Clinical aspects

Description

Vectors for gene therapy of neurodegenerative disorders. A variety of viral as well as nonviral vectors are available. Adeno-associated virus (AAV) vectors are particularly useful for neurodegenerative diseases and have been used in preclinical studies as well as in human clinical trials.

Nanoparticles and other nanostructures can be used for refining gene delivery. Targeted and effective delivery of therapeutic genes is facilitated by polymeric nanoparticles as carriers to cross the blood-brain barrier for treating neurodegenerative disorders (03). Nanobiotechnology is required to develop a tailor-made system that may not only cross this barrier but also effectively modulate the expression of disease-causing genes.

A challenge in the translation of the promising preclinical gene therapies under development for neurodegenerative disorders is to achieve the most complete transduction of the target structure while avoiding leakage into neighboring regions or perivascular spaces. Interventional MRI-guided convection-enhanced delivery has become the gold standard for verifying accurate vector delivery in real time (28).

Genome editing. Tools for editing such as CRISPR (clustered regularly interspaced short palindromic repeats) can be used in gene therapy strategies for the treatment of neurodegenerative disorders (12). CRISPR/Cas9 gene editing technology provides an opportunity to establish animal models of neurodegenerative disorders with high accuracy and facilitates research for the development of new therapies for neurodegenerative diseases (04).

Techniques of gene therapy for Parkinson disease. Gene therapy for Parkinson disease is the most important potential application of the technology to neurologic disorders. A classification of various gene therapy techniques applicable to Parkinson disease is shown in Table 1.

Table 1. Gene Therapy Techniques Applicable to Parkinson Disease

Herpes simplex virus-1 vectors in Parkinson disease. The most relevant vector of this category is a defective herpes simplex virus-1 vector containing a gene encoding human tyrosine hydroxylase, the rate-limiting enzyme in dopamine biosynthesis. Several studies have shown that augmenting the activity of tyrosine hydroxylase will increase levodopa and subsequent dopamine production, as well as its release into the extracellular fluid compartment, bypassing the dopaminergic nigral afferent input. This would provide a more physiological dopamine delivery than is possible with any of the other methods described in the previous section. A schematic effect of tyrosine hydroxylase gene delivery on dopamine levels is shown.

Long-term (1 year) behavioral and biochemical recovery has been demonstrated in parkinsonian rats by a herpes simplex virus vector expressing tyrosine hydroxylase. Later work by the same authors showed that genetically engineered cells transplanted into the brains of monkeys caused a 40% improvement in the symptoms of neurotoxin-induced parkinsonism. A criticism of this work was that striatal injection of a gene-delivery herpes amplicon system can damage the neurons and cross synapses in the striatum. One explanation that has been offered for the improvement of parkinsonism is that it is due to surgical intervention involved in cell transplantation rather than due to alteration of specific function by the gene delivery. This explanation is unlikely because the damage around the site of injection of the vector is limited, and improvement is not seen with implantation of the vector without tyrosine hydroxylase gene in control animals.

A complimentary approach to transmitter enzyme replacement is the strategy to alter the function of surviving dopaminergic neurons and improve dopaminergic transmission. The key mediators in these pathways are signal-transduction proteins. Defective herpes simplex virus-1 vectors containing genetic sequences encoding catalytic domains of adenylate cyclase, protein kinase C, and calcium calmodulin protein kinase 2 can direct the stable expression of these enzymes. Nigrostriatal cells can be specifically targeted by using a combination of stereotactic surgery directed at substantia nigra with further cell specificity, which is obtained by using the tyrosine hydroxylase promoter driving the expression of a signal-transduction enzyme.

Adeno-associated virus vectors in Parkinson disease. An adeno-associated virus vector (AAVgdnf) has been developed that contains the cDNA for glial cell line-derived neurotrophic factor. AAVgdnf introduced unilaterally prior to 6-hydroxydopamine administration results in neuroprotection in a rat model of Parkinson disease.

A bicistronic construct, AAVthIRESaadc, which contains both the human tyrosine hydroxylase and AADC genes, has also been tested. AADC is responsible for the conversion of levodopa to dopamine in dopaminergic neurons. Elevated levels of dopamine could be demonstrated near injection tracts following the intrastriatal injection of bicistronic vector in MPTP-treated monkeys. Convection-enhanced, MRI-guided delivery of an AAV vector with human AADC gene bilaterally to the putamen has been completed in a phase I trial in Parkinson disease patients (31). PET uptake correlated with hAADC transgene expression and confirmed the findings of T2 hyperintensity on MRI and hAADC immunohistochemistry. Long-term evaluation of a phase 1 study of AADC gene therapy for Parkinson disease using AAV2-hAADC vector has shown stable transgene expression over 4 years after vector delivery and continued safety (19).

A gene therapy candidate consisting of the GDNF gene carried by an AAV-2 vector has been granted FDA approval to enable the start of clinical trials.

