Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches.

Since its discovery in 2012, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system has supposed a promising panorama for developing novel and highly precise genome editing-based gene therapy (GT) alternatives, leading to overcoming the challenges associated with classical GT. Classical GT aims to deliver transgenes to the cells via their random integration in the genome or episomal persistence into the nucleus through lentivirus (LV) or adeno-associated virus (AAV), respectively. Although high transgene expression efficiency is achieved by using either LV or AAV, their nature can result in severe side effects in humans. For instance, an LV (NCT03852498)- and AAV9 (NCT05514249)-based GT clinical trials for treating X-linked adrenoleukodystrophy and Duchenne Muscular Dystrophy showed the development of myelodysplastic syndrome and patient's death, respectively. In contrast with classical GT, the CRISPR/Cas9-based genome editing requires the homologous direct repair (HDR) machinery of the cells for inserting the transgene in specific regions of the genome. This sophisticated and well-regulated process is limited in the cell cycle of mammalian cells, and in turn, the nonhomologous end-joining (NHEJ) predominates. Consequently, seeking approaches to increase HDR efficiency over NHEJ is crucial. This manuscript comprehensively reviews the current alternatives for improving the HDR for CRISPR/Cas9-based GTs.

Bone Growth Induction in Mucopolysaccharidosis IVA Mouse.

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is caused by a deficiency of the N-acetylgalactosamine-6-sulfate-sulfatase (GALNS) enzyme, leading to the accumulation of glycosaminoglycans (GAG), keratan sulfate (KS) and chondroitin-6-sulfate (C6S), mainly in cartilage and bone. This lysosomal storage disorder (LSD) is characterized by severe systemic skeletal dysplasia. To this date, none of the treatment options for the MPS IVA patients correct bone pathology. Enzyme replacement therapy with elosulfase alpha provides a limited impact on bone growth and skeletal lesions in MPS IVA patients. To improve bone pathology, we propose a novel gene therapy with a small peptide as a growth-promoting agent for MPS IVA. A small molecule in this peptide family has been found to exert biological actions over the cardiovascular system. This work shows that an AAV vector expressing a C-type natriuretic (CNP) peptide induces bone growth in the MPS IVA mouse model. Histopathological analysis showed the induction of chondrocyte proliferation. CNP peptide also changed the pattern of GAG levels in bone and liver. These results suggest the potential for CNP peptide to be used as a treatment in MPS IVA patients.

The landscape of CRISPR/Cas9 for inborn errors of metabolism.

Since its discovery as a genome editing tool, the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) system has opened new horizons in the diagnosis, research, and treatment of genetic diseases. CRISPR/Cas9 can rewrite the genome at any region with outstanding precision to modify it and further instructions for gene expression. Inborn Errors of Metabolism (IEM) are a group of more than 1500 diseases produced by mutations in genes encoding for proteins that participate in metabolic pathways. IEM involves small molecules, energetic deficits, or complex molecules diseases, which may be susceptible to be treated with this novel tool. In recent years, potential therapeutic approaches have been attempted, and new models have been developed using CRISPR/Cas9. In this review, we summarize the most relevant findings in the scientific literature about the implementation of CRISPR/Cas9 in IEM and discuss the future use of CRISPR/Cas9 to modify epigenetic markers, which seem to play a critical role in the context of IEM. The current delivery strategies of CRISPR/Cas9 are also discussed.

CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors.

The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the ß-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the ß-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay-Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the ß-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the ß-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.

Iduronate-2-sulfatase interactome: validation by yeast two-hybrid assay.

Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome, is a rare X-linked recessive disease caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS), which activates intracellular accumulation of nonmetabolized glycosaminoglycans such as heparan sulfate and dermatan sulfate. This accumulation causes severe damage to several tissues, principally the central nervous system. Previously, we identified 187 IDS-protein interactions in the mouse brain. To validate a subset of these interactions, we selected and cloned the coding regions of 10 candidate genes to perform a targeted yeast two-hybrid assay. The results allowed the identification of the physical interaction of IDS with LSAMP and SYT1. Although the physiological relevance of these complexes is unknown, recent advances allow us to point out that these interactions could be involved in vesicular trafficking of IDS through the interaction with SYT1, as well as to the ability to form a transcytosis module between the cellular components of the blood-brain-barrier (BBB) through its interaction with LSAMP. These results may shed light on the role of IDS on cellular homeostasis and may also contribute to the understanding of MPS II physiopathology and the development of novel therapeutic strategies to transport recombinant IDS through the brain endothelial cells toward the brain parenchyma.

