Safety and Efficacy of Intravenous and Intrathecal Delivery of AAV9-Mediated ARSA in Minipigs.

Metachromatic leukodystrophy (MLD) is a hereditary neurodegenerative disease characterized by demyelination and motor and cognitive impairments due to deficiencies of the lysosomal enzyme arylsulfatase A (ARSA) or the saposin B activator protein (SapB). Current treatments are limited; however, gene therapy using adeno-associated virus (AAV) vectors for ARSA delivery has shown promising results. The main challenges for MLD gene therapy include optimizing the AAV dosage, selecting the most effective serotype, and determining the best route of administration for ARSA delivery into the central nervous system. This study aims to evaluate the safety and efficacy of AAV serotype 9 encoding ARSA (AAV9-ARSA) gene therapy when administered intravenously or intrathecally in minipigs, a large animal model with anatomical and physiological similarities to humans. By comparing these two administration methods, this study contributes to the understanding of how to improve the effectiveness of MLD gene therapy and offers valuable insights for future clinical applications.

Various AAV Serotypes and Their Applications in Gene Therapy: An Overview.

Despite scientific discoveries in the field of gene and cell therapy, some diseases still have no effective treatment. Advances in genetic engineering methods have enabled the development of effective gene therapy methods for various diseases based on adeno-associated viruses (AAVs). Today, many AAV-based gene therapy medications are being investigated in preclinical and clinical trials, and new ones are appearing on the market. In this article, we present a review of AAV discovery, properties, different serotypes, and tropism, and a following detailed explanation of their uses in gene therapy for disease of different organs and systems.

Gene Therapy of Sphingolipid Metabolic Disorders.

Sphingolipidoses are defined as a group of rare hereditary diseases resulting from mutations in the genes encoding lysosomal enzymes. This group of lysosomal storage diseases includes more than 10 genetic disorders, including GM1-gangliosidosis, Tay-Sachs disease, Sandhoff disease, the AB variant of GM2-gangliosidosis, Fabry disease, Gaucher disease, metachromatic leukodystrophy, Krabbe disease, Niemann-Pick disease, Farber disease, etc. Enzyme deficiency results in accumulation of sphingolipids in various cell types, and the nervous system is also usually affected. There are currently no known effective methods for the treatment of sphingolipidoses; however, gene therapy seems to be a promising therapeutic variant for this group of diseases. In this review, we discuss gene therapy approaches for sphingolipidoses that are currently being investigated in clinical trials, among which adeno-associated viral vector-based approaches and transplantation of hematopoietic stem cells genetically modified with lentiviral vectors seem to be the most effective.

Gene and Cell Therapy for Epilepsy: A Mini Review.

Epilepsy is a chronic non-infectious disease of the brain, characterized primarily by recurrent unprovoked seizures, defined as an episode of disturbance of motor, sensory, autonomic, or mental functions resulting from excessive neuronal discharge. Despite the advances in the treatment achieved with the use of antiepileptic drugs and other non-pharmacological therapies, about 30% of patients suffer from uncontrolled seizures. This review summarizes the currently available methods of gene and cell therapy for epilepsy and discusses the development of these approaches. Currently, gene therapy for epilepsy is predominantly adeno-associated virus (AAV)-mediated delivery of genes encoding neuro-modulatory peptides, neurotrophic factors, enzymes, and potassium channels. Cell therapy for epilepsy is represented by the transplantation of several types of cells such as mesenchymal stem cells (MSCs), bone marrow mononuclear cells, neural stem cells, and MSC-derived exosomes. Another approach is encapsulated cell biodelivery, which is the transplantation of genetically modified cells placed in capsules and secreting various therapeutic agents. The use of gene and cell therapy approaches can significantly improve the condition of patient with epilepsy. Therefore, preclinical, and clinical studies have been actively conducted in recent years to prove the benefits and safety of these strategies.

Mesenchymal Stem Cell-Based Therapy for Lysosomal Storage Diseases and Other Neurodegenerative Disorders.

Lysosomal storage diseases (LSDs) are a group of approximately 50 genetic disorders caused by mutations in genes coding enzymes that are involved in cell degradation and transferring lipids and other macromolecules. Accumulation of lipids and other macromolecules in lysosomes leads to the destruction of affected cells. Although the clinical manifestations of different LSDs vary greatly, more than half of LSDs have symptoms of central nervous system neurodegeneration, and within each disorder there is a considerable variation, ranging from severe, infantile-onset forms to attenuated adult-onset disease, sometimes with distinct clinical features. To date, treatment options for this group of diseases remain limited, which highlights the need for further development of innovative therapeutic approaches, that can not only improve the patients' quality of life, but also provide full recovery for them. In many LSDs stem cell-based therapy showed promising results in preclinical researches. This review discusses using mesenchymal stem cells for different LSDs therapy and other neurodegenerative diseases and their possible limitations.

Current Strategies for the Gene Therapy of Autosomal Recessive Congenital Ichthyosis and Other Types of Inherited Ichthyosis.