Adenovirus vectors in Parkinson disease. A recombinant adenovirus vector expressing tyrosine hydroxylase, administered by intracerebral injection in the 6-hydroxydopamine rodent model of Parkinson disease, decreases rotational asymmetry. Adenoviral-mediated glial cell line-derived neurotropic factor injection into the rat striatum was shown to reduce loss of dopamine neurons. In another study, adenoviral mediated glial cell line-derived neurotropic factor injected into the 6-hydroxydopamine rat model of Parkinson disease not only prevented dopaminergic neuronal degeneration but also improved motor function. These studies indicate that similar benefits may occur in patients with Parkinson disease.

A tropism-modified adenovirus5 vector that contains the fiber knob domain from canine adenovirus serotype 2 has been constructed, and delivery to the substantia nigra or striatum showed that this vector produced a neuronally restricted pattern of gene expression (14). Besides precise control of transgene expression, this vector can be further developed to be a high-capacity vector, which can maintain transgene expression in the CNS for over a year. Targeted stereotactic delivery to substantia nigra or striatum is possible for enabling clinical trials in human patients.

Lentiviral vectors. These vectors have been used to carry the cDNA for neublastin (a neurotrophic factor of GDNF family). Neuroprotective effect has been reported following injection of these vectors into the striatum and ventral midbrain of animal models of Parkinson disease.

Plasmid DNA-lipofectin complex. Plasmid-DNA-transfected brain cells can generate levodopa locally in the striatum in quantities that are sufficient to partially compensate for loss of intrinsic striatal dopaminergic input. Controlled and prolonged delivery of genes in vivo is possible by direct intracerebral tyrosine hydroxylase and L-amino acid decarboxylase gene transfer with injection of plasmid DNA-liposome complex. Injection of such a gene construct into striatum of a rat model of Parkinson disease can lead to expression of tyrosine hydroxylase.

Novel vector system. As an aid in the development of vector systems for use in gene therapy paradigms of CNS disorders such as Parkinson disease, L-Dopa or dopamine-producing gene cassettes have been developed. Specifically, a human tyrosine hydroxylase cDNA (HTH-2) is rendered constitutively active by truncation of the N-terminal regulatory domain. In addition, a bicistronic construct capable of directing the production of dopamine is created by inserting an internal ribosome entry site downstream of N-terminal regulatory domain followed by the coding sequences of aromatic amino acid decarboxylase. All 3 constructs generated immunoreactive peptides of the predicted size, were enzymatically active, and produced L-Dopa (HTH-2, N-terminal regulatory domain) or dopamine (bicistronic construct) following transient transfection of COS-7 cells. These constructs, in conjunction with viral or nonviral expression systems, may be efficacious in gene therapy approaches to Parkinson disease. Regulatable systems enable the expression of the introduced gene to be adjusted or stopped by changing the dose of an oral inducer drug and provide an ideal method for safely delivering effective, flexible gene therapy treatments over the long course of Parkinson disease.

Ex vivo gene therapy of Parkinson disease using genetically engineered cells. Various cells have been used for ex vivo gene therapy of experimental Parkinson disease. Differences between direct gene transfer into striatal cells and cell transplantation (grafting) are:

Some of the ex vivo approaches are described briefly here:

Fibroblasts. A well-established experimental approach uses fibroblasts transduced with retroviral vectors encoding the human tyrosine hydroxylase 2 and human glutamyl transpeptidase cyclohydrolase 1 genes. This results in an increase of levodopa production, but no improvement of symptoms. Fibroblasts, even though they do not integrate with the brain, are preferred cells for implantation because they remain where they are inserted and their location can be verified by a suitable marker and positron emission tomography scanning.

Bone marrow stromal cells. These can be transduced by retroviral vectors to produce levodopa. Intrastriatal injection of such vectors into 6-hydroxydopamine lesioned rats leads to engraftment and dopamine production.

Astrocytes. When genetically engineered by viral vector-mediated tyrosine hydroxylase gene transfer, astrocytes secrete high levels of levodopa. Astrocytes can also be engineered to produce brain-derived neurotrophic factor. The efficacy of a technique for expressing GDNF exclusively in astrocytes was evaluated in the mouse MPTP model of Parkinson disease (08). The efficacy was equal to that of neuron-derived GDNF, but this technique was safer as unilateral striatal GDNF expression in astrocytes did not result in delivery of GDNF to the contralateral hemispheres as was the case when GDNF was expressed in neurons. Astrocytes, transfected with an adenoviral vector encoding human tyrosine hydroxylase under the negative control of a tetracycline-based regulatory system, can synthesize large amounts of tyrosine hydroxylase and synthesize levodopa as shown in vitro, and have the potential for transplantation for Parkinson disease.

Because they integrate with brain tissues, astrocytes are promising material for the gene therapy approach to Parkinson disease. However, a potential drawback is that that these cells wander off in the brain after implantation, and it is difficult to predict their final location.

