Advances in gene therapy technology are showing increasing promise for the effective treatment of rare neurological disorders. The most recent and notable demonstration of this is the FDA approval of Zolgensma for spinal muscular atrophy (SMA), with more advancements on the horizon for other disorders such as Rett syndrome (RTT), a rare neurodevelopmental disorder that exhibits X chromosome linkage and is the predominant cause of severe mental retardation in girls. Incidence is approximately 1 in 10,000 female births worldwide.1 The disease is characterized by developmental regression, with symptoms typically becoming apparent by the age of 3 months.

Caused by loss-of-function mutations in a transcription regulator gene that encodes methyl CpG-binding protein 2 (MECP2), currently no effective treatment exists for RTT.

In the case of RTT, the therapeutic potential of viral vector–mediated MECP2 gene transfer has been demonstrated in mouse models during preclinical trials.2 Many preclinical studies involving other rare neurological diseases show similar promise, and a few clinical trials have demonstrated, for the first time, real therapeutic benefit for patients.

Vectors for Gene Therapy Solutions

Several viral vectors have been investigated as possible mechanisms for transgene expression in gene therapy solutions, including adeno-viral vectors, adeno-associated viral (AAV) vectors, and retroviral vectors such as lentiviral vectors. “Viral vectors are efficient vehicles to deliver genes for replacing or silencing a defective gene,” Vibha Jawa, PhD, director of Risk Assessment and Clinical Immunogenicity Strategy for Biologics and Vaccines at Merck, told NeurologyLive. “The capsids of viral vectors have a good tropism for most of the cells in the central nervous system (CNS) and can efficiently deliver the gene to the cell and express the protein of interest.”

AAV vectors are established as the most promising gene therapy platforms for the treatment of rare neurological disease.3 According to Mariana Bravo-Hernández, PhD, a postdoctoral researcher at the University of California, San Diego, “The [major] advantages of the AAV vectors versus the lentivirus are: the high titers that you can obtain; the availability of different serotypes that allow the transduction of specific cell types in the CNS; that AAV viruses do not integrate in the host genome and allow for long-term gene expression; that they are not associated with any human disease; and that they produce a minimal immune response.”

A large body of evidence has accumulated to demonstrate the ability of AAV vectors to introduce transgenes to the CNS effectively4:
• Various routes of administration have been shown to achieve therapeutic levels of gene expression.
• Transgenes introduced thus far have been shown to successfully encode a range of products, including therapeutic proteins, microRNAs, and antibodies.
• Gene-editing machinery has also been incorporated within AAV vectors.
• Clinical studies have demonstrated encouraging safety and efficacy, as well as the durability of transgene expression.

Perhaps the most exciting recent development has been the ability to engineer novel AAV capsids with an unprecedented capacity to transfer genes to the CNS of animal models following standard administrations. Research shows a more than 40-fold enhancement relative to the AAV9 capsid, the current “gold standard” for CNS gene delivery.5

Clinical Success Stories

Spinal Muscular Atrophy (SMA)
SMA is inherited as an autosomal recessive trait. It is a neurodegenerative disorder with an incidence of 1 in 10,000 births and is caused by mutations of the survival motor neuron 1 (SMN1) gene.6 The condition is associated with progressive loss of motor neurons of the brainstem and spinal cord, and it is characterized by muscular atrophy.

Four subtypes of SMA (types 1-4) are recognized, based upon motor milestone achievement and the age at which symptoms become apparent. The phenotype is dependent on the number of copies of a modifier gene, the survival motor neuron 2 (SMN2) gene. This is similar to SMN1 but encodes the production of a small but functionally inadequate fraction of SMN protein. SMA type 1, the most severe form of the disease, accounts for 60% of all cases.6 Children with SMA type 1 often die from respiratory complications within the first 2 years of life.

