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There is a considerable interest in developing gene therapy for neurogenetic disorders because the conventional treatment is inadequate and unsatisfactory. Diseases most amenable to gene therapy would be those where a single gene product is missing and the gene expression has a simple mechanism, particularly several hereditary metabolic disorders involving the nervous system. The aim of somatic gene therapy in neurogenetic disorders is to replace the missing enzyme and improve the clinical symptoms. Gene therapy cannot eradicate the disease or prevent its transmission to offspring; this can be achieved only by germline gene therapy but it is currently not permitted on ethical grounds.
• By changing the pathophysiological concepts and classification of several diseases, molecular diagnostics may pave the way for gene therapy of neurogenetic diseases.
• Identification and replacement of defective genes in monogenic genetic disorders are ideal therapies, but they are not essential.
• Gene therapy has been shown to be effective in laboratory models of disease, but the long-term efficacy in human patients remains to be demonstrated.
• Gene editing techniques such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas enables in vivo correction of some neurogenetic disorders.
• Clinical trials in humans have taken place in Gaucher disease and Canavan disease, in which the safety of the procedure has been established.
Gene therapy was originally defined as the transfer of defined genetic material to specific target cells of a patient, instead of just the products of cells with altered genes, for the ultimate purpose of preventing or altering a disease state. Carriers or delivery vehicles for therapeutic genetic material are called “vectors”; these vectors are usually viral, but several nonviral techniques are being used as well. The history and basics of gene therapy are described in the introductory article on gene therapy.
The term "genetic engineering" applies to genetic manipulation of living cells and implantation of genetically engineered cells into the living body and can be considered as a form of gene therapy. Neurosurgeons refer to gene therapy as "cellular and molecular" neurosurgery.
Application of gene therapy to neurogenetic disorders will be described here. This will include inherited disorders of metabolism as well. Gene therapy of Duchenne muscular dystrophy is the topic of a separate MedLink article. With few exceptions, no rational therapies exist for any of these diseases. Molecular diagnostics have changed the pathophysiological concepts and classification of several neurologic disorders and may pave the way for gene therapy of several presently incurable neurologic diseases.
• Some adeno-associated virus vectors can be transported along neuronal pathways from the injected region for wide distribution for correction of a neurogenetic disease.
• Gene therapy has potential indications in several monogenetic hereditary metabolic disorders involving the nervous system.
• In lysosomal storage disease, gene correction limited to the most severely affected organ may be insufficient and should be combined with generalized enzyme replacement.
The use of gene therapy to target the inherited neurogenetic disorders of the central nervous system presents specific technical and biological challenges. Neurogenetic disorders are usually associated with widespread involvement of cells throughout the brain. Most of the gene therapy approaches with viral vectors correct the abnormality at site of injection and multiple injections are required for widespread delivery of the normal gene and/or protein. Because some adeno-associated virus vectors can be transported along neuronal pathways from the injected region, widespread vector distribution may take place for correction of a neurogenetic disease.
Gene therapy is being investigated for the treatment of several genetic disorders, some of which involve the nervous system. The aim of somatic gene therapy in neurogenetic disorders is to provide a gene-based approach for correcting the enzymatic defect responsible for the disease and to improve the clinical symptoms. Somatic gene therapy cannot eradicate the disease or prevent its transmission to offspring; this can be achieved only by germline gene therapy, but it is currently not permitted on ethical grounds.
Some of the gene therapy techniques are still in development in experimental models of disease whereas others are in clinical trials. Criteria for patient selection are specified for each clinical trial.
Trinucleotide repeat disorders. Friedreich ataxia is an autosomal recessive neurogenetic disease caused by a GAA triplet repeat in the first intron of the frataxin gene that is mainly associated with atrophy of the spinal cord and progressive neurodegeneration in the cerebellum. There is a decreased level of frataxin protein, which results in mitochondrial dysfunction. There is no effective treatment but gene therapy has the potential. Gene therapy approaches to deliver frataxin and/or brain-derived neurotrophic factor to treat models of Friedreich ataxia have been revised (20). This has involved optimization of the viral vector to reduce immunotoxicity and the reduction of toxicity associated with both frataxin and neurotrophins by modifying dose and route of delivery. Huntington disease, another triple repeat disorder, is described in the MedLink Neurology article Gene therapy of neurodegenerative disorders.
Hereditary metabolic disorders. There is a resurgence of interest in the development of gene-based approaches for inherited neurometabolic disorders with severe neurologic involvement, which are monogenetic diseases caused by enzymatic or structural deficiencies affecting the lysosomal or peroxisomal metabolic activity leading to neuronal death, microglial activation, inflammatory demyelination, and axonal degeneration (23). Gene therapy has potential indications in several hereditary metabolic disorders involving the nervous system, discussed below:
Purine and pyrimidine metabolism. An example of this type of disorder is Lesch-Nyhan syndrome. There is an underlying deficiency of hypoxanthine guanine phosphoribosyl transferase. The potential gene therapy for this disorder involves hypoxanthine guanine phosphoribosyl transferase deficient fibroblasts transfected with retroviral vectors, carrying hypoxanthine guanine phosphoribosyl transferase cDNA; this can produce an active hypoxanthine guanine phosphoribosyl transferase enzyme.