Genetically engineered cells producing neurotrophic factors. The basic fibroblast growth factor is known to enhance the survival of dopaminergic neurons in vitro. Cells that have been genetically modified to produce basic fibroblast growth factor can improve the functional efficacy of dopaminergic neurons following implantation in the brains of animal models of Parkinson disease. Interest has also been focused on the development of proliferating cell lines for transplantation that can be genetically manipulated to produce other neurotrophic factors. Cells for transplantation can also carry genes for dopamine-synthesizing enzymes.

Stem cells. Neural stem cells are transduced with tyrosine hydroxylase and glutamyl transpeptidase cyclohydroxylase 1 genes, which produce important enzymes in dopamine biosynthesis and can be successfully transplanted into the brains of rat models of Parkinsonian disease. Marrow stromal cells can be transfected with tyrosine hydroxylase gene by an adeno-associated virus vector and transplanted into the striatum of rat model of Parkinson disease.

Glutamic acid decarboxylase (GAD) gene therapy. Glutamic acid decarboxylase delivered by an adeno-associated virus vector (NLX-P101) was shown to be a safe and efficient method of reducing motor deficits in a primate model of Parkinson disease. As glutamic acid decarboxylase potentiates inhibitory neurotransmission from the subthalamic nucleus, sustained expression of glutamic acid decarboxylase in the nucleus by direct delivery of NLX-P101 is an explanation of the beneficial effects (07).

Delivery of parkin gene. Mutations in parkin gene have been linked to familial Parkinson disease. The loss of parkin’s E3-ligase activity leads to dopaminergic neuronal degeneration in early-onset autosomal recessive juvenile parkinsonism, suggesting a key role of parkin for dopamine neuron survival. Animals overexpressing parkin showed significant reductions in alpha-synuclein-induced neuropathology, including preservation of tyrosine hydroxylase-positive cell bodies in the substantia nigra and sparing of tyrosine hydroxylase-positive nerve terminals in the striatum. Overexpression of parkin has complementary effects to those described with use of neurotrophic factors for Parkinson disease.

Targeting alpha-synuclein gene expression by RNA interference. Overexpression of wild-type alpha-synuclein is considered to confer susceptibility to Parkinson disease. Use of short interfering RNAs to target and knock down alpha-synuclein gene expression has been shown to be feasible in animal experimental studies and has the potential for developing therapeutics to treat Parkinson disease. Another approach is in vivo transfer of genes encoding beta-synuclein, the nonamyloidogenic homologue of alpha-synuclein, which binds to alpha-synuclein, reducing its aggregation and accumulation in the synaptic membrane. This might be an alternative gene therapy approach for Parkinson disease.

Targeted editing of DNA methylation for downregulation of alpha-synuclein expression. A gene editing strategy based on CRISPR-deactivated Cas9 has been developed for the downregulation of alpha-synuclein expression by targeting DNA methylation using a lentiviral vector system (11). Application of the system to human induced pluripotent stem cell-derived dopaminergic neurons from a Parkinson disease patient with the alpha-synuclein triplication resulted in fine downregulation of alpha-synuclein mRNA and protein, indicating its potential as a novel epigenetic-based therapeutic approach for Parkinson disease.

Prospects of gene therapy for Parkinson disease. The technique of direct delivery of the vector to the striatum or substantia nigra is a standard neurosurgical procedure. However, for the diffuse neurologic manifestations of Parkinson disease, global delivery of the vector to the brain may be preferred. Intraventricular delivery has also been tried and may result in preferential expression in ependymal and circumventricular tissues.

Among the ex vivo approaches, the most important is implantation of genetically engineered cells releasing neurotrophic factors (protective and regenerative) and among the in vivo approaches is the delivery of tyrosine hydroxylase gene (neurotransmitter restoration). The ideal approach may be a combination of these 2 therapies.

RNA interference can theoretically be applied to Parkinson disease because overexpression of various proteins is known to kill the dopamine neurons of the substantia nigra. RNA interference has been combined with lentiviral vector-based gene therapy to turn off alpha-synuclein in dopamine neurons. Use of a dual cassette lentivirus, which coexpresses an alpha-synuclein-targeting short hairpin RNA, enables delivery of RNAi to the brain of experimental animals and turn off the alpha-synuclein protein in neurons. This technique has the potential for clinical development of Parkinson disease treatment.

Clinical trials of gene therapy for Parkinson disease. Several approaches have been used in clinical trials for gene therapy of Parkinson disease:

The first 2 therapies focus on increasing dopamine production via direct delivery of genes involved in neurotransmitter synthesis (amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase 1). To bypass the degenerating nigrostriatal pathway, another approach uses adeno-associated viral vectors to deliver glutamic acid decarboxylase to the subthalamic nucleus, converting a subset of excitatory neurons to GABA-producing cells. The third approach is aimed at protecting the degenerating nigrostriatum by striatal delivery of adeno-associated viral vectors harboring the neuroprotective gene, neurturin, aiming to slow disease progression by enhancing neuronal survival. The fourth approach involves an equine infectious anemia virus-based lentiviral system encoding amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I in a single transcriptional unit (21).

