No abstract

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.

Tay-Sachs Disease (TSD) is an inherited neurological disorder caused by deficiency of hexosaminidase A (HexA). Preclinical work demonstrated safety and efficacy of CNS gene therapy using AAVrh8-HEXA/HEXB. Here we describe an expanded access trial in two patients with infantile TSD (IND 18225).Case TSD-001 demonstrated neurodevelopmental regression by 8 months of age and severe seizures by 1 year was treated at 30 months. An equimolar mix of AAVrh8-HEXA and AAVrh8-HEXB (now AXO-AAV-GM2) was administered intrathecally (IT), with 75% of the dose (1x10 14 vg) delivered to the cisterna magna and 25% at the thoraco-lumbar junction. The second patient (TSD-002) was treated at 7 months of age with 4•2x10 13 vg by a combination of bilateral thalamic (0•18 mL; 1•5x10 12 vg per thalamus), and IT infusion (3•9x10 13 vg). Both patients underwent immunosuppression with sirolimus, corticosteroids, and rituximab.Injection procedures were well tolerated and have shown no vector-related adverse events to date. CSF HexA activity nearly doubled from baseline and remained stable. In TSD-002 (now 16 months of age), MRI showed stabilization of disease by 3 months post-injection; there now appeared to temporarily deviate from the natural history of infantile TSD but declined again 6 months post-treatment. TSD-001 (now 4.5 years of age remains seizure-free on the same anticonvulsant therapy as pre-therapy, but TSD-002 developed seizures between 13 and 17 months post-treatment (by 2 years of age).Administration of AXO-AAV-GM2 by IT and thalamic injections was safe, HexA activity increased in CSF and ongoing myelination was apparent in the younger patient treated at an early symptomatic stage. This study provides early safety and proof-of-concept in humans for treatment of TSD patients by AAV gene therapy.

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 muscledirected 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.

No abstract

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.

Tay-Sachs Disease (TSD) is an inherited neurological disorder caused by deficiency of hexosaminidase A (HexA). Preclinical work demonstrated safety and efficacy of CNS gene therapy using AAVrh8-HEXA/HEXB. Here we describe an expanded access trial in two patients with infantile TSD (IND 18225). Case TSD-001 demonstrated neurodevelopmental regression by 8 months of age and severe seizures by 1 year was treated at 30 months. An equimolar mix of AAVrh8-HEXA and AAVrh8-HEXB (now AXO-AAV-GM2) was administered intrathecally (IT), with 75% of the dose (1x1014vg) delivered to the cisterna magna and 25% at the thoraco-lumbar junction. The second patient (TSD-002) was treated at 7 months of age with 4.2x1013 vg by a combination of bilateral thalamic (0.18 mL; 1.5x1012vg per thalamus), and IT infusion (3.9x1013vg). Both patients underwent immunosuppression with sirolimus, corticosteroids, and rituximab. Injection procedures were well tolerated and have shown no vector-related adverse events to date. CSF HexA activity nearly doubled from baseline and remained stable. In TSD-002 (now 16 months of age), MRI showed stabilization of disease by 3 months post-injection and appeared to temporarily deviate from the natural history of infantile TSD but declined again 6 months post-treatment. TSD-001 (now 4.5 years of age remains seizure-free on the same anti-convulsant therapy as pre-therapy, but TSD-002 developed seizures between 13 and 17 months posttreatment (by 2 years of age). Administration of AXO-AAV-GM2 by IT and thalamic injections was safe, HexA activity increased in CSF and ongoing myelination was apparent in the younger patient treated at an early symptomatic stage. This study provides early safety and proof-of-concept in humans for treatment of TSD patients by AAV gene therapy.

No abstract