Aminoacidopathies. An example of this type of disorder is phenylketonuria. Phenylalanine hydroxylase deficiency causes an inability to convert phenylalanine to tyrosine. The potential gene therapy for this disorder involves the following: a recombinant adenoviral vector containing the human phenylalanine hydroxylase cDNA was constructed and administered to phenylalanine hydroxylase-deficient mice with restoration of deficiency.
Carbohydrate metabolism. An example of this type of disorder is galactosemia. There is a deficiency of the enzyme involved in the conversion of galactose to glucose. The potential gene therapy for this disorder involves the liver as a target tissue for the introduction and expression of genes for deficient enzymes.
Urea cycle. An example of this type of disorder is ornithine transcarbamylase deficiency. There is a lack of ornithine transcarbamylase as a catalyst in the second step of the urea cycle. The potential gene therapy for this disorder involves beta-galactosidase and ornithine transcarbamylase gene expression in human and mouse primary hepatocytes in vitro. Gene expression was mediated by adenoviral vectors.
Phenylketonuria. This is due to deficiency of phenylalanine hydroxylase, causing an inability to convert phenylalanine to tyrosine. A variant form of it, due to tetrahydrobiopterin deficiency, produces severe neurologic deficits and is discussed separately.
Gene therapy is feasible for phenylketonuria because the gene for phenylalanine hydroxylase is localized to chromosome 12q22-q24. Phenylalanine hydroxylase is expressed in liver cells so the liver is the most reasonable target tissue for gene therapy. Phenylalanine hydroxylase cDNA gene has been introduced into murine fibroblasts and hepatocytes in vitro, and expression has been documented by detection of phenylalanine hydroxylase-specific mRNA and active protein. Recombinant adenoviral vector containing the human phenylalanine hydroxylase cDNA, when administered to phenylalanine hydroxylase-deficient mice, completely normalizes the phenotype within 1 week of treatment, but effects do not persist. A more persistent vector system needs to be developed for application of gene therapy for human phenylketonuria. Complete and persistent phenotypic correction of phenylketonuria in mouse models can be achieved by site-specific genome integration of murine phenylalanine hydroxylase cDNA. Elevated gene expression levels in the phenylketonuria-affected brain in mouse models can be normalized by human phenylalanine hydroxylase gene delivery by an AAV vector. Among all the techniques tried so far, liver-directed AAV-mediated gene transfer has been the most effective gene therapy approach in murine phenylketonuria, although issues of immunogenicity and potential insertional mutagenesis with risk of development of hepatic carcinoma must be resolved prior to clinical use. Complete phenotypic correction of murine phenylketonuria for the longest duration reported to date has been achieved by using the scAAV8 vector, which overcomes some of the disadvantages of AAV-mediated liver transduction (30).
Aromatic L-amino acid decarboxylase (AADC) deficiency. This hampers the body’s ability to make dopamine and serotonin and results in developmental delays as well as a range of motor and behavioral symptoms. The gene therapy uses a viral vector to carry DNA-expressing AADC to the brain. In a clinical trial on children aged 4 to 9 years with AADC deficiency (NCT02852213), convection-enhanced delivery of AAV2-hAADC to the bilateral substantia nigra resulted in increase of dopamine metabolism in all subjects with enhancement of FDOPA uptake within the midbrain and the striatum, which led to clinical improvements in symptoms as well as motor function (22).
Tetrahydrobiopterin deficiency. Tetrahydrobiopterin is required as a cofactor for several enzymes such as aromatic amino acid hydroxylases and nitric oxide syntheses. Tetrahydrobiopterin deficiency causes a variant form of hyperphenylalaninemia that, unlike classical phenylketonuria, cannot be treated by dietary restriction of phenylalanine. The most frequent genetic defects responsible for deficiency of tetrahydrobiopterin biosynthesis are autosomal recessive mutations in the gene encoding 6-pyruvoyl-tetrahydropterin synthase, located on the chromosome 11q22.3-q23.3. Mutations leading to tetrahydrobiopterin deficiency impair hepatic phenylalanine degradation and neuronal monoamine neurotransmitter biosynthesis, thus, severely affecting brain development and function. Tetrahydrobiopterin is required not only for the catabolic phenyl-hydroxy hydroxylase, but also for tyrosine and tryptophan hydroxylases, the rate-limiting enzymes for dopamine and serotonin biosynthesis, respectively. Synthetic tetrahydrobiopterin replacement is complex and only partially effective.
In vitro correction of tetrahydrobiopterin deficiency by using retrovirus-mediated transfer of 6-pyruvoyl-tetrahydropterin synthase cDNA into primary fibroblast cultures established from different patients has been demonstrated. This indicates the potential usefulness of the somatic gene therapy for this condition.
Ornithine transcarbamylase deficiency. Ornithine transcarbamylase deficiency is an inborn error of urea synthesis that has been considered as a model for liver-directed gene therapy because current treatment has failed to avert a high mortality or morbidity from hyperammonemic coma. Restoration of enzyme activity in the liver should suffice to normalize metabolism.