Several clinical trials of gene therapy for Parkinson disease have been conducted during the past decade. As of August 2021, 25 clinical trials are registered on the U.S. Government clinical trials web site: https://clinicaltrials.gov/ct2/results?cond=Parkinson+Disease&term=gene+therapy&cntry1=&state1=&Search=Search.

The first in vivo gene therapy for Parkinson disease was administered to the brain of a patient in 2003 at a medical center in New York. The therapy delivered the glutamic acid decarboxylase (GAD) gene (NLX-P10) using an adeno-associated viral vector via 90-minute infusion to a target identified by CT scan and MRI. A phase 1 clinical trial of AAV-GAD gene therapy delivered to the subthalamic nucleus showed that the procedure is safe and well tolerated by patients with advanced Parkinson disease. A double-blind phase 2 trial completed all the surgical procedures in 2009, but there has been no further clinical development.

In an open-label clinical trial, patients with moderately advanced Parkinson disease received bilateral stereotactic injections of the gene therapy vector AAV2 expressing the neurotrophic factor neurturin (CERE-120) into the putamen and substantia nigra (NCT00985517). Analyses of autopsied brains from subjects previously administered AAV2-neurturin gene transfer in clinical trials indicate that optimizing the effects of neurotrophic factors in Parkinson disease likely requires delivery to both the degenerating cell bodies in substantia nigra and their terminals in striatum (05). Experiments in animal models indicate that high-dose delivery to substantia nigra was responsible for weight loss seen as an adverse effect. A low dose of neurturin delivered with the modified technique in ongoing clinical trials is expected to be safer. Five-year follow-up data showed that the procedure was well-tolerated with no serious adverse events and provided class IV evidence that the procedure is safe as well as feasible for clinical application (17).

In another clinical trial, patients with moderately advanced Parkinson disease received bilateral intraputaminal infusion of an adeno-associated viral type 2 vector (AAV2) containing the gene for human aromatic L-amino acid decarboxylase (AADC), the enzyme that converts levodopa into dopamine (06). The gene therapy resulted in improvements of scores of motor scales and was well tolerated, but intracranial hemorrhages followed the operative procedure in 3 of 10 patients in the study. An open-label phase 1 safety and efficacy study that started in 2016 tested AAV2-hAADC administered by MRI-guided convective infusion into the putamen of subjects with Parkinson disease who have fluctuating responses to levodopa (NCT01973543). The study was completed in January 2020, but the results have not been published yet.

Results of a phase II trial to evaluate ProSavin gene therapy in patients on levodopa therapy, which was designed to deliver key enzymes involved in the synthesis of dopamine by a lentiviral vector bilaterally in the putamen, showed that it was safe and effective with improvement in motor behavior in all patients (24). ProSavin remained safe and well tolerated in patients with Parkinson disease and moderate improvements in motor behavior over baseline continued to be reported in most of the patients who could still be evaluated with up to 5 years of follow-up (23).

Data from clinical trials suggest that gene therapy targeting multiple brain regions including the striatum and substantia nigra can be safe and well tolerated in Parkinson disease patients (02). There are still challenges, including how to modulate gene expression and how to determine the optimal target, dose, and selection of patient population for study in future gene therapy trials.

Gene therapy for Alzheimer disease. Retroviral vectors have been used to genetically modify primary autologous primate fibroblasts ex vivo to produce and secrete nerve growth factor. These cells were then grafted to the basal forebrain region of adult rhesus monkeys after fornix transection. Cholinergic neuronal degeneration could be prevented for extended periods in vivo; this method has the potential to be a treatment for Alzheimer disease. A phase I clinical trial of Alzheimer disease uses adeno-associated viral vector.

Preventive gene therapy with nerve growth factor might be useful for persons at high risk of developing Alzheimer disease. One advantage of this approach is that it provides a method for selective delivery of therapeutic genes and their products to afflicted areas of the brain.

Lentiviral vectors engineered to overexpress the glial cell-derived neurotrophic factor (GDNF) gene in hippocampal astrocytes of 3xTg-AD mice in vivo were shown to be safe and effective, both as a potential gene therapy as well as a tool to uncover the mechanisms of GDNF neuroprotection in Alzheimer disease (27).

As in the case of other neurodegenerative disorders, it is possible that gene therapy can be combined with systemic cholinomimetic therapy. If amyloid deposit is a primary event, it will provide a strong rationale to target amyloid deposits for therapeutic intervention. Several amyloid-based approaches are in the preclinical stage and are expected to advance to clinical trials in the next few years. In 1 of these projects, transgenic neurons expressing human Bcl-2 were partially protected against amyloid beta-peptide-induced neuronal death. This neuroprotection appears to be related to the complete inhibition of apoptosis induced by both amyloid beta-peptides, which can form the basis of gene therapy for Alzheimer disease.

