Molecular therapy and nucleic acid adeno-associated virus-based gene therapy delivering combinations of two growth-associated genes to MPS IVA mice.

Mucopolysaccharidosis type IVA (MPS IVA) is caused by a deficiency of the galactosamine (N-acetyl)-6-sulfatase (GALNS) enzyme responsible for the degradation of specific glycosaminoglycans (GAGs). The progressive accumulation of GAGs leads to various skeletal abnormalities (short stature, hypoplasia, tracheal obstruction) and several symptoms in other organs. To date, no treatment is effective for patients with bone abnormalities. To improve bone pathology, we propose a novel combination treatment with the adeno-associated virus (AAV) vectors expressing GALNS enzyme and a natriuretic peptide C (CNP; NPPC gene) as a growth-promoting agent for MPS IVA. In this study, an MPS IVA mouse model was treated with an AAV vector expressing GALNS combined with another AAV vector expressing NPPC gene, followed for 12 weeks. After the combination therapy, bone growth in mice was induced with increased enzyme activity in tissues (bone, liver, heart, lung) and plasma. Moreover, there were significant changes in bone morphology in CNP-treated mice with increased CNP activity in plasma. Delivering combinations of CNP and GALNS gene therapies enhanced bone growth in MPS IVA mice more than in GALNS gene therapy alone. Enzyme expression therapy alone fails to reach the bone growth region; our results indicate that combining it with CNP offers a potential alternative.

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.

Sex Difference Leads to Differential Gene Expression Patterns and Therapeutic Efficacy in Mucopolysaccharidosis IVA Murine Model Receiving AAV8 Gene Therapy.

Adeno-associated virus (AAV) vector-based therapies can effectively correct some disease pathology in murine models with mucopolysaccharidoses. However, immunogenicity can limit therapeutic effect as immune responses target capsid proteins, transduced cells, and gene therapy products, ultimately resulting in loss of enzyme activity. Inherent differences in male versus female immune response can significantly impact AAV gene transfer. We aim to investigate sex differences in the immune response to AAV gene therapies in mice with mucopolysaccharidosis IVA (MPS IVA). MPS IVA mice, treated with different AAV vectors expressing human N-acetylgalactosamine 6-sulfate sulfatase (GALNS), demonstrated a more robust antibody response in female mice resulting in subsequent decreased GALNS enzyme activity and less therapeutic efficacy in tissue pathology relative to male mice. Under thyroxine-binding globulin promoter, neutralizing antibody titers