Current Clinical Applications Of In Vivo Gene Therapy With AAVs Part 2

Solid Biosciences using SGT-001 (ClinicalTrials.gov: NCT03368742) initiated a phase 1/2 open-label clinical trial using AAV9 and a CK8 muscle-specific promoter (SGT-001) targeting skeletal and cardiac muscle, the IGNITE DMD study. 

The relationship between myocardium and memory is a research field that has received much attention in recent years. Myocardium refers to cardiac muscle tissue. As the main part of the heart, it not only plays the role of pumping blood but also has an important impact on human health. Memory is one of the foundations of human consciousness and behavior, and its importance is self-evident. So what is the connection between myocardium and memory?

First of all, there is a close relationship between myocardium and memory. Studies have found that patients with heart disease will experience cognitive dysfunction such as memory loss. This is because heart disease can cause harm to the human body, leading to problems such as poor blood flow or hypoxia in the brain, which affects people's memory. Therefore, keeping the myocardium healthy is of great significance for maintaining normal memory and cognitive function.

Myocardial exercise has a positive effect on improving memory. Exercise can improve the health of the human myocardium, increase the elasticity and flexibility of the myocardium, enhance the heart's ability to pump blood, improve the stability and tolerance of the myocardium, and help the myocardium better transport blood to the brain, thereby maintaining the health of the brain. In addition, exercise can enhance the function of vascular endothelial cells, increase blood flow to the brain, and help promote the growth and repair of brain neurons, thereby improving people's memory.

In summary, there is indeed a certain relationship between myocardium and memory. Therefore, we should always pay attention to the health of our myocardium and actively participate in healthy activities such as exercise to improve the stability and tolerance of the myocardium and to fundamentally maintain our memory and cognitive function. Only in this way can we better enjoy the beauty of life and welcome a better tomorrow. It can be seen that we need to improve our memory. Cistanche can significantly improve memory because Cistanche has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidative and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche can also promote the growth and repair of nerve cells, thereby enhancing the connectivity and function of neural networks. These effects can help improve memory, learning ability, and thinking speed, and can also prevent the occurrence of cognitive dysfunction and neurodegenerative diseases.

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This cassette includes the neuronal nitric oxide synthase (nNOS)-binding domain, encoded by SRs 16/17, to enhance muscle perfusion.29,30 Six subjects have been enrolled and treated to date at the time of this review: low dose (5.0
1013 mg/kg, n = 3) and high dose (2.0
1014 mg/kg, n = 3). 

There have been repeated problems with complement activation resulting in 2 clinical holds by the FDA. The first in 2018 was related to thrombocytopenia. The second was in October 2019 because of more widespread complement activation affecting red blood cells (RBCs) and causing renal damage and cardiopulmonary insufficiency. 

Clinical results for the Solid trial were reported from the first cohort treated at low doses. In a single patient, microdystrophin was detected via WB below the 5% level of quantification and via IF in approximately 10% of fibers. 

Colocalization of nNOS and beta-sarcoglycan were also reported. In the other two subjects, microdystrophin was detected at very minimal levels by IF and none by WB. 

In March 2020, it was announced that 3 months post-treatment, the third patient, dosed at 2
1014 mg/kg, revealed 50%–70% of muscle fibers expressed micro-dystrophin, with WB detection at 8% of normal. The clinical hold for the Solid trial was lifted as of October 1, 2020.

XLMTM

The XLMTM is caused by mutations in the myotubularin 1 (MTM1) gene, located at Xq28.31 Myotubularin, a ubiquitously expressed phosphoinositide phosphatase, functions as a membrane enzyme and plays a role in skeletal muscle development and homeostasis.32 

Mutations in the MTM1, resulting in loss of function, impact the excitation-coupling contraction mechanisms by disrupting the function and organization of the T-tubule network.33 Severe XLMTM, the most common form, presents at birth with hypotonia, external ophthalmoplegia, skeletal muscle weakness, and respiratory insufficiency.34 

Signs of antenatal onset include reduced fetal movements and polyhydramnios. The muscle biopsy shows a uniform appearance of small muscle fibers with large centrally placed nuclei. The RECENSUS natural history study reported nearly all patients requiring respiratory support at birth with a 64% mortality at %18 months of age. 