Genetically engineered cells producing nerve growth factor when implanted into basal cerebral structures have been shown to reverse cognitive changes associated with aging in rhesus monkeys. Aging is associated with a significant 25% reduction in cortical innervation by cholinergic systems in rhesus monkeys and this was restored by nerve growth factor. These findings are relevant to potential clinical use of nerve growth factor in Alzheimer disease. Postmortem examination of brains from Alzheimer disease patients treated in clinical trials of nerve growth factor (NGF) gene therapy and survived for 1 to 10 years showed that neurons exhibiting tau pathology, as well as neurons free of tau, expressed NGF, indicating that degenerating cells can be infected with therapeutic genes, with resultant activation of cell signaling (30). A multicenter phase 2 randomized clinical trial (NCT00876863) demonstrated the feasibility and safety of sham-surgery-controlled stereotactic NGF gene delivery using a AAV2 vector in patients with Alzheimer disease but did not affect clinical outcomes (26). Pathological confirmation of accurate gene targeting is needed for better evaluation of the results.

Clinical trials of gene therapy in Alzheimer disease. As of August 2021, 26 clinical trials of gene therapy for Alzheimer disease are listed on the U.S. Government website: https://clinicaltrials.gov. Only 6 of these trials involve gene therapy according to the definition of this term. Three of the 6 have been completed, information is insufficient on 1 trial, and 2 are recruiting patients. One of these, a phase 1 trial that started in 2018, is assessing the safety and toxicity of intracisternal administration of AAVrh.10hAPOE2, gene transfer vector expressing the cDNA coding for human apolipoprotein E2 (APOE2), directly to the CNS/CSF of APOE4 homozygotes with Alzheimer disease (NCT03634007). The second trial, a phase 1 starting in 2019, is treating subjects with hTERT (telomerase) delivered via transduction using AAV to extend the telomeres to prevent, delay, or even reverse the development of the pathology of Alzheimer disease (NCT04133454).

Neprilysin gene therapy. Neprilysin, a substance naturally found in the brain, alters amyloid beta proteins in ways that lead to their destruction. A form of gene therapy appears to reduce amyloid beta in the brains of mice with Alzheimer disease pathology by increasing brain levels of neprilysin. However, some challenges of neprilysin gene therapy include secondary toxicity, activation of the immune response, and low efficiency (15). Therefore, safe and efficient neprilysin delivery systems with high transfection efficiency are needed gene therapy of Alzheimer disease.

Fibroblast growth factor (FGF) gene therapy. FGF2 gene was delivered bilaterally to the hippocampi of mouse models of Alzheimer disease via an AAV2/1 vector, resulting in significantly improved spatial learning as well as clearance of amyloid beta peptides from the hippocampi (13). This method has potential as an alternative therapy of Alzheimer disease and possibly other neurocognitive disorders.

RNAi-based therapy for Alzheimer disease. Cyclin-dependent kinase 5 (CDK5) is a key mediator of tau hyperphosphorylation and neurofibrillary pathology. Use of RNAi for CDK5 silencing has been proposed as a targeted and specific therapeutic alternative for Alzheimer disease (16).

Development of gene therapy for Huntington disease. Advances in understanding of the pathogenesis of Huntington disease have resulted in the discovery of new therapeutic targets, some of which are now in clinical trials. Focus is on RNA- and DNA-based gene therapies for the reduction of mutant huntingtin (mHTT) protein. Modification of genetic cause of Huntington disease and functional restoration of mHtt protein can be achieved by using targeted multimodal gene therapy (10). Various methods of gene therapy that are under investigation for Huntington disease include the following:

Encapsulated genetically engineered cellular implants. Implants of encapsulated recombinant human ciliary neurotrophic factor-producing fibroblasts have been shown to prevent behavioral deficits and striatal degeneration in a rodent model of Huntington disease.

Other neurotrophic factors have been shown to protect vulnerable striatal neurons in animal models of Huntington disease. The mechanisms of protection and prevention of behavioral deficits are complex and unrelated to the trophic factor binding to its receptor. For example, cells genetically modified to secrete nerve growth factor or ciliary neurotrophic factor protect degenerating striatal neurons that do not express receptors for these factors.

Viral vector mediated administration of neurotrophic factors. Neuroprotective effect of a ciliary neurotrophic factor-expressing lentiviral vector was demonstrated in a rat model of Huntington disease. Lentiviral-mediated tetracycline-regulated gene transfer is feasible in the brain, and this vector has been used to investigate the dose-dependent neuroprotective effect of human ciliary neurotrophic factor in the quinolinic acid model of Huntington disease.

Adeno-associated viral-mediated gene transfer of glial cell line-derived neurotrophic factor into the striatum provides neuroanatomical and behavioral protection in a rodent model of Huntington disease. Further studies in mouse models support the concept that viral vector delivery of glial cell line-derived neurotrophic factor may be a viable treatment for patients suffering from Huntington disease.

Antisense therapy for Huntington disease. Modified 2'-O-methyl phosphorothioate triplet-repeat antisense oligonucleotides have been shown to effectively reduce mutant huntingtin transcript and protein levels in patient-derived Huntington disease fibroblasts and lymphoblasts (09).