Injection of an adenoviral vector expressing ornithine transcarbamylase can correct the metabolic defect rapidly in an ornithine transcarbamylase-deficient newborn mouse model. Thus, gene therapy is feasible in this disorder and may be lifesaving in patients suffering from severe hyperammonemic crisis. Fetal gene therapy for ornithine transcarbamylase deficiency by intrahepatic plasmid DNA-micro-bubble injection combined with hepatic ultrasound insonation significantly lowers levels of blood ammonia as well as urinary orotic acid (21). This approach might provide more time for ornithine transcarbamylase-deficient infants until liver transplantation.
In an earlier pilot trial, a third-generation adenovirus vector containing human ornithine transcarbamylase cDNA was infused into the right hepatic artery in adults with partial ornithine transcarbamylase deficiency and resulted in death of 1 of the patients due to hepatotoxicity (26). The trial was stopped, and the whole event was thoroughly investigated due to wide publicity of the first death due to gene therapy, which set back further trials of gene therapy.
Lesch-Nyhan syndrome. The Lesch-Nyhan syndrome occurs because of a deficiency of hypoxanthine-guanine phosphoribosyltransferase, resulting in an increase of purine biosynthesis. There is no effective therapy for the central nervous system disorder in this syndrome, although allopurinol can control the manifestations of gout.
Gene therapy is feasible for this syndrome. Hypoxanthine-guanine phosphoribosyltransferase deficient fibroblasts transfected with retroviral vectors carrying hypoxanthine-guanine phosphoribosyltransferase cDNA can produce an active hypoxanthine-guanine phosphoribosyltransferase enzyme. A mouse model of the disease is available and hypoxanthine-guanine phosphoribosyltransferase activity is detectable in the mouse bone marrow after transfection with hypoxanthine-guanine phosphoribosyltransferase cDNA carrying retroviral vectors. This technique has been used in a 22-year-old patient via bone marrow transplantation, but the results were equivocal. The percentage of cells that need to be successfully transfected for a clinically effective gene expression is not known.
Canavan disease. This is an autosomal recessive disorder caused by aspartoacylase (ASPA) deficiency. The gene for ASPA has been cloned, and mutations that lead to deficiency of ASPA have been identified. The gene has been localized to the terminal end of the short arm of chromosome 17. There is no cure for this disease. Symptomatic management includes treatment of seizures and general rehabilitation measures. The first in vivo gene transfer study was carried out in 2 children with Canavan disease to assess the in vivo toxicity and efficacy of ASPA gene delivery (13). The results suggested that low protein diet with pAAVaspa is well tolerated in human subjects, and is associated with biochemical, radiological, and clinical changes. The vector used had 3 elements: (1) adeno-associated virus (AAV) plasmid, (2) gene expressing ASPA, and (3) a low-risk artificial structure (including liposome and polymer). A prospective cohort study of safety and efficacy of gene therapy with an AAV carrying the ASPA gene resulted in a decrease in elevated NAA in the brain and slowed progression of brain atrophy with some improvement in seizure frequency (14).
Adrenoleukodystrophy. The main biochemical defect is an impaired oxidation of saturated very-long-chain fatty acids and is related to deletions and mutations of the adrenoleukodystrophy gene that has been cloned. Metabolic abnormality is due to defects of a 75-kd peroxisomal integral membrane protein that is a member of the adenosine triphosphate-binding cassette transporter family. Mutations in X-adrenoleukodystrophy gene have been found in all X-adrenoleukodystrophy patients.
X-adrenoleukodystrophy is a promising condition for gene therapy because it can be diagnosed years before neurologic damage occurs. In mouse models of X-linked adrenoleukodystrophy, viral vector-mediated, long-lasting delivery of insulin-like growth factor-1 and neurotrophin-3 significantly halts the progression of the disease and protects against the demyelination process. Two gene therapies methods are being evaluated for clinical application.
(1) The first aims at replacing the endogenous brain microglia from patients by autotransplantation of genetically corrected hematopoietic stem cells using a lentiviral vector. Therapeutic success using this approach has been obtained in X-linked adrenoleukodystrophy and metachromatic leukodystrophy (01). It showed efficacy in 2 boys with cerebral adrenoleukodystrophy, and at 3-year follow-up, the hematopoiesis remained polyclonal with 7% to 14% of granulocytes, monocytes, and T and B lymphocytes expressing the lentivirally encoded ALD protein, with arrest of cerebral demyelination in both patients (06). A single-group, open-label, phase 2/3 safety and efficacy study is evaluating infusion of autologous CD34+ cells transduced with the elivaldogene tavalentivec (Lenti-D) lentiviral vector in boys with cerebral adrenoleukodystrophy (NCT01896102). Early results of this study suggest that Lenti-D gene therapy may be a safe and effective alternative to allogeneic stem-cell transplantation in boys with early-stage cerebral adrenoleukodystrophy, but additional follow-up is needed to fully assess the duration of response and long-term safety (08).
(2) The second strategy aims at targeting directly the adrenoleukodystrophy gene into brain glial cells using stereotactic injections of viral vectors. No clinical results are available for this approach.
