (miRNA) that targeted SOD1 In a superoxide dismutase 1 (SOD1)-mediated mouse model of ALS, we have previously demonstrated that SOD1 silencing delayed disease onset, increased survival time, and reduced muscle loss and motor and respiratory impairments. Here, we describe the preclinical characterization of this approach in cynomolgus macaques (Macaca fascicularis) using an AAV serotype for delivery that has been shown to be safe in clinical trials. We optimized AAV delivery to the spinal cord by preimplantation of a catheter and placement of the subject with head down at 30° during intrathecal infusion. We compared different promoters for the expression of artificial miRNAs directed against mutant SOD1 Results demonstrated efficient delivery and effective silencing of the SOD1 gene in motor neurons. These results support the notion that gene therapy with an artificial miRNA targeting SOD1 is safe and merits further development for the treatment of mutant SOD1-linked ALS.