RNA interference therapy (RNAi) of Huntington disease. This method, based on blocking expression of mutant huntingtin gene, is a promising therapeutic strategy. A high-capacity adenoviral vector, expressing a short hairpin RNA targeted to exon 1 of the huntingtin gene, was shown to efficiently inhibit huntingtin expression in neuronal and nonneuronal cell lines.

Intraventricular infusion of single-stranded (ss) siRNA produced selective silencing of the mutant huntingtin (mHTT) allele throughout the brain in a mouse model of Huntington disease (33). These results demonstrate that chemically modified ss-siRNAs, functioning through the RNAi pathway, provide allele-selective compounds for clinical development.

Although RNAi-based approaches for lowering mHTT expression have been efficacious in mouse models, basal mutant protein levels are still detectable. A strategy has been developed for allele-specific genome-editing of mHTT based on CRISPR/Cas9 technology, which takes advantage of highly prevalent single nucleotide polymorphisms in the HTT locus for guiding mutant allele-specific cleavage and reducing the expression from mHTT alleles in human Huntington disease fibroblasts and mouse brain (20). A randomized, double-blind, multiple-ascending-dose, phase 1 to 2a trial of intrathecal administration of HTT_Rx to patients with early Huntington disease led to dose-dependent reductions in concentrations of mutant huntingtin and was not accompanied by serious adverse events (29).

MicroRNA-based gene therapy of Huntington disease. AM-130 is composed of a small portion of synthetic genetic material ‒ microRNA (miRNA) ‒ which is carried and inserted into cells using an AAV vector. Once inside a cell, the miRNA targets the RNA molecule that carries instructions to produce the huntingtin protein and marks it for degradation. Thus, AMT-130 should lower the production of abnormal huntingtin protein.

A phase I/II, randomized, multicenter, dose escalation, double-blind, imitation surgery, first-in-human study (NCT04120493) is designed to establish safety and proof-of-concept of single dose AMT-130 gene therapy, which lowers huntingtin protein and improves signs of Huntington disease in animal models.

Technique of gene therapy of amyotrophic lateral sclerosis. Various neurotrophic factors have been administered using genetically engineered cells. Neurotrophic factor genes may be transduced using viral vectors.

Clinical trials of gene therapy of amyotrophic lateral sclerosis. As of August 2021, only a few trials of gene therapy for amyotrophic lateral sclerosis are listed on the web site: http://clinicaltrials.gov.

Ciliary neurotrophic factor. Only 1 trial has been conducted with gene therapy; it involved intrathecal implantation of polymer-encapsulated genetically engineered cells by CRIBTM secreting recombinant human ciliary neurotrophic factor (01). In this phase 1 study, 6 patients with amyotrophic lateral sclerosis received genetically modified ciliary neurotrophic factor-secreting baby hamster cells via CRIBTM introduced into the lumbar intrathecal space. These cells were capable of secreting 1.0 µg of human ciliary neurotrophic factor per day and nanogram levels of ciliary neurotrophic factor were measured in the patients' CSF for at least 17 weeks posttransplantation. Prior to this treatment, ciliary neurotrophic factor was not detectable in the CSF of these patients. This study demonstrated for the first time that significant and measurable doses of neurotrophic factors can be delivered by this approach without the deleterious side effects observed with peripheral administration. This study also demonstrated the pharmacological dose control of a neurotrophic factor by gene therapy; the authors have suggested further clinical trials with multiple neurotrophic factors using this method of delivery. The results were satisfactory and phase 2 studies are planned. Use of multiple neurotrophic factors will be considered. Gene therapy is anticipated to be used mainly in the form of genetically engineered encapsulated cells for the in vivo production and delivery of neurotrophic factors. Because of the multifactorial pathogenesis of amyotrophic lateral sclerosis, various combination therapies are foreseen; administration of neurotrophic factors by gene therapy may be combined with riluzole.

Glial cell line-derived neurotrophic factor. Using retroviral vectors, myoblasts have been genetically modified to secrete glial cell line-derived neurotrophic factor. Use of these myoblasts has prolonged the onset of disease and delayed deterioration in mouse models of familial amyotrophic lateral sclerosis. Glial cell line-derived neurotrophic factor gene therapy in mouse models of familial amyotrophic lateral sclerosis promote the survival of motoneurons, suggesting that a similar approach might delay the progression of neurodegeneration of amyotrophic lateral sclerosis. Adeno-associated virus-mediated glial cell line-derived neurotrophic factor delivery to the muscle is a promising means of gene therapy for amyotrophic lateral sclerosis.

Vascular endothelial growth factor. Vascular endothelial growth factor has been implicated in neuroprotection and has a therapeutic potential for the treatment of amyotrophic lateral sclerosis. Although a single injection of a vascular endothelial growth factor-expressing lentiviral vector into various muscles has been shown to delay onset and slow progression of amyotrophic lateral sclerosis in mouse models engineered to overexpress the gene coding for the mutated form of the superoxide dismutase-1, this method has not been developed further.