Lysosomal storage disorders. There are about 50 distinct genetic disorders described as lysosomal storage diseases. They are mainly due to abnormalities in the catabolism of macromolecules such as glycolipids, glycoproteins, and glycosaminoglycans and result in intracellular accumulation of the storage material. The most severely affected organs are the kidney, the heart, and the central nervous system. Identification of the deficient enzyme responsible for each lysosomal storage disease enables early diagnosis, even in the prenatal stage. Most of the genes have been cloned, and pathophysiology has been partially clarified.
Main forms of therapy are enzyme replacement, bone marrow transplantation, and gene therapy. The following points should be considered in formulating therapeutic strategies for lysosomal storage disease:
• The ubiquitous nature of lysosomal enzymes that are expressed in all body tissues, except erythrocytes.
• Tissue specificity in the pathological accumulation of the substrate.
• Lysosomal enzymes are partially secreted outside the cell and can be taken up by the adjacent cells by endocytosis.
• There is a direct enzyme transfer system by cell-to-cell contact.
• A low level (10% to 15%) of the enzyme activity may be compatible with a normal life.
• Transplantation of a healthy organ in a patient with lysosomal storage disease will lead to accumulation of the substrate with eventual destruction of the transplanted organ.
In generalized diseases such as lysosomal storage disease, gene correction limited to the most severely affected organ may be insufficient and would need to be combined with generalized enzyme replacement to ensure an optimal homeostatic threshold. Several techniques have been tested in experimental animals, and clinical trials are in progress in some of the diseases. Retroviral vectors (and adeno-associated vectors and lentiviral vectors) have been extensively tested in lysosomal disorders with some success.
Because the blood-brain barrier hinders the uptake of lysosomal enzymes from the peripheral circulation into the brain, direct gene delivery to the brain has been considered as a therapeutic option. In a mouse model of mucopolysaccharidosis type IIIA, which occurs due to an inherited deficiency of the lysosomal hydrolase sulphamidase, lentiviral-mediated intraventricular sulphamidase gene delivery led to improvements in behavior (17).
Batten disease (late infantile neuronal ceroid lipofuscinosis) has been treated with gene therapy. In an open-label clinical trial, an adeno-associated viral vector expressing the human CLN2 cDNA was injected into several locations in the brains of 10 children with Batten disease (28). There were some unexplained adverse effects not related to the viral vector, and results suggest a slowing of progression of Batten disease in the treated children. On this basis, additional studies have been proposed to assess the safety and efficacy of this approach. One of the 2 ongoing clinical trials is testing the safety and efficacy of a virus called AAVrh.10 as the gene delivery system and is slightly different from the vector used in previous trials (NCT01414985). This third protocol of phase 2 study will use the same virus AAVrh.10 as the gene delivery system and has expanded the eligibility criteria.
Gaucher disease. Gene therapy may become the treatment of choice for patients with Gaucher disease in the future. Various approaches to gene therapy are as follows:
• Because the disease can be corrected by transplantation of allogeneic hematopoietic cells, introduction of a wild-type of glucocerebrosidase gene into autologous hematopoietic stem cells and infusion of these genetically engineered cells into the patient is an attractive prospect for the patient. Results obtained in animal and cell culture model systems have provided the scientific basis for clinical trials (that are now in progress) of gene transfer to hematopoietic stem cells in Gaucher disease.
• Cells expressing human glucocerebrosidase from a retroviral vector repopulate macrophages and central nervous system microglia after murine bone marrow transplantation. This suggests that gene therapy for the neuropathic form of this disease is feasible.
• Type 2 Gaucher cells infected with the retrovirus carrying the human cDNA have been shown to be corrected to normal levels of glucocerebrosidase activity.
• A phase 1 trial for Gaucher disease is in progress wherein the glucocerebrosidase gene is introduced into the patient's hematopoietic stem cells. The retroviral vector carrying the Gaucher cerebrosidase gene will be used to transfer this gene to the purified stem cells in the laboratory; the gene will then be transfused into patients.
• The recombinant DNA Advisory Committee has approved a protocol involving an ex vivo gene transfer and autologous transplantation of CD34+ cells.
Hunter syndrome. Hunter syndrome is mucopolysaccharidosis type 2 involving deficiency of iduronate-2-sulfatase. The enzyme hydrolyzes 2-sulfate groups from nonreducing terminal iduronide moieties of glycosaminoglycan molecules resulting in a characteristic phenotype.
Attempts at systemic therapy have been based on early in vitro observations showing that normal glycosaminoglycan metabolism was restored when iduronate-2-sulfatase-deficient fibroblasts were cocultured with cells having normal enzyme activity. Clinical trials of bone marrow transplantation have demonstrated a therapeutic response in some tissues. The limitations are the usual problems associated with bone marrow transplantation, but this experience has suggested that gene transfer targeted at autologous blood cells might provide a means of treatment of this disease.
Clinical trials of bone marrow transplantation have demonstrated a therapeutic response in some tissues. The limitations are the usual problems associated with bone marrow transplantation, but this experience has suggested that gene transfer targeted at autologous blood cells might provide a means of treatment of this disease.
