Intrathecal Gene Therapy for Giant Axonal Neuropathy.

BACKGROUND: Giant axonal neuropathy is a rare, autosomal recessive, pediatric, polysymptomatic, neurodegenerative disorder caused by biallelic loss-of-function variants in GAN, the gene encoding gigaxonin. METHODS: We conducted an intrathecal dose-escalation study of scAAV9/JeT-GAN (a self-complementary adeno-associated virus-based gene therapy containing the GAN transgene) in children with giant axonal neuropathy. Safety was the primary end point. The key secondary clinical end point was at least a 95% posterior probability of slowing the rate of change (i.e., slope) in the 32-item Motor Function Measure total percent score at 1 year after treatment, as compared with the pretreatment slope. RESULTS: One of four intrathecal doses of scAAV9/JeT-GAN was administered to 14 participants - 3.5×1013 total vector genomes (vg) (in 2 participants), 1.2×1014 vg (in 4), 1.8×1014 vg (in 5), and 3.5×1014 vg (in 3). During a median observation period of 68.7 months (range, 8.6 to 90.5), of 48 serious adverse events that had occurred, 1 (fever) was possibly related to treatment; 129 of 682 adverse events were possibly related to treatment. The mean pretreatment slope in the total cohort was -7.17 percentage points per year (95% credible interval, -8.36 to -5.97). At 1 year after treatment, posterior mean changes in slope were -0.54 percentage points (95% credible interval, -7.48 to 6.28) with the 3.5×1013-vg dose, 3.23 percentage points (95% credible interval, -1.27 to 7.65) with the 1.2×1014-vg dose, 5.32 percentage points (95% credible interval, 1.07 to 9.57) with the 1.8×1014-vg dose, and 3.43 percentage points (95% credible interval, -1.89 to 8.82) with the 3.5×1014-vg dose. The corresponding posterior probabilities for slowing the slope were 44% (95% credible interval, 43 to 44); 92% (95% credible interval, 92 to 93); 99% (95% credible interval, 99 to 99), which was above the efficacy threshold; and 90% (95% credible interval, 89 to 90). Between 6 and 24 months after gene transfer, sensory-nerve action potential amplitudes increased, stopped declining, or became recordable after being absent in 6 participants but remained absent in 8. CONCLUSIONS: Intrathecal gene transfer with scAAV9/JeT-GAN for giant axonal neuropathy was associated with adverse events and resulted in a possible benefit in motor function scores and other measures at some vector doses over a year. Further studies are warranted to determine the safety and efficacy of intrathecal AAV-mediated gene therapy in this disorder. (Funded by the National Institute of Neurological Disorders and Stroke and others; ClinicalTrials.gov number, NCT02362438.).

AAV9-mediated FIG4 delivery prolongs life span in Charcot-Marie-Tooth disease type 4J mouse model.

Charcot-Marie-Tooth disease type 4J (CMT4J) is caused by recessive, loss-of-function mutations in FIG4, encoding a phosphoinositol(3,5)P2-phosphatase. CMT4J patients have both neuron loss and demyelination in the peripheral nervous system, with vacuolization indicative of endosome/lysosome trafficking defects. Although the disease is highly variable, the onset is often in childhood and FIG4 mutations can dramatically shorten life span. There is currently no treatment for CMT4J. Here, we present the results of preclinical studies testing a gene-therapy approach to restoring FIG4 expression. A mouse model of CMT4J, the Fig4-pale tremor (plt) allele, was dosed with a single-stranded adeno-associated virus serotype 9 (AAV9) to deliver a codon-optimized human FIG4 sequence. Untreated, Fig4plt/plt mice have a median survival of approximately 5 weeks. When treated with the AAV9-FIG4 vector at P1 or P4, mice survived at least 1 year, with largely normal gross motor performance and little sign of neuropathy by neurophysiological or histopathological evaluation. When mice were treated at P7 or P11, life span was still significantly prolonged and peripheral nerve function was improved, but rescue was less complete. No unanticipated adverse effects were observed. Therefore, AAV9-mediated delivery of FIG4 is a well-tolerated and efficacious strategy in a mouse model of CMT4J.

Gene Transfer Therapy for Neurodevelopmental Disorders.