Adenoviral-mediated transfer of neurotrophic factors for amyotrophic lateral sclerosis. Pretreatment with brain-derived neurotrophic factor or glial cell-line derived neurotrophic factor using adenovirus-mediated gene transfer by intramuscular injection has been shown to prevent the massive death of motor neurons that normally follows axotomy in the neonatal period. Another study using adenoviral-mediated transfer of brain-derived neurotrophic factor or ciliary neurotrophic factor into the lesioned nerves showed that survival of axotomized motor neurons was maintained for at least 5 weeks in rats. However, overexpression of muscle-derived neurotrophin-3, although it prevented degeneration of axons at motor end plates, had little effect on the number of motor neuron cell bodies in a murine model of motor neuron disease. Techniques suitable for tonic exposure to both nerve and muscle-derived neurotrophic factors may have implications for the design of future therapeutic strategies. Advances in gene transfer techniques may enable transduction of sufficient numbers of cerebral, brainstem, and spinal cord neurons for therapeutic applications. It may be possible to repopulate lost corticospinal and lower motor neurons by transplanting stem cells or stimulating native progenitor populations.

Retrograde delivery of gene therapy for amyotrophic lateral sclerosis. Although chronic delivery of molecules to the CNS is difficult, an adeno-associated virus can be retrogradely transported efficiently from muscle to motor neurons of the spinal cord after intramuscular delivery. Insulin-like growth factor 1-AAV complex, when injected intramuscularly, prolongs life and delays disease progression in mouse models of amyotrophic lateral sclerosis.

Use of lentiviral vectors for gene therapy of amyotrophic lateral sclerosis. Numerous studies in animal models have shown that lentiviral vectors are suitable for gene therapy of neurodegenerative diseases such as amyotrophic lateral sclerosis (22).

Antisense therapy for amyotrophic lateral sclerosis. An antisense oligonucleotide targeting SOD1 was tested in a phase 1 human clinical trial and provided encouraging safety data; the drug was not clinically advanced because the next-generation SOD1 antisense oligonucleotides more potently reduced SOD1 mRNA and reversed initial loss of compound muscle action, and extended survival by more than 50 days in SOD1G93A rats and by almost 40 days in SOD1G93A mice (18). Rise of serum phospho-neurofilament heavy chain levels, a promising biomarker for amyotrophic lateral sclerosis, is stopped by SOD1 antisense oligonucleotide therapy. These results indicated that this SOD1 antisense oligonucleotide is ready for human clinical trials. Tofersen, an antisense oligonucleotide that targets a mutation in the SOD1 gene is in a phase 3 clinical trial for presymptomatic adult carriers of SOD1 mutation with elevated neurofilament (NCT04856982).

BIIB105, an antisense oligonucleotide administered intrathecally that binds the mRNA for ataxin-2 gene to mediate its degradation resulting in lower ataxin-2 protein levels is in a phase 1 trial for amyotrophic lateral sclerosis (NCT04494256).

RNAi therapy for amyotrophic lateral sclerosis. RNAi, using recombinant adenovirus and AAV2 vectors, has been tested in a transgenic mouse model that overexpresses mutant Cu, Zn superoxide dismutase and causes amyotrophic lateral sclerosis by a gained toxic property (32). Nerve injection of viral vectors efficiently transferred transgenes into motor neurons with modest therapeutic efficacy.

Spinal muscular atrophy. Spinal muscular atrophy type 1 (SMA1) is a neurodegenerative, autosomal recessive disorder with an onset during infancy that results in failure to achieve motor milestones and in death or the need for mechanical ventilation by 2 years of age. It is a monogenic motor neuron disease with deletion of exon 7 of the SMN1 gene that predominantly affects the anterior horn cells. Nusinersen, a splice-modulating antisense oligonucleotide indicated for the treatment of spinal muscular atrophy, is described in a separate article in MedLink.

Onasemnogene abeparvovec is a gene therapy for delivery of a functional copy of the human survival motor neuron (SMN) gene to the motor neuron cells of patients with spinal muscular atrophy type 1 and was the first gene therapy to be approved for the treatment of spinal muscular atrophy in the United States in 2019. In August 2021, a new study of onasemnogene in older patients with spinal muscular atrophy started following the Food and Drug Administration’s clearance of administration via a spinal infusion, which had been suspended earlier due to safety concerns.

Indications

Indications for gene therapy of neurodegenerative disorders are mainly for the following conditions:

The techniques are mostly in preclinical development. Clinical trials are also in progress and criteria for patient selection are determined for each clinical trial.

Contraindications

No contraindications have been identified.

Results

Most of the gene therapy experiments done in animal models have demonstrated the safety and efficacy of gene therapy. Clinical trials in humans are in early stages and have demonstrated safety, but efficacy requires long-term, later-stage clinical trials.

Adverse effects

Adverse effects of gene therapy are described in the article on gene therapy techniques.