Approximately 74% of patients surviving >18 months require tracheostomy and mechanical ventilation.34 Disruption of Mtm1 in mice resembles human XLMTM, with similar pathology and early mortality.35 Intramuscular injections of the Mtm1 delivered by AAV rescued muscle function, indicating that restoration of functional myotubularin could ameliorate the disease phenotype.36 

Subsequently, a series of studies from Childers' lab 37–39 showed long-term therapeutic efficacy using systemic administration of an AAV8 vector expressing Mtm1 using the muscle-specific desmin (DES) promoter. 

In the Mtm1-deficient mice, rAAV8.- desmin.Mtm1, at 3
1013 vg/kg corrected muscle pathology and prolonged survival throughout the 6-month study. Low dose was less effective in the mouse. 

The canine model carrying the MTM1 mutation improved function from locoregional vascular delivery. These studies prepared the way for a more extensive study in the canine model with whole-body correction of myotubular myopathy.39 

In a dose-escalating intravenous study of rAAV8.desmin.cMTM1 (0.3
1014, 2
1014, and 5
1014 mg/kg) in the canine model (n = 3 per dose) at 10 weeks of age, with the comparison of saline-treated, age-matched mutants and normal littermates as controls, the two higher doses led to a reversal of the disease with clinical findings indistinguishable from normal without additional safety concerns. 

Together, these preclinical studies showed the feasibility, safety, and efficacy of gene therapy with AAV8 for long-term correction of XLMTM.37,38 The ASPIRO trial is a phase 1/2 open-label, randomized, ascending-dose study evaluating the safety and efficacy of AT132 (resamirigene bilparvovec). 

In this two-part gene-transfer study, a single intravenous dose is delivered in part 1, assessing two doses for safety and efficacy: 1
1014 mg/kg and 3
1014 mg/kg (ClinicalTrials.gov: NCT03199469). In part 2 of the study, eight subjects are randomized to either a treatment arm at 3
1014 mg/kg or to a delayed treatment control arm. 

As of August 2019, 12 patients were enrolled in the study with six (cohort 1) receiving a low dose and cohort 2 (n = 4) treated at a high dose compared to untreated controls. 

Preliminary results shared by Audentes showed a favorable response in safety and efficacy and follow-up ranging between 4 and 72 weeks. All treated subjects but one had a clinically meaningful improvement in respiratory function, with a reduction in daily hours of ventilatory requirement as well as an improvement in the mean inspiratory pressure (MIP). 

A positive response in motor function measured by CHOP-INTEND was also seen. Muscle biopsies available for 9 subjects demonstrated robust dose-dependent tissue transduction and myotubularin protein expression with an improvement in overall muscle histology. 

This contrasts with natural history data wherein XLMTM subjects had a 2.7-point annual decline from baseline in CHOP-INTEND scores and a reduction in MIP over 12 months.40 In 2018, following an acknowledgment of the progress made in this trial, the FDA granted AT132 the regenerative medicine advanced therapy (RMAT) designation, equivalent to the FDA's Fast Track and Breakthrough Therapy. 

Unfortunately, as the trial appeared to be proceeding favorably, in May 2020, Audentes, the trial sponsor, reported in a letter to patient groups that a participant in the trial died after receiving AT132 at the higher dose of 3
1014 mg/kg. Subsequently, in June 2020, Audentes shared further details when a second of 3 in the high-dose cohort also died. 

The two deaths followed a similar clinical course, each suffering from progressive liver disease 46 weeks after receiving the gene therapy. The direct cause of death was generalized sepsis following the liver complications. 

None of the six patients who received the lower dose experienced serious AEs (SAEs), despite four of them having a history of hepatobiliary disease. Notable features among the three patients with these SAEs include older age, heavier weight, evidence of pre-existing hepatobiliary disease, and dosing with the higher dose of 3
1014 mg/kg. 

The XLMTM trial is now on clinical hold by the FDA and searching for further explanation. Active data collection is ongoing. In conclusion, in this review, we highlight gene-therapy clinical trials in three neuromuscular disorders to illustrate the potential for the broader application of systemic delivery. Undoubtedly, significant advances have been made in systemic gene delivery; however, several limitations remain to be resolved. 

The efficacy and safety of each disease require further analysis and long-term follow-up. The severe complications in the XLMTM trial require further study.