A phase 1/2 study to determine the safety of retroviral-mediated transfer of the iduronate-2-sulfatase gene into lymphocytes of patients with mucopolysaccharidosis 2 (mild Hunter syndrome) was done several years ago, but results have not been published. A dose ranging phase 1/2 study of RGX-121, a gene therapy, which is intended to deliver a functional copy of the iduronate-2-sulfatase gene to the central nervous system, is ongoing to determine safety in patients with Hunter syndrome (NCT03566043).
Metachromatic leukodystrophy. Patients with metachromatic leukodystrophy have a deficiency of arylsulfatase A, a lysosomal enzyme that hydrolyzes cerebroside sulfate. This single gene defect leads to lysosomal storage in the central nervous system and peripheral nerves, leading to demyelination as well as to a range of neurologic deficits that are often lethal. Cloning of the human cDNA and the genomic DNA of arylsulfatase has enabled the identification and characterization of the mutations and correlation of the genotype with the phenotype. A low level of enzyme activity is sufficient to prevent lysosomal storage of the sulfatides and partial correction of the gene defect may suffice for therapeutic benefit.
Successful transduction of mouse hematopoietic stem cells and their different progeny with a retroviral vector (MFG-arylsulfatase) incorporating arylsulfatase has been achieved. In vivo transfer of arylsulfatase gene by lentiviral vectors reverses the disease phenotype in mouse model of metachromatic leukodystrophy. Following transplantation of bone marrow cells overexpressing arylsulfatase A from a retroviral vector, the enzyme-secreting cells are endocytosed by recipient cells. The enzyme transfer results in the metabolic cross-correction of recipient cells and the improvement of biochemical, clinical manifestations. Intrathecal injection of an adeno-associated viral vector expressing arylsulfatase A in mouse models of metachromatic leukodystrophy is a useful and non-invasive method for widespread gene delivery to the brain and dorsal root ganglia.
A lentiviral vector was used to transfer a functional ARSA gene into hematopoietic stem cells from 3 presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile metachromatic leukodystrophy that prevented manifestation or progression of the disease, indicating therapeutic potential of this method (04).
Globoid cell leukodystrophy (Krabbe disease). The most common form of Krabbe disease is the infantile form, which presents before the age of 1 with muscle weakness, feeding difficulties, and delayed mental as well as physical development. It is caused by deficient activity of the hydrolytic enzyme galactosylceramidase (GALC). As the disease progresses, muscles continue to weaken with difficulty in swallowing and breathing. Affected infants may also experience vision loss and seizures. They rarely survive beyond the age of 2. Presymptomatic hematopoietic stem cell transplantation is the only treatment for infantile Krabbe disease, but the results are poor, and procedure-related morbidity is high.
A study in the naturally occurring canine model of Krabbe disease has shown that presymptomatic monotherapy with intrathecal AAV9 encoding canine GALC administered into the cisterna magna increased GALC enzyme activity, improved myelination, and attenuated inflammation in the nervous system as well as the manifestations (05). Moreover, treated dogs survived beyond 2.5 years of age, more than 7 times longer than untreated dogs, indicating the potential for therapy of infantile Krabbe disease in humans.
Mucopolysaccharidosis type 1 (Hurler syndrome). Mucopolysaccharidosis type 1 (Hurler syndrome) is due to a mutation in the gene encoding alpha-(L)-iduronidase. The gene defect causes a specific lysosomal enzyme deficiency, resulting in intracellular accumulation and storage of unprocessed glycosaminoglycans, dermatan sulfate, and heparan sulfate.
The uptake of recombinant alpha-(L)-iduronidase into glial and neuronal cells, produced by retrovirally transduced fibroblasts, is followed by high levels of expression of alpha-(L)-iduronidase. Brain cells take up the alpha-(L)-iduronidase through a cation-independent mannose-6-phosphate receptor-mediated pathway, and this uptake is higher in actively dividing or immature brain cells. Neuronal metabolic disturbance in the brain can be corrected, provided the genetically engineered cells are provided by bone marrow transplantation.
Canine alpha-(L)-iduronidase deficiency is a model of Hurler syndrome. In this model, genetically corrected hematopoietic stem cells have been transferred at the fetal stage with resulting engraftment. The therapeutic potential of this method was limited by the low level of gene expression.
Sanfilippo type B syndrome. This lysosomal storage disease is also known as mucopolysaccharidosis type IIIB syndrome and results in progressive deterioration of cognitive acquisition after 2 to 4 years of age. No treatment is available for the neurologic manifestations of the disease. A phase 1/2 clinical trial of intracerebral rAVV2/5 encoding human α-N-acetylglucosaminidase gene was well tolerated and induced sustained enzyme production in the brain (24). Initial immune response subsided later with acquired immunological tolerance. There was improved cognitive development, which needs to be confirmed with long-term follow-up.