A recent open-label study assessed the health outcomes of 12 patients with homozygous deletions of SMN1 and 2 copies of SMN2. Patients were given a single intravenous infusion of an AAV9 vector called Zolgensma (AveXis), which contains the human survival motor neuron gene. The outcomes included decreased respiratory and nutritional complications, improved motor milestone achievements, and fewer hospitalizations.7

Recently reported data from the Zolgensma clinical trial program show a clinically transformative impact across a broad spectrum of patients with SMA.

“These include the first interim data from the phase 1 STRONG trial in SMA type 2, new interim data from the phase 3 STR1VE study in SMA type 1, preliminary data from the phase 3 SPR1NT trial in presymptomatic SMA patients, and data from the ongoing long-term follow-up study for the phase 1 START trial in SMA type 1,” AveXis representative Farah Speer shared. “These data are part of a growing body of evidence that supports the use of Zolgensma as the foundational therapy for the treatment of SMA.”

As a result of that data, the FDA recently approved Zolgensma in May 2019 for the treatment of SMA in patients <2 years of age.

Aromatic L-Amino Acid Decarboxylase (AADC) Deficiency
AADC deficiency is an inherited disorder in which symptoms typically present within the first year of life and can result in mortality before age 10 years.8 The condition is caused by mutations in the AADC gene, which is necessary to manufacture dopamine and serotonin. Currently, no approved therapies exist to treat AADC deficiency, and best practice is to inhibit monoamine oxidase and to directly stimulate dopamine receptors using dopamine agonists. However, this treatment has little benefit for patients.

A recent open-label phase 1/2 clinical study describes therapeutic benefits arising from a novel gene therapy treatment for AADC deficiency.9 The study involved 6 adolescent patients, all of whom received AGIL-AADC, an AAV vector harboring the DDC gene that is delivered with bilateral intraputaminal infusions. Motor function was remarkably improved for up to 2 years following treatment. Positron emission tomography using a specific tracer for AADC showed increased uptake of the enzyme within the putamen. Further, the restoration of dopamine synthesis in the putamen provided transformative medical benefit across all patients.

Current Preclinical and Clinical Trials

Mucopolysaccharidosis (MPS)
MPS is a group of lysosomal storage disorders, almost all of which are inherited as recessive autosomal traits; the exception is MPS II, which shows X chromosome linkage. The total incidence of all forms has been estimated to be 1 in 25,000 births.10 Three forms of MPS are particularly promising as targets for gene therapy solutions: MPS I, MPS II, and MPS III (types A and B).

MPS III, for example, has 4 subtypes (A, B, C, and D) distinguished by deficiencies in different enzymes. For MPS IIIA, preclinical studies have shown that systemic delivery of an AAV9 vector encoding human N-sulfoglucosamine sulfohydrolase resulted in the elevation of lysosomal enzyme activity in MPS IIIA mouse models. Behavioral performance and survival rate improved; improvements were most pronounced when the vector was administered at an early stage, but phenotypes were also partially corrected at intermediate disease progressions.11 

Intravenous administration of an AAV9 vector containing the gene for human α-N-acetylglucosaminidase has also been shown to correct glycosaminoglycan accumulation within the CNS and somatic tissues in MPS IIIB mouse models. This treatment also provides long-term neurological improvements and correction of metabolomic impairments corresponding to functional benefit (TABLE).12
Duchenne Muscular Dystrophy (DMD)
DMD is a genetic disorder characterized by debilitating and progressive muscle weakness caused by recessive X-linked mutations in the DMD gene, preventing the production of functional dystrophin. Symptoms of the disease, which almost exclusively affects males with a prevalence of 1 in 3500 to 5000 live male births, typically become apparent in early childhood, with loss of ambulation in adolescence.13 Beyond progressive muscle wasting, patients may also experience respiratory, orthopedic, and cardiac complications.