Gene Therapy for CNS Diseases

Gene therapy for CNS diseases using AAV vectors was initiated nearly 2 decades ago using stereotaxic intracerebral delivery of AAV2 vectors. Early pioneering clinical trials were for CD, AD, and PD. 

Whereas the approaches for AD and PD were focal bilateral injections, the clinical trial for CD attempted stereotaxic AAV2 administration to 12 distinct sites to maximize vector distribution throughout the brain. 

AADC deficiency disorder followed, again using AAV2, and was a pivotal demonstration of disease-altering efficacy in a CNS gene therapy trial. These early CNS gene therapy trials demonstrated the feasibility of AAV-based gene transfer to treat the CNS safely.

AD

AD is a neurodegenerative disorder that is the leading cause of age-related dementia. A deep understanding of AD and the associated neuropathology has led to the development of numerous viral-mediated gene-transfer approaches for AD, as reviewed by Raikwar et al.41 

One approach that has entered clinical trials is the delivery of nerve growth factor (NGF), which is hypothesized to promote the survival of cholinergic neurons.42,43 A phase 1 study of bilateral intracerebral delivery AAV2-NGF to the basal forebrain of patients with mild to moderate AD-associated dementia showed promising results. 

The surgical delivery was safe and well tolerated, and there was a lack of clinically relevant progression of disease 2 years postinjection.44 However, in the subsequent randomized controlled phase 2 study (n = 49), efficacy endpoints were not met.45 Despite this early failure, there are numerous additional applications in the gene therapy pipeline for AD.

PD

PD is a neurodegenerative movement disorder characterized by bradykinesia, gait impairment, and later cognitive decline that is caused by a loss of dopaminergic neurons in the basal ganglia. 

Viral-mediated gene therapy approaches aimed at modulating GABAergic neuronal signaling (AAV2-GAD [AAV2-glutamic acid decarboxylase]) and increasing dopamine production (AAV2-hAADC, amino acid decarboxylase) have been studied in early-phase clinical trials. 

The initial open-label, dose-escalation phase 1 study explored the delivery of AAV-GAD injected unilaterally into the subthalamic nucleus of 12 PD patients. There were no treatment-related AEs, and all subjects demonstrated improvements in motor functioning with diminished thalamic metabolisms within the treated hemisphere.46 

In the phase 2 randomized controlled trial, patients with advanced PD received either a sham procedure (n = 23) or bilateral infusion of AAV2-GAD to the subthalamic nuclei (n = 22). 

The procedure was again well tolerated, and patients in the treatment group demonstrated improvement in motor function.47 These results were sustained out to 12 months postinjection.48 

It is postulated that delivery of GAD to this brain region leads to the formation of new functional pathways between the subthalamic neurons and motor cortical regions, termed "GAD-related pathways," which correlate to clinical improvements in the treatment group.49 

In an alternative approach, AADC, an enzyme involved in the synthesis of dopamine, is delivered to the putamen of PD patients, to increase dopamine production. In a phase 1 study of bilateral intrastriatal infusion of AAV-hAADC in five patients with moderately advanced PD, there was a 30% increase in AADC expression in the putamen and a modest clinical improvement in patients.50 

A subsequent dose-escalation study demonstrated similar results with a 30% increase in AADC expression on positron emission tomography (PET) scan in the low-dose cohort and a 75% increase in the high-dose cohort.51 

In this study, three patients had a postoperative intracranial hemorrhage (one symptomatic, two asymptomatic).51 In long-term follow-up, effects persisted for up to 96 weeks.52 Most recently, in phase 1 gene-delivery optimization study, gadoteridol is coadministered with AAV2-AADC via MRI-guided infusion to the putamen to facilitate visualization of the vector spread and coverage. 

In this three-level dose-escalation study (n = 5 per dosing level), there was a dose-responsive increase in AADC activity ranging from 13% to 79% that correlated to improvements in motor outcomes, including increased response to levodopa without dyskinesia.53 Both approaches demonstrated favorable safety profiles and promising clinical response, but larger, well-controlled studies are needed.

CD

CD is a leukodystrophy caused by pathogenic variants of the aspartoacylase gene (ASPA). ASPA is expressed by oligodendrocytes and is responsible for the degradation of N-acetyl aspartate (NAA) through deacetylation. 