Sly syndrome. This is the name given to mucopolysaccharidosis 7 associated with deficiency of the enzyme beta-glucuronidase. Enzyme replacement therapy has not been developed for this disease, as it is rare (less than 100 known patients), and the clinical manifestations are too variable to allow controlled clinical trials. Mice with mucopolysaccharidosis 7 respond well to bone marrow transplantation from normal syngeneic donors, although there is little reduction in brain storage vesicles.
The therapeutic gene has been transferred to hematopoietic stem cells and to fibroblasts by retroviral vectors ex vivo, and the genetically modified cells have been implanted in mice with resulting improvement of visceral lesions but no alterations in brain pathology. Fibroblasts overexpressing enzyme beta-glucuronidase have been shown to reduce lysosomal storage vesicles in the brains of mice with Sly syndrome. Recombinant adenovirus that encodes beta-glucuronidase, delivered intravenously and into the brain parenchyma of mucopolysaccharidosis type 7 mice, has been shown to provide long-term transgene expression and correction of lysosomal distension. Adult mucopolysaccharidosis type 7 mice treated by injection of herpes simplex virus-1 vector expressing beta-glucuronidase into a single site on each side of the brain were shown to have correction of storage lesions in a large volume of brain (02). These findings are important for future treatment of a lysosomal storage disease in the adult brain by gene therapy.
Tay-Sachs disease. It is a variant of GM2 gangliosidosis with infantile onset due to hexosaminidase alpha-subunit deficiency that begins within the first few months of life. There is no effective treatment for this disease. Attempts at enzyme replacement, cellular infusions, and bone marrow transplantation have proven unsuccessful. Gene therapy is feasible for Tay-Sachs disease.
Intravenous administration of adenoviral vectors, coding for both alpha-subunits and beta-subunits, in hexosaminidase A-deficient knock-out mice resulted in the highest HexA activity in the liver. The liver, therefore, is the preferential target organ to deliver a large amount of secreted proteins. The enzymatic correction in these animal models was likely due to direct cellular transduction by adenoviral vectors and uptake of secreted HexA by different organs.
Fabry disease. This is an X-linked lysosomal storage disease caused by a deficiency in the lysosomal enzyme α-galactosidase (α-GAL). Clinical manifestations are progressive kidney disease, peripheral neuropathy, early-onset cerebrovascular disease, gastrointestinal symptoms, hypertrophic cardiomyopathy, arrhythmias, corneal whorls, and angiokeratomas. Usual treatment of this disease is intravenous replacement of the deficient enzyme with agalsidase alpha or agalsidase beta. Now migalastat, an oral molecular chaperone, has been approved. Gene therapy, through both ex vivo and in vivo methods, is in development (09).
In an ex vivo approach, CD34-positive hematopoietic stem cells are harvested and modified through recombinant lentivirus-mediated gene transfer of the alpha-GAL gene. These cells are infused into autologous recipients with alpha-GAL production at 1 year and 2 years (18). The first patient in the clinical trial has now discontinued enzyme replacement and is currently undergoing long-term follow-up study.
In the in vivo approach, infusion of AAV-mediated gene transfer increases enzyme activity levels as shown in preclinical studies on alpha-GAL knock-out mice.
Another approach is through the administration of alpha-GAL mRNA encapsulated in nanocapsules to help stimulate production of alpha-GAL, which is another unique form of therapy (07). Although all these approaches increase alpha-GAL enzyme activity, antibody generation can inhibit enzyme activity with aggravation of clinical outcomes. It is currently unknown whether the introduction of sustained enzyme production will lead to increased immunogenicity with associated complications.
Leber hereditary optic neuropathy. This disorder is characterized by severe and rapidly progressive visual loss caused by a mutation in the mitochondrial gene encoding NADH:ubiquinone oxidoreductase subunit 4 (ND4). Preliminary results of a phase 1 open-label dose escalation trial have determined the safety of an adeno-associated virus vector (scAAV2-P1ND4v2) injected unilaterally into the eyes of patients with Leber hereditary optic neuropathy (10).
Hereditary transthyretin amyloidosis. This is also called ATTR amyloidosis, and is characterized by accumulation in tissues of amyloid fibrils composed of misfolded transthyretin protein. Hereditary ATTR (hATTR) amyloidosis can be triggered by more than 100 different pathogenic mutations in TTR, has an autosomal dominant pattern of inheritance, and has a clinical phenotype dominated by amyloid polyneuropathy or cardiomyopathy, with most patients having a combination of the 2. Currently available treatments are not satisfactory. Inhibition of TTR protein synthesis with inotersen or patisiran by mRNA targeting-based gene silencing produces symptomatic relief and functional improvement but is limited by the need for long-term administration to maintain TTR knockdown.
NTLA-2001 is a CRISPR-Cas9-based in vivo gene editing therapy, administered by intravenous infusion using a lipid nanoparticle delivery system with liver tropism, carrying a single guide RNA that targets human TTR. It leads to greater TTR knockdown than currently available therapies. In a clinical trial on patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR (11).
None have yet been identified.
Gene therapy has been shown to be effective in the laboratory models of disease, but the long-term efficacy in human patients remains to be demonstrated. Clinical trials in humans in several neurogenetic disorders have shown that gene therapy can be performed safely and effectively.