Special considerations

Gene therapy can be carried out safely during pregnancy, but neurodegenerative diseases covered in this article do not usually occur during pregnancy.

Scientific basis

Gene mutations are relevant to etiology of neurodegenerative disorders and several gene therapy approaches are directed to these mutations.

Mutations in the ATXN2 gene (CAG expansions) can be a rare cause of Parkinson disease and amyotrophic lateral sclerosis and the stress granule protein Staufen1 (STAU1) is overabundant in the neurodegenerative disorder spinocerebellar ataxia type 2. STAU1 overabundance in neurodegeneration is a common phenomenon and targeting it with antisense oligonucleotides or miRNA viral vectors may represent a novel, efficacious therapy for neurodegenerative diseases (25).

Rationale of gene therapy for Parkinson disease. This remains to be the most important area for development of gene therapy for neurologic disorders. Parkinson disease is an ideal candidate; the neurologic abnormalities are known to result from the degeneration of dopaminergic neurons and nigrostriatal pathways. Clinical signs of the disease can be ameliorated by the replacement of levodopa. The availability of well-characterized animal models facilitates the evaluation of gene therapy. Discovery of a genetic locus for Parkinson disease is a big step in our understanding of the disease, even though there may be no genetic contribution or multiple genes involved in the majority (98%) of patients with Parkinson disease. Several problems, however, need to be addressed. These include the following:

The introduction of functional genes into the brains of patients with Parkinson disease may, for example, prove useful to replace a defective gene, introduce a potentially neuroprotective or neurorestorative protein, or permit the physiological delivery of a deficient neurotransmitter. Observations suggest that the oral administration of currently available dopaminomimetics to relatively advanced parkinsonian patients leads to nonphysiological intermittent stimulation of striatal neurons that express dopamine receptors. Resultant activation of signal transduction pathways from these dopaminergic receptors on medium-sized GABAergic neurons apparently induces long-term potentiation of adjacent glutamatergic receptors of the N-methyl-D-aspartate subtype. Thus, the effects of dopaminergic drugs become modified in ways that favor the clinical appearance of response fluctuations and peak-dose dyskinesias. In parkinsonian models, as well as in patients with Parkinson disease, continuous dopaminergic replacement tends to prevent or alleviate these adverse effects. By continuously maintaining appropriate cerebral dopamine concentrations, molecular techniques that stimulate an increase in the intrastriatal activity of tyrosine hydroxylase (the rate-limiting enzyme for dopamine synthesis) might be expected to palliate parkinsonian symptoms with less risk of the disabling consequences of current therapy. Clinical study of these approaches could also serve as initial, relatively simple, proof-of-principle evaluations of the safety and efficacy of genetic approaches to the treatment of basic disease processes in Parkinson disease.

Among the various neurotrophic factors, glial cell line-derived neurotrophic factor is receiving the most attention for Parkinson disease therapy; its administration by gene therapy would be an ideal method of delivery. Glial cell line-derived neurotrophic factor mRNA is mainly expressed in neurons of the cortex, hippocampus, striatum, substantia nigra, thalamus, cerebellum, and spinal cord. Its synthesis is not restricted to dopaminergic areas. The widespread expression of glial cell line-derived neurotrophic factor throughout the adult brain suggests that its administration in Parkinson disease should be restricted to the altered structures in order to avoid possible deleterious side effects.

The strategies for the application of gene therapy techniques to Parkinson disease treatment have expanded beyond the classical dopamine replacement toward the use of neurotrophic factors in enhancing cell function or preventing cell death.

Rationale of gene therapy for Alzheimer disease. Potential gene therapy approaches to Alzheimer disease are based on neurotrophic factors and not neurotransmitters. The optimal method of gene therapy would be ex vivo involving implantation of genetically engineered cells secreting nerve growth factor. Naturally occurring age-related memory loss in rats can be reversed by grafting cells engineered to secrete nerve growth factor directly into the nucleus basalis magnocellularis. Grafting of nerve growth factor-producing fibroblasts has been shown to reduce behavioral deficits associated with lesions of the nucleus basalis magnocellularis in rats. Intraventricular transplants of polymer encapsulated fibroblasts secreting human nerve growth factor have been shown to provide trophic support to basal forebrain neurons in cynomolgus monkeys with unilateral transection of fornix.

Rationale for gene therapy of Huntington disease. It has been reported that intrastriatal implantation of genetically engineered nerve growth factor-producing fibroblasts has a protective effect against excitotoxic insults.

Rationale for gene therapy of amyotrophic lateral sclerosis. In cases associated with mutations in superoxide dismutase gene, the rational approach would be replacement of the defective gene by transfer of a wild-type superoxide dismutase gene. The patients with such genetic defects constitute only 5% of the cases, and no gene therapy has yet been developed. The strategies for gene therapy are based on the beneficial effects of neurotrophic factors in amyotrophic lateral sclerosis, and gene therapy by implantation of genetically engineered cells secreting neurotrophic factors is the best strategy at present.