Solid Biosciences is currently conducting a phase 1/2 dose-ascending clinical trial (IGNITE DMD, NCT03368742) evaluating the safety and efficacy of SGT-001 microdystrophin gene transfer therapy in this population. SGT-001 is a novel, AAV9 vector-mediated gene transfer that delivers microdystrophin, a synthetic dystrophin transgene, that encodes for a functional protein surrogate expressed in muscles. Preclinical studies showed that SGT-001 has the potential to slow or halt disease progression regardless of mutation or disease stage. The current trial will ultimately enroll 16 patients aged 4 to 17 years who will be randomly assigned to an active treatment group or delayed treatment control group (TABLE).

Preliminary interim results announced earlier this year14 demonstrated low levels of microdystrophin protein expression in muscle fibers in patients who received the lowest outlined dose in the study protocol. With the safety profile remaining positive, the investigators plan to expedite the planned dose escalation strategy so that efficacy can be evaluated at higher doses.

Similarly, Sarepta Therapeutics is conducting a phase 2 clinical trial (NCT03769116) of its exogenous gene therapy SRP-9001, which utilizes the unique AAV vector AAVrh74 to transport the transgene that will express functional microdystrophin. The trial will evaluate the safety and efficacy of a single intravenous infusion of SRP-9001 in 24 male patients age 4 to 7 with an established diagnosis of DMD. The trial will consist of a 48-week randomized, double-blind, placebo-controlled period and a 96-week, double-blind, extension period (TABLE).

Interim results of an earlier phase 1/2a trial presented in June 2018 indicated positive results in 3 patients enrolled in the trial, whom demonstrated robust microdystrophin gene expression in muscle tissue and a significant decrease in serum creatine kinase 3 months after treatment. Of note, the AAVrh74 vector has a particular affinity for delivering the transgene to diaphragm and cardiac tissue, progressive weakness of which is a leading cause of mortality in this population.

Batten Disease (BD)
BD is an autosomal recessive condition composed of a group of genetic neurodegenerative disorders also known as neuronal ceroid lipofuscinoses. These are a subset of lysosomal storage diseases that cause myoclonic epilepsy, loss of cognitive and motor function, and premature mortality. To date, 14 subtypes of the disease are recognized (based on mutations in 14 genes; CLN1 through CLN14), with most caused by loss-of-function mutations in genes encoding lysosomal proteins or transmembrane proteins.

Biotechnology company Regenxbio is developing a novel, 1-time gene therapy solution for CLN2 (late infantile) BD, among the most common forms of the disease. CLN2 is caused by mutations in the TPP1 gene, which encodes the enzyme tripeptidyl peptidase 1. Studies in animal models of CLN2 disease show that a single intracisternal injection of RGX-181 led to a wide expression of the TPP1 enzyme throughout the CNS. The therapy significantly improved neurobehavior and extended survival (TABLE).16

Giant Axonal Neuropathy
Giant axonal neuropathy is a neurodegenerative disorder that shows autosomal recessive inheritance and is caused by loss-of-function mutations in the GAN gene. The prevalence of the condition is uncertain and the carrier frequency unknown. The disease typically presents in early childhood and results in mortality within the first 30 years of life.17 The GAN gene encodes the protein gigaxonin, which is involved in the regulation of neurofilaments. The condition is characterized by giant axonal swellings filled with disorganized aggregates of neurofilaments.

Preclinical trials demonstrate that treatment with AAV9/JeT-GAN, an AAV9 vector carrying a normal copy of the human GAN transgene, restores the normal configuration of neurofilaments within 4 weeks in GAN knockout mouse models. Intrathecal delivery of AAV9/JeT-GAN in aged GAN knockout mice reduced the accumulation of neurofilaments within neurons and conferred sustained wild-type GAN expression across the CNS and parasympathetic nervous system for at least 12 months (TABLE).18

Amyotrophic Lateral Sclerosis (ALS)
ALS is a neurodegenerative condition with an incidence of 2 in 100,000. Within the inherited version of this condition, 20% of individuals have a mutation in the SOD1 gene.19 Currently no effective therapy exists for this familial form or for the more common sporadic version of ALS. Both have a median survival of 3 to 5 years following symptom onset.20

Preclinical trials have introduced a transgene that silences SOD1 into an AAV vector delivered by a spinal subpial route.21 According to Hernandez, “The principle of the subpial technique is to deliver the viral vectors on top of the spinal cord, avoiding all membranes and allowing the vectors to penetrate the parenchyma of the spinal cord. The technique can be used in adult rodents (mouse and rat) and large animal models (mini-pigs and nonhuman primates) with no neurological deficits [associated] with the surgery” (TABLE).