Gene therapy has been studied extensively in animal experiments and human clinical trials. The record of safety has been well maintained. The following are some of the adverse effects that have been observed.
Retroviral vectors. Although the viruses are made replication-defective, the possibility of producing a replication-competent virus remains. Stable transformation of target cells makes it is difficult to reverse the treatment if appearance of undesirable side effects requires that the treatment be stopped.
Herpes simplex virus vectors. The issue of neuronal cytotoxicity remains unsolved. Novel recombinant herpes simplex virus-1 vectors that appear to reduce the risk of neurovirulence and cytotoxicity also are less effective. Further studies on the source of herpes simplex virus-1 cytotoxicity and improved vector development are required.
Adenoviral vectors. Most of the adverse effects of viral vectors are associated with adenoviruses and some of these are inflammatory effects. These have been observed mostly in the respiratory tract after local administration of adenoviral vectors.
Induction of immune response. One major concern about using adenoviral vectors for repetitive gene delivery is the induction of immune response to the vector that impedes effective gene transduction. Alternate use of adenovirus vectors from different serotypes within the same subgroup can circumvent anti-adenovirus humoral immunity to permit effective gene transfer after repeat administration; however, the chronicity of the expression is limited by cellular immune processes directed both against the transgene and the viral gene products expressed by the vector. Animal experimental data show the potential of immunosuppression in the management of immune reactions triggered by adenoviral vectors by use of monoclonal antibodies.
Insertional mutagenesis. Adverse events observed during some of the hematopoietic stem cell gene therapy clinical trials are linked to insertional activation of proto-oncogenes by integrated proviral vectors leading to clonal expansion and eventual development of leukemia (29). Research is ongoing to further elucidate the mechanisms of insertional mutagenesis in hematopoietic stem cells’ progenitor cells due to integrating gene transfer vectors. Assays are available for predicting genotoxicity and mapping vector integration sites, and some approaches are available for minimizing genotoxicity.
Toxicity of lipopolysaccharides. The presence of lipopolysaccharides as a contaminant in plasmid prepared from Escherichia coli is well documented. Lipopolysaccharides are internalized during adenovirus-mediated gene transfer and can generate toxicity in primary cell types. Other commonly used nonviral methods of gene transfer can also generate toxicity in some primary cell types. Toxicity has been found to be due to lipid A component of lipopolysaccharides. The lipopolysaccharides-chelating antibiotic polymyxin B, when present at concentration of 10 to 30 µg/ml, can block this activity.
Clinical trials of gene therapy. As of August 2021, the U.S. government clinical trials U.S. government clinical trials website lists 266 studies for gene therapy of genetic disorders involving the nervous system.
Performing gene therapy in utero would allow early correction prior to disease onset and, thus, is 1 of the few therapeutic modalities that could promise the birth of a healthy infant. In utero gene therapy has been carried out in animal models and is technically feasible.
In some lysosomal storage diseases, considerable alterations of the central nervous system occur prior to birth, and the disease progresses rapidly after birth when no effective treatment is available. Treatment may need to be initiated before birth to prevent the onset or progression of irreversible neurologic changes. Brain-directed in utero gene therapy through an intraventricular route would be an effective strategy to treat some lysosomal storage diseases with central nervous system involvement. AAV9-intrauterine gene therapy in late gestation in nonhuman primates was shown to efficiently transduce both the central and peripheral nervous systems with genes of translational relevance to neurogenetic disorders characterized by perinatal onset of neuropathology (16).
Advances in molecular genetics have had more impact on neurology than on any other branch of medicine. The brain is the most complex organ in the body, and it is not surprising that 75% of the human genes are expressed in the brain, including 25% of the "housekeeping" genes that have metabolic functions common to all cells. More than 150 genetic disorders involving the nervous system have been defined at the molecular level, and this number is increasing. Genetic lesions that have been discovered include gene deletions, translocations that interrupt the gene, point mutations, and abnormal replications of trinucleotide repeats. In addition to single gene disorders, several diseases are polygenic and occur due to interaction of genetic and exogenous factors such as environmental toxins.
Current treatment of neurogenetic diseases, in general, is inadequate and offers the prospect of normal life only for a handful of these diseases. Present treatment of genetic disease does not remove the cause, but only corrects symptoms or secondary biochemical defects. Diseases most amenable to gene therapy would be those where a single gene product is missing, and the gene expression has a simple mechanism. Genetic defects in several neurogenetic disorders have been identified. Mouse models are available for testing various therapies for these disorders (Table 1).
Gene defect or manipulation
Human disease equivalent
Gene therapy outcomes in murine models are difficult to translate into human clinical application. Therefore, large animal models of neurologic disorders are being developed.
Gene therapy. Mutations of several autosomal dominant neurologic disorders appear to result in the gain of gene function where the defective gene overrides the effects of its wild-type homologue. In these diseases, the goal of gene therapy is the replacement of the defective gene by its wild type. The main problem is the need to replace the gene, rather than just add copies of the wild-type gene. Another approach would be to inactivate the defective gene while keeping its wild counterpart active. Because the difference between the wild-type and the mutant type is only a single base pair, the methods applied should use this difference to achieve inactivation. This may be possible with a ribozyme approach that cleaves the defective RNA while keeping the wild-type intact. Although identification and replacement of defective genes in monogenic genetic disorders may be the ideal therapies, they are not essential.