Mutations in genes such as transglutaminase-1 (TGM1), which are responsible for the formation and normal functioning of a lipid barrier, lead to the development of autosomal recessive congenital ichthyosis (ARCI). ARCIs are characterized by varying degrees of hyperkeratosis and the presence of scales on the body surface since birth. The quality of life of patients is often significantly affected, and in order to alleviate the manifestations of the disease, symptomatic therapy with moisturizers, keratolytics, retinoids and other cosmetic substances is often used to improve the condition of the patients' skin. Graft transplantation is commonly used to correct defects of the eye. However, these approaches offer symptomatic treatment that does not restore the lost protein function or provide a long-term skin barrier. Gene and cell therapies are evolving as promising therapy for ARCIs that can correct the functional activity of altered proteins. However, these approaches are still at an early stage of development. This review discusses current studies of gene and cell therapy approaches for various types of ichthyosis and their further prospects for patient treatment.

Functionality of a bicistronic construction containing HEXA and HEXB genes encoding ß-hexosaminidase A for cell-mediated therapy of GM2 gangliosidoses.

Tay-Sachs disease and Sandhoff disease are severe hereditary neurodegenerative disorders caused by a deficiency of ß-hexosaminidase A (HexA) enzyme, which results in the accumulation of GM2 gangliosides in the nervous system cells. In this work, we analyzed the efficacy and safety of cell-mediated gene therapy for Sandhoff disease and Sandhoff disease using a bicistronic lentiviral vector encoding cDNA of HexA α- and ß-subunit genes separated by the nucleotide sequence of a P2A peptide (HEXA-HEXB). The functionality of the bicistronic construct containing the HEXA-HEXB genetic cassette was analyzed in a culture of HEK293T cells and human umbilical cord blood mononuclear cells (hUCBMCs). Our results showed that the enzymatic activity of HexA in the conditioned medium harvested from genetically modified HEK293T-HEXA-HEXB and hUCBMCs-HEXA-HEXB was increased by 23 and 8 times, respectively, compared with the conditioned medium of native cells. Western blot analysis showed that hUCBMCs-HEXA-HEXB secreted both completely separated HEXA and HEXB proteins, and an uncleaved protein containing HEXA + HEXB linked by the P2A peptide. Intravenous injection of genetically modified hUCBMCs-HEXA-HEXB to laboratory Wistar rats was carried out, and the HexA enzymatic activity in the blood plasma of experimental animals, as well as the number of live cells of immune system organs (spleen, thymus, bone marrow, lymph nodes) were determined. A significant increase in the enzymatic activity of HexA in the blood plasma of laboratory rats on days 6 and 9 (by 2.5 and 3 times, respectively) after the administration of hUCBMCs-HEXA-HEXB was shown. At the same time, the number of live cells in the studied organs remained unchanged. Thus, the functionality of the bicistronic genetic construct encoding cDNA of the HEXA and HEXB genes separated by the nucleotide sequence of the P2A peptide was shown in vitro and in vivo. We hypothesize that due to the natural ability of hUCBMCs to overcome biological barriers, such a strategy can restore the activity of the missing enzyme in the central nervous system of patients with GM2 gangliosidoses. Based on the obtained data, it can be concluded that intravenous administration of hUCBMCs with HexA overexpression is a promising method of the therapy for GM2 gangliosidoses. The animal protocol was approved by the Animal Ethics Committee of the Kazan Federal University (No. 23) on June 30, 2020.

Serum Cytokine Profile, Beta-Hexosaminidase A Enzymatic Activity and GM2 Ganglioside Levels in the Plasma of a Tay-Sachs Disease Patient after Cord Blood Cell Transplantation and Curcumin Administration: A Case Report.

Tay-Sachs disease (TSD) is a progressive neurodegenerative disorder that occurs due to a deficiency of a ß hexosaminidase A (HexA) enzyme, resulting in the accumulation of GM2 gangliosides. In this work, we analyzed the effect of umbilical cord blood cell transplantation (UCBCT) and curcumin administration on the course of the disease in a patient with adult TSD. The patient's serum cytokine profile was determined using multiplex analysis. The level of GM2 gangliosides in plasma was determined using mass spectrometry. The enzymatic activity of HexA in the plasma of the patient was assessed using a fluorescent substrate assay. The HexA α-subunit (HexA) concentration was determined using ELISA. It was shown that both UCBCT and curcumin administration led to a change in the patient's cytokine profile. The UCBCT resulted in an increase in the concentration of HexA in the patient's serum and in an improvement in the patient's neurological status. However, neither UCBCT nor curcumin were able to alter HexA activity and the level of GM2 in patient's plasma. The data obtained indicate that UCBCT and curcumin administration can alter the immunity of a patient with TSD, reduce the level of inflammatory cytokines and thereby improve the patient's condition.

Metachromatic Leukodystrophy: Diagnosis, Modeling, and Treatment Approaches.