In ALS mice, no detectable ALS disease-associated symptoms were seen for the duration of the study (~470 days) while the sibling sham-operated animals reached the end stage at ~390 days of age. The treatment conferred widespread transgene expression and resulting neuroprotection throughout the spinal parenchyma, including in both white and grey matter as well as brain motor centers.

“The main advantage of the subpial delivery versus the intrathecal, intracerebroventricular, or systemic delivery is the widespread gene expression achieved in adult animals with relatively low injection volume,” Hernandez told NeurologyLive. “This will, for example, allow the delivery of vectors into the CNS of adult ALS patients carrying the SOD1 mutation.”

Exploring Fetal Intervention
Simon Waddington, PhD, professor of gene therapy at University College London, and colleagues are currently developing a gene therapy approach for early-onset childhood genetic diseases such as neuronopathic Gaucher disease (nGD), a rare neurological disorder with an incidence of approximately 1 in 100,000 births.22 The team has used a mouse model of nGD to develop a procedure to deliver an AAV vector that reconstitutes glucocerebrosidase expression to the developing fetal brain. Mice treated in this way lived for at least 18 weeks and were fertile and fully mobile.23 The team has also demonstrated the potential of using ultrasound to guide global AAV gene transfer in the brains of fetal macaques.

“Fetal injection delivers the transgene before the disease has already caused irreparable damage,” Waddington said in describing the advantages of their novel delivery system. “Secondly, the fetus is small and so there is a better spread of the vector, which makes the treatment more effective. Thirdly, the fetus will not yet have an immune response to the archetypal virus.”

Future Challenges
Rare monogenetic disorders currently dominate research into potential gene therapy solutions for neurological disorders, with AAV vectors emerging as the most promising treatment platform. Much progress has been made and, for the first time, significant therapeutic benefits for patients are forthcoming from clinical trials.

However, challenges remain regarding the long-term efficacy of such treatments, because successful clinical trials are very recent and long- term safety and efficacy are currently impossible to determine. The potential long-term toxic effects from gene replacement therapy are largely unknown. The “Goldilocks effect” is well understood from research into Rett syndrome, in which overexpression of the therapeutic transgene in target tissues, or expression within nontarget tissues, can ultimately cause toxic effects in animals. However, as Waddington pointed out, “Even if the porridge is too hot or too cold, it’s better than none at all.”

In addition, future research needs to consider systemic administration for CNS diseases in older children and adults, as higher doses may be required for gene transfer to the adult CNS. “This is, by far, the most important limitation to clinical translation of gene therapy,” Waddington said. He also emphasized the relative lack of validated biomarkers or the ability to biopsy CNS tissue, which makes it difficult to monitor the efficacy of treatments.

“Preexisting immunogenicity24 to AAV capsids can impact exposure and/or efficacy,” according to Jawa. “However, the likelihood of a trans- gene-specific immune response is low, especially with a single dose administration. The route of delivery can impact the immune response to both capsid and transgene, and it can potentially impact safety if the viral vector delivery is not directly into the CNS and there is a potential for exposure in the periphery.”

Despite these limitations, gene therapy represents a vast area of promise for the treatment of rare neurological diseases. As Speer noted, “Gene therapies have the potential to offer life-changing benefits to patients with genetic diseases, as acknowledged by the regulatory bodies across the world who are granting them priority and accelerated reviews. We believe that therapies like Zolgensma will be a clinically important treatment option in many rare monogenic disorders.”
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