Transplantation of autologous hematopoietic stem cells genetically modified to express the missing protein is effective for the treatment of Hurler syndrome, metachromatic leukodystrophy, Krabbe disease, and X-linked adrenoleukodystrophy.
Scientific basis of gene therapy for lysosomal storage diseases. Murine models of lysosomal storage disease with beta-glucuronidase deficiency (mucopolysaccharidosis type VII) are available. Remarkable results are obtained in vivo by using implants of genetically modified cells. A retroviral vector containing cDNA for human beta-glucuronidase has been used to transfer the gene into cutaneous fibroblasts, bone marrow cells, and myoblasts; these were then implanted into murine models. Fibroblasts, introduced into collagen gel were used to constitute an "organoid" that was implanted into the animal's peritoneal cavity and expressed beta-glucuronidase for 150 days. Considerable enzymatic activity was observed in various organs before signs of lysosomal overload disappeared. Ventricular vector delivery of a lentiviral vector expressing murine beta-glucuronidase cleared lysosomal storage within the CNS more effectively than intravenous administration in a mouse model of mucopolysaccharidosis type VII, making this a reasonable, but more challenging, therapeutic option (03).
Mucopolysaccharidosis type II (MPS II) is a neuropathic lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase, which leads to the accumulation of glycosaminoglycans. An experimental study on MPS II mice has shown that lentiviral hematopoietic stem cell gene therapy is a promising approach for the treatment of central nervous system lesions in MPS II (25). These experiments demonstrate the basis for clinical application of gene therapy for lysosomal storage disorders. The efficacy and safety of gene therapy has been demonstrated in numerous preclinical studies, and promising clinical results suggest that gene therapy treatment for several lysosomal storage disease is a real possibility (19).
The goal of gene therapy is to provide therapeutic levels of the deficient enzymatic activity to treat both systemic and neurologic defects due to lysosomal storage diseases. Because these disorders are monogenic, it is theoretically possible to correct the disease by correcting the gene deficiency. Involvement of multiple systems in these disorders could be a limiting factor because it is difficult to genetically modify all the affected cells. Combination of various strategies may be required to achieve full correction in lysosomal storage diseases with neurologic involvement (12). Several methods are being considered for the delivery of gene therapy for lysosomal storage disorders, including intraventricular, intrathecal, intranasal, and systemic administrations (27). Systemic delivery by intravenous infusion can also achieve widespread delivery to the central nervous system; however, the distribution to the brain is greatly dependent on the ability of the vector to cross the blood-brain barrier. Some subtypes of adeno-associated virus vectors are more effective than others in crossing the blood-brain barrier. Among those for direct introduction of gene therapeutic into the brain, the intraventricular route provides the most widespread distribution of gene therapy vectors to the brain.
AAV vector-mediated gene transfer strategies currently being developed for MPSIII (Sanfilippo syndrome) rely on local delivery of AAV vectors to the CNS, either through direct intraparenchymal injection at several sites or through delivery to the CSF, or exploit the properties of certain AAV serotypes capable of crossing the blood-brain barrier on systemic administration. Although studies in animal models of MPSIII, which primarily affects the brain and spinal cord, have provided evidence supporting the efficacy and safety of all these strategies, there are considerable differences between the different routes of administration in terms of procedure-associated risks, vector dose requirements, sensitivity to the effect of circulating neutralizing antibodies that block AAV transduction, and potential toxicity (15). Ongoing clinical studies should shed light on which gene transfer strategy leads to the highest clinical benefits while minimizing risks.
Scientific basis for gene therapy of trinucleotide repeat disorders. Trinucleotide or (“triple repeats") are dynamic mutations because they show instability of alleles through generations, and the mutation rate is related to the copy number of repeats. The mutability of 1 of these sequences in the offspring is different from that in the parents. A distinctive feature of all these diseases is a phenomenon termed “anticipation," whereby the symptoms appear at earlier ages and with greater severity in the successive generation. This phenomenon is due to the instability of the expanded DNA sequence as it passes through the germ line and the number of repeats increases. Several neurologic diseases due to the expansion of trinucleotide repeats have been described. Currently, only 1 of these, Huntington disease, is a candidate for gene therapy (see Gene therapy of neurodegenerative disorders). Friedrich ataxia is another potential candidate for gene therapy.
RNA interference, which is a method for inhibiting target gene expression, provides another promising tool for attacking the fundamental problem in trinucleotide repeat disorders directly.
Gene editing in neurogenetic disorders by CRISPR-Cas9 system. CRISPR/Cas9 system, in combination with delivery vectors engineered to target disease tissue, has enabled in vivo correction of mutations in disease models of ornithine transcarbamylase deficiency, lysosomal storage disorders, and hereditary transthyretin amyloidosis.
K K Jain MD†
Dr. Jain was a consultant in neurology and had no relevant financial relationships to disclose.See Profile
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