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.

Iron oxide-coupled CRISPR-nCas9-based genome editing assessment in mucopolysaccharidosis IVA mice.

Mucopolysaccharidosis (MPS) IVA is a lysosomal storage disorder caused by mutations in the GALNS gene that leads to the lysosomal accumulation of keratan sulfate (KS) and chondroitin 6-sulfate, causing skeletal dysplasia and cardiopulmonary complications. Current enzyme replacement therapy does not impact the bone manifestation of the disease, supporting that new therapeutic alternatives are required. We previously demonstrated the suitability of the CRISPR-nCas9 system to rescue the phenotype of human MPS IVA fibroblasts using iron oxide nanoparticles (IONPs) as non-viral vectors. Here, we have extended this strategy to an MPS IVA mouse model by inserting the human GALNS cDNA into the ROSA26 locus. The results showed increased GALNS activity, mono-KS reduction, partial recovery of the bone pathology, and non-IONPs-related toxicity or antibody-mediated immune response activation. This study provides, for the first time, in vivo evidence of the potential of a CRISPR-nCas9-based gene therapy strategy for treating MPS IVA using non-viral vectors as carriers.

Mucopolysaccharidosis IVA: Current Disease Models and Drawbacks.

Mucopolysaccharidosis IVA (MPS IVA) is a rare disorder caused by mutations in the N-acetylgalactosamine-6-sulfate-sulfatase (GALNS) encoding gene. GALNS leads to the lysosomal degradation of the glycosaminoglyccreasans keratan sulfate and chondroitin 6-sulfate. Impaired GALNS enzymes result in skeletal and non-skeletal complications in patients. For years, the MPS IVA pathogenesis and the assessment of promising drugs have been evaluated using in vitro (primarily fibroblasts) and in vivo (mainly mouse) models. Even though value information has been raised from those studies, these models have several limitations. For instance, chondrocytes have been well recognized as primary cells affected in MPS IVA and responsible for displaying bone development impairment in MPS IVA patients; nonetheless, only a few investigations have used those cells to evaluate basic and applied concepts. Likewise, current animal models are extensively represented by mice lacking GALNS expression; however, it is well known that MPS IVA mice do not recapitulate the skeletal dysplasia observed in humans, making some comparisons difficult. This manuscript reviews the current in vitro and in vivo MPS IVA models and their drawbacks.

Regulation of Molecular Targets in Osteosarcoma Treatment.

The most prevalent malignant bone tumor, osteosarcoma, affects the growth plates of long bones in adolescents and young adults. Standard chemotherapeutic methods showed poor response rates in patients with recurrent and metastatic phases. Therefore, it is critical to develop novel and efficient targeted therapies to address relapse cases. In this regard, RNA interference technologies are encouraging options in cancer treatment, in which small interfering RNAs regulate the gene expression following RNA interference pathways. The determination of target tissue is as important as the selection of tissue-specific promoters. Moreover, small interfering RNAs should be delivered effectively into the cytoplasm. Lentiviral vectors could encapsulate and deliver the desired gene into the cell and integrate it into the genome, providing long-term regulation of targeted genes. Silencing overexpressed genes promote the tumor cells to lose invasiveness, prevents their proliferation, and triggers their apoptosis. The uniqueness of cancer cells among patients requires novel therapeutic methods that treat patients based on their unique mutations. Several studies showed the effectiveness of different approaches such as microRNA, drug- or chemotherapy-related methods in treating the disease; however, identifying various targets was challenging to understanding disease progression. In this regard, the patient-specific abnormal gene might be targeted using genomics and molecular advancements such as RNA interference approaches. Here, we review potential therapeutic targets for the RNA interference approach, which is applicable as a therapeutic option for osteosarcoma patients, and we point out how the small interfering RNA method becomes a promising approach for the unmet challenge.

Hematological Findings in Lysosomal Storage Disorders: A Perspective from the Medical Laboratory.

Lysosomal storage disorders (LSDs) are a group of rare and genetic diseases produced by mutations in genes coding for proteins involved in lysosome functioning. Protein defect leads to the lysosomal accumulation of undegraded macromolecules including glycoproteins, glycosaminoglycans, lipids, and glycogen. Depending on the stored substrate, several pathogenic cascades may be activated leading to multisystemic and progressive disorders affecting the brain, eye, ear, lungs, heart, liver, spleen, kidney, skin, or bone. In addition, for some of these disorders, hematological findings have been also reported. In this paper, we review the major hematological alterations in LSDs based on 56 case reports published between 2010 and 2020. Hematological alterations were reported in sphingolipidosis, mucopolysaccharidoses, mucolipidoses, neuronal ceroid lipofuscinosis, glycogenosis, glycoproteinosis, cystinosis, and cholesteryl ester storage disease. They were reported alterations in red cell linage and leukocytes, such as anemia and morphology changes in eosinophils, neutrophils, monocytes, and lymphocytes. In addition, changes in platelet counts (thrombocytopenia) and leukocyte abnormalities on non-peripheral blood samples were also reported for some LSDs. Although in most of the cases these hematological alterations are not pathognomonic of a specific disease or group of LSDs, since they can be easily identified in general clinical laboratories, their identification may contribute to the diagnosis of these disorders. In this sense, we hope that this review contributes to the awareness of the importance of hematological alterations in the diagnosis of LSDs.

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.

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.

Lysosomal storage diseases: current therapies and future alternatives.

Lysosomal storage disorders (LSDs) are a group of monogenic diseases characterized by progressive accumulation of undegraded substrates into the lysosome, due to mutations in genes that encode for proteins involved in normal lysosomal function. In recent years, several approaches have been explored to find effective and successful therapies, including enzyme replacement therapy, substrate reduction therapy, pharmacological chaperones, hematopoietic stem cell transplantation, and gene therapy. In the case of gene therapy, genome editing technologies have opened new horizons to accelerate the development of novel treatment alternatives for LSD patients. In this review, we discuss the current therapies for this group of disorders and present a detailed description of major genome editing technologies, as well as the most recent advances in the treatment of LSDs. We will further highlight the challenges and current bioethical debates of genome editing.