Elevations of NAA in the CNS have variable downstream effects that may explain the underlying pathophysiology of CD, including aberrant myelination, parenchymal edema, and vacuolation of the white matter (reviewed by Leone et al.54). 

The clinical presentation of CD varies based on the residual activity levels of ASPA and corresponding concentrations of NAA. It is a neurodegenerative disorder with hydrocephalus due to progressive spongy neurodegeneration, progressive neurologic disability, intractable epilepsy, feeding intolerance, and premature death in adolescence or early adulthood. 

The AAV2 approach evolved from earlier studies of gene transfer using a lipid-entrapped polycation-condensed delivery system (LPD) in conjunction with AAV-based plasmids containing rASPA.55 

Whereas this approach was well tolerated and led to both biochemical and clinical improvements, improved viral-mediated gene-transfer technologies using AAV2 were studied in a phase 1 study of intracranial infusions via six cranial burr holes in patients with CD (n = 13).56 The procedure was well tolerated with only minimal systemic inflammation and demonstrated, promising long-term safety and clinical efficacy. 

Global CNS concentrations of NAA, as measured by magnetic resonance (MR) spectroscopy, were decreased after AAV2-ASPA delivery, especially in the basal ganglia. 

Similarly, the T1 relaxation time decreased in white-matter tracts, especially the splenium of the corpus callosum, and some patients demonstrated a halting or reversal of brain atrophy. 

Finally, improvement in these biochemical and imaging biomarkers was accompanied by stabilization to improvements in gross motor functioning, although social and cognitive recovery was less consistent. 

The reversal of biochemical and structural biomarkers suggests that the process is reversible; however, earlier intervention is suspected to lead to a more robust clinical response.54 New technologies using AAV-ASPA targeting oligodendrocytes are now being studied.57 

AADC Deficiency

After promising results in the AAV2-AADC studies for PD, investigators sought to apply this technology to a rare neurogenetic disorder of childhood. AADC deficiency disorder is a rare inherited disorder of neurotransmitter synthesis caused by biallelic variants in the dopa decarboxylase (DDC) gene on chromosome 7.58 

Although the clinical spectrum can vary, 80% of patients are classified as severe and present in infancy with hypotonia, growth retardation, and marked motor deficits. 

They never attain proper head control or the ability to sit independently and suffer from frequent episodes of dystonia and oculogyric crises. Autonomic dysfunction and severe emotional irritability are frequently reported.58 

Individuals typically die by the age of 5 years old.59 In 2012, the initial gene-transfer compassionate-use clinical trial of AAV2/hAADC was performed in Taiwan. Hwu et al.59 selected four subjects with confirmed diagnoses of AADC from the twenty known living patients in Taiwan. 

On baseline assessment, all patients were bedridden, lacked head control, and were unable to speak. Oculogyric crises were reported every 2 to 3 days, and caregivers reported marked irritability, excessive sweating, and unstable body temperatures. Patients received AAV2/hAADC by direct bilateral intraluminal injection and followed for up to 24 months. 

Transient dyskinesias sufficient to interfere with feeding were reported in two of four subjects, and one subject had significant apneic events that subsided within 10 months of dosing. 

All four subjects demonstrated improvements across both motor and cognitive developmental outcomes, and caregivers reported a decrease in the frequency and intensity of oculogyric crises, diminished irritability, and increased temperature stability. 

Further, biomarker data were compelling, demonstrating a 45% to 86% increase in uptake of dopamine on PET scan as compared to baseline measurements, and all demonstrated an increase in cerebrospinal fluid (CSF) levels of dopamine and serotonin metabolites.59 

In the first phase 1/2, an open-label clinical trial at the National Taiwan University Hospital (Taipei, Taiwan), an additional ten children, ages 24 months and up, with confirmed AADC deficiency diagnoses were dosed with intraluminal AAV2-hAADC. 

One subject died from an unrelated cause, but the remaining survivors demonstrated remarkable improvements in motor functioning (an increase of 62 points on the Peabody Developmental Motor Scale). 

Reported AEs included pyrexia and transient orofacial dyskinesia that resolved with risperidone.60 Similar results were found in a second open-label phase 1/2 study of a more genetically diverse population (n = 6).61 

Within 2 months, all had improvement in their voluntary movements, two were weaned off mechanical ventilation, four regained the ability to eat by mouth, and all showed improvements in dystonic episodes, irritability, and autonomic dysfunction.61

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