Metachromatic leukodystrophy is a lysosomal storage disease, which is characterized by damage of the myelin sheath that covers most of nerve fibers of the central and peripheral nervous systems. The disease occurs due to a deficiency of the lysosomal enzyme arylsulfatase A (ARSA) or its sphingolipid activator protein B (SapB) and it clinically manifests as progressive motor and cognitive deficiency. ARSA and SapB protein deficiency are caused by mutations in the ARSA and PSAP genes, respectively. The severity of clinical course in metachromatic leukodystrophy is determined by the residual ARSA activity, depending on the type of mutation. Currently, there is no effective treatment for this disease. Clinical cases of bone marrow or cord blood transplantation have been reported, however the therapeutic effectiveness of these methods remains insufficient to prevent aggravation of neurological disorders. Encouraging results have been obtained using gene therapy for delivering the wild-type ARSA gene using vectors based on various serotypes of adeno-associated viruses, as well as using mesenchymal stem cells and combined gene-cell therapy. This review discusses therapeutic strategies for the treatment of metachromatic leukodystrophy, as well as diagnostic methods and modeling of this pathology in animals to evaluate the effectiveness of new therapies.

Extracellular vesicles in the diagnosis and treatment of central nervous system diseases.

Extracellular vesicles, including exosomes and microvesicles, play a fundamental role in the activity of the nervous system, participating in signal transmission between neurons and providing the interaction of central nervous system with all body systems. In many neurodegenerative diseases, neurons pack toxic substances into vesicles and release them into the extracellular space, which leads to the spread of misfolded neurotoxic proteins. The contents of neuron-derived extracellular vesicles may indicate pathological changes in the central nervous system, and the analysis of extracellular vesicle molecular content contributes to the development of non-invasive methods for the diagnosis of many central nervous system diseases. Extracellular vesicles of neuronal origin can be isolated from various biological fluids due to their ability to cross the blood-brain barrier. Today, the diagnostic potential of almost all toxic proteins involved in nervous system disease pathogenesis, specifically α-synuclein, tau protein, superoxide dismutase 1, FUS, leucine-rich repeat kinase 2, as well as some synaptic proteins, has been well evidenced. Special attention is paid to extracellular RNAs mostly associated with extracellular vesicles, which are important in the onset and development of many neurodegenerative diseases. Depending on parental cell type, extracellular vesicles may have different therapeutic properties, including neuroprotective, regenerative, and anti-inflammatory. Due to nano size, biosafety, ability to cross the blood-brain barrier, possibility of targeted delivery and the lack of an immune response, extracellular vesicles are a promising vehicle for the delivery of therapeutic substances for the treatment of neurodegenerative diseases and drug delivery to the brain. This review describes modern approaches of diagnosis and treatment of central nervous system diseases using extracellular vesicles.

Production and Application of Multicistronic Constructs for Various Human Disease Therapies.

The development of multicistronic vectors has opened up new opportunities to address the fundamental issues of molecular and cellular biology related to the need for the simultaneous delivery and joint expression of several genes. To date, the examples of the successful use of multicistronic vectors have been described for the development of new methods of treatment of various human diseases, including cardiovascular, oncological, metabolic, autoimmune, and neurodegenerative disorders. The safety and effectiveness of the joint delivery of therapeutic genes in multicistronic vectors based on the internal ribosome entry site (IRES) and self-cleaving 2A peptides have been shown in both in vitro and in vivo experiments as well as in clinical trials. Co-expression of several genes in one vector has also been used to create animal models of various inherited diseases which are caused by mutations in several genes. Multicistronic vectors provide expression of all mutant genes, which allows the most complete mimicking disease pathogenesis. This review comprehensively discusses multicistronic vectors based on IRES nucleotide sequence and self-cleaving 2A peptides, including its features and possible application for the treatment and modeling of various human diseases.

New Approaches to Tay-Sachs Disease Therapy.

Tay-Sachs disease belongs to the group of autosomal-recessive lysosomal storage metabolic disorders. This disease is caused by ß-hexosaminidase A (HexA) enzyme deficiency due to various mutations in α-subunit gene of this enzyme, resulting in GM2 ganglioside accumulation predominantly in lysosomes of nerve cells. Tay-Sachs disease is characterized by acute neurodegeneration preceded by activated microglia expansion, macrophage and astrocyte activation along with inflammatory mediator production. In most cases, the disease manifests itself during infancy, the "infantile form," which characterizes the most severe disorders of the nervous system. The juvenile form, the symptoms of which appear in adolescence, and the most rare form with late onset of symptoms in adulthood are also described. The typical features of Tay-Sachs disease are muscle weakness, ataxia, speech, and mental disorders. Clinical symptom severity depends on residual HexA enzymatic activity associated with some mutations. Currently, Tay-Sachs disease treatment is based on symptom relief and, in case of the late-onset form, on the delay of progression. There are also clinical reports of substrate reduction therapy using miglustat and bone marrow or hematopoietic stem cell transplantation. At the development stage there are methods of Tay-Sachs disease gene therapy using adeno- or adeno-associated viruses as vectors for the delivery of cDNA encoding α and ß HexA subunit genes. Effectiveness of this approach is evaluated in α or ß HexA subunit defective model mice or Jacob sheep, in which Tay-Sachs disease arises spontaneously and is characterized by the same pathological features as in humans. This review discusses the possibilities of new therapeutic strategies in Tay-Sachs disease therapy aimed at preventing neurodegeneration and neuroinflammation.