Neurodevelopmental disorders (NDDs) include a broad spectrum of disorders that disrupt normal brain development. Though some NDDs are caused by acquired insults (i.e., toxic or infectious encephalopathy) or may be cryptogenic, many NDDs are caused by variants in a single gene or groups of genes that disrupt neuronal development or function. In this review, we will focus on those NDDs with a genetic etiology. The exact mechanism, timing, and progression of the molecular pathology are seldom well known; however, the abnormalities in development typically manifest in similar patterns such as delays or regression in motor function, social skills, and language or cognitive abilities. Severity of impairment can vary widely. At present, only symptomatic treatments are available to manage seizures and behavioral problems commonly seen in NDDs. In recent years, there has been a rapid expansion of research into gene therapy using adeno-associated viruses (AAVs). Using AAVs as vectors to replace the non- or dysfunctional gene in vivo is a relatively simple model which has created an unprecedented opportunity for the future of NDD treatment. Advances in this field are of paramount importance as NDDs lead to a massive lifelong burden of disease on the affected individuals and families. In this article, we review the unique advantages and challenges of AAV gene therapies. We then look at potential applications of gene therapy for 3 of the more common NDDs (Rett syndrome, fragile X syndrome, and Angelman syndrome), as well as 2 less common NDDs (SLC13A5 deficiency disorder and SLC6A1-related disorder). We will review the available natural history of each disease and current state of preclinical studies including a discussion on the application of AAV gene therapies for each disease.

Comparison of high-dose intracisterna magna and lumbar puncture intrathecal delivery of AAV9 in mice to treat neuropathies.

Gene therapy clinical trials for neurological disorders are ongoing using intrathecal injection of adeno-associated virus (AAV) vector directly into the cerebral spinal fluid. Preliminary findings from these trials and results from extensive animal studies provides compelling data supporting the safety and benefit of intrathecal delivery of AAV vectors for inherited neurological disorders. Intrathecal delivery can be achieved by a lumbar puncture (LP) or intracisterna magna (ICM) injection, although ICM is not commonly used in clinical practice due to increased procedural risk. Few studies directly compared these delivery methods and there are limited reports on transduction of the PNS. To further test the utility of ICM or LP delivery for neuropathies, we performed a head to head comparison of AAV serotype 9 (AAV9) vectors expressing GFP injected into the cisterna magna or lumbar subarachnoid space in mice. We report that an intrathecal gene delivery of AAV9 in mice leads to stable transduction of neurons and glia in the brain and spinal cord and has a widespread distribution that includes components of the PNS. Vector expression was notably higher in select brain and PNS regions following ICM injection, while higher amounts of vector was found in the lower spinal cord and peripheral organs following LP injection. These findings support that intrathecal AAV9 delivery is a translationally relevant delivery method for inherited neuropathies.

Development of Intrathecal AAV9 Gene Therapy for Giant Axonal Neuropathy.

An NIH-sponsored phase I clinical trial is underway to test a potential treatment for giant axonal neuropathy (GAN) using viral-mediated GAN gene replacement (https://clinicaltrials.gov/ct2/show/NCT02362438). This trial marks the first instance of intrathecal (IT) adeno-associated viral (AAV) gene transfer in humans. GAN is a rare pediatric neurodegenerative disorder caused by autosomal recessive loss-of-function mutations in the GAN gene, which encodes the gigaxonin protein. Gigaxonin is involved in the regulation, turnover, and degradation of intermediate filaments (IFs). The pathologic signature of GAN is giant axonal swellings filled with disorganized accumulations of IFs. Herein, we describe the development and characterization of the AAV vector carrying a normal copy of the human GAN transgene (AAV9/JeT-GAN) currently employed in the clinical trial. Treatment with AAV/JeT-GAN restored the normal configuration of IFs in patient fibroblasts within days in cell culture and by 4 weeks in GAN KO mice. IT delivery of AAV9/JeT-GAN in aged GAN KO mice preserved sciatic nerve ultrastructure, reduced neuronal IF accumulations and attenuated rotarod dysfunction. This strategy conferred sustained wild-type gigaxonin expression across the PNS and CNS for at least 1 year in mice. These results support the clinical evaluation of AAV9/JeT-GAN for potential therapeutic outcomes and treatment for GAN patients.

Methods for gene transfer to the central nervous system.

Gene transfer is an increasingly utilized approach for research and clinical applications involving the central nervous system (CNS). Vectors for gene transfer can be as simple as an unmodified plasmid, but more commonly involve complex modifications to viruses to make them suitable gene delivery vehicles. This chapter will explain how tools for CNS gene transfer have been derived from naturally occurring viruses. The current capabilities of plasmid, retroviral, adeno-associated virus, adenovirus, and herpes simplex virus vectors for CNS gene delivery will be described. These include both focal and global CNS gene transfer strategies, with short- or long-term gene expression. As is described in this chapter, an important aspect of any vector is the cis-acting regulatory elements incorporated into the vector genome that control when, where, and how the transgene is expressed.

Akt and CHIP coregulate tau degradation through coordinated interactions.