Mucopolysaccharidoses: Cellular Consequences of Glycosaminoglycans Accumulation and Potential Targets.

Mucopolysaccharidoses (MPSs) constitute a heterogeneous group of lysosomal storage disorders characterized by the lysosomal accumulation of glycosaminoglycans (GAGs). Although lysosomal dysfunction is mainly affected, several cellular organelles such as mitochondria, endoplasmic reticulum, Golgi apparatus, and their related process are also impaired, leading to the activation of pathophysiological cascades. While supplying missing enzymes is the mainstream for the treatment of MPS, including enzyme replacement therapy (ERT), hematopoietic stem cell transplantation (HSCT), or gene therapy (GT), the use of modulators available to restore affected organelles for recovering cell homeostasis may be a simultaneous approach. This review summarizes the current knowledge about the cellular consequences of the lysosomal GAGs accumulation and discusses the use of potential modulators that can reestablish normal cell function beyond ERT-, HSCT-, or GT-based alternatives.

Evaluation of HIV-1 derived lentiviral vectors as transductors of Mucopolysaccharidosis type IV a fibroblasts.

Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal storage disease produced by the deficiency of the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme, leading to glycosaminoglycans (GAGs) accumulation. Since currently available treatments remain limited and unspecific, novel therapeutic approaches are essential for the disease treatment. In an attempt to reduce treatment limitations, gene therapy rises as a more effective and specific alternative. We present in this study the delivery assessment of GALNS and sulfatase-modifying factor 1 (SUMF1) genes via HIV-1 derived lentiviral vectors into fibroblasts from MPS IVA patients. After transduction, we determined GALNS enzymatic activity, lysosomal mass change, and autophagy pathway impairment. Additionally, we computationally assessed the effect of mutations over the enzyme-substrate interaction and phenotypic effects. The results showed that the co-transduction of MPS IVA fibroblasts with GALNS and SUMF1 cDNAs led to a significant increase in GALNS enzyme activity and a reduction of lysosomal mass. We show that patient-specific differences in cellular response are directly associated with the set of mutations on each patient. Lastly, we present new evidence supporting autophagy impairment in MPS IVA due to the presence and changes in autophagy proteins in treated MPS IVA fibroblasts. Our results offer new evidence that demonstrate the potential of lentiviral vectors as a strategy to correct GALNS deficiency.

GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies.

GM2 gangliosidoses are a group of pathologies characterized by GM2 ganglioside accumulation into the lysosome due to mutations on the genes encoding for the ß-hexosaminidases subunits or the GM2 activator protein. Three GM2 gangliosidoses have been described: Tay-Sachs disease, Sandhoff disease, and the AB variant. Central nervous system dysfunction is the main characteristic of GM2 gangliosidoses patients that include neurodevelopment alterations, neuroinflammation, and neuronal apoptosis. Currently, there is not approved therapy for GM2 gangliosidoses, but different therapeutic strategies have been studied including hematopoietic stem cell transplantation, enzyme replacement therapy, substrate reduction therapy, pharmacological chaperones, and gene therapy. The blood-brain barrier represents a challenge for the development of therapeutic agents for these disorders. In this sense, alternative routes of administration (e.g., intrathecal or intracerebroventricular) have been evaluated, as well as the design of fusion peptides that allow the protein transport from the brain capillaries to the central nervous system. In this review, we outline the current knowledge about clinical and physiopathological findings of GM2 gangliosidoses, as well as the ongoing proposals to overcome some limitations of the traditional alternatives by using novel strategies such as molecular Trojan horses or advanced tools of genome editing.