A hallmark of the pathology of Alzheimer's disease is the accumulation of the microtubule-associated protein tau into fibrillar aggregates. Recent studies suggest that they accumulate because cytosolic chaperones fail to clear abnormally phosphorylated tau, preserving a pool of toxic tau intermediates within the neuron. We describe a mechanism for tau clearance involving a major cellular kinase, Akt. During stress, Akt is ubiquitinated and degraded by the tau ubiquitin ligase CHIP, and this largely depends on the Hsp90 complex. Akt also prevents CHIP-induced tau ubiquitination and its subsequent degradation, either by regulating the Hsp90/CHIP complex directly or by competing as a client protein with tau for binding. Akt levels tightly regulate the expression of CHIP, such that, as Akt levels are suppressed, CHIP levels also decrease, suggesting a potential stress response feedback mechanism between ligase and kinase activity. We also show that Akt and the microtubule affinity-regulating kinase 2 (PAR1/MARK2), a known tau kinase, interact directly. Akt enhances the activity of PAR1 to promote tau hyperphosphorylation at S262/S356, a tau species that is not recognized by the CHIP/Hsp90 complex. Moreover, Akt1 knockout mice have reduced levels of tau phosphorylated at PAR1/MARK2 consensus sites. Hence, Akt serves as a major regulator of tau biology by manipulating both tau kinases and protein quality control, providing a link to several common pathways that have demonstrated dysfunction in Alzheimer's disease.

Organic anion transporter 3 (Oat3/Slc22a8) knockout mice exhibit altered clearance and distribution of penicillin G.

The interaction of renal basolateral organic anion transporter 3 (Oat3) with commonly used pharmacotherapeutics (e.g., NSAIDs, beta-lactams, and methotrexate) has been studied extensively in vitro. However, the in vivo role of Oat3 in drug disposition, in the context of other transporters, glomerular filtration, and metabolism, has not been established. Moreover, recent investigations have identified inactive human OAT3 polymorphisms. Therefore, this investigation was designed to elucidate the in vivo role of Oat3 in the disposition of penicillin G and prototypical substrates using an Oat3 knockout mouse model. Oat3 deletion resulted in a doubling of penicillin's half-life (P < 0.05) and a reduced volume of distribution (P < 0.01), together yielding a plasma clearance that was one-half (P < 0.05, males) to one-third (P < 0.001, females) of that in wild-type mice. Inhibition of Oat3 abolished the differences in penicillin G elimination between genotypes. Hepatic accumulation of penicillin was 2.3 times higher in male knockouts (P < 0.05) and 3.7 times higher in female knockouts (P < 0.001). Female knockouts also exhibited impaired estrone-3-sulfate clearance. Oat3 deletion did not impact p-aminohippurate elimination, providing correlative evidence to studies in Oat1 knockout mice that suggest Oat1 governs tubular uptake of p-aminohippurate. Collectively, these findings are the first to indicate that functional Oat3 is necessary for proper elimination of xenobiotic and endogenous compounds in vivo. Thus Oat3 plays a distinct role in determining the efficacy and toxicity of drugs. Dysfunctional human OAT3 polymorphisms or instances of polypharmacy involving OAT3 substrates may result in altered systemic accumulation of beta-lactams and other clinically relevant compounds.

The prolyl hydroxylase oxygen-sensing pathway is cytoprotective and allows maintenance of mitochondrial membrane potential during metabolic inhibition.

The cellular oxygen sensor is a family of oxygen-dependent proline hydroxylase domain (PHD)-containing enzymes, whose reduction of activity initiate a hypoxic signal cascade. In these studies, prolyl hydroxylase inhibitors (PHIs) were used to activate the PHD-signaling pathway in cardiomyocytes. PHI-pretreatment led to the accumulation of glycogen and an increased maintenance of ATP levels in glucose-free medium containing cyanide. The addition of the glycolytic inhibitor 2-deoxy-d-glucose (2-DG) caused a decline of ATP levels that was indistinguishable between control and PHI-treated myocytes. Despite the comparable levels of ATP depletion, PHI-preconditioned myocytes remained significantly protected. As expected, mitochondrial membrane potential (DeltaPsi(mito)) collapses in control myocytes during cyanide and 2-DG treatment and it fails to completely recover upon washout. In contrast, DeltaPsi(mito) is partially maintained during metabolic inhibition and recovers completely on washout in PHI-preconditioned cells. Inclusion of rotenone, but not oligomycin, with cyanide and 2-DG was found to collapse DeltaPsi(mito) in PHI-pretreated myocytes. Thus, continued complex I activity was implicated in the maintenance of DeltaPsi(mito) in PHI-treated myocytes, whereas a role for the "reverse mode" operation of the F(1)F(0)-ATP synthase was ruled out. Further examination of mitochondrial function revealed that PHI treatment downregulated basal oxygen consumption to only approximately 15% that of controls. Oxygen consumption rates, although initially lower in PHI-preconditioned myocytes, recovered completely upon removal of metabolic poisons, while reaching only 22% of preinsult levels in control myocytes. We conclude that PHD oxygen-sensing mechanism directs multiple compensatory changes in the cardiomyocyte, which include a low-respiring mitochondrial phenotype that is remarkably protected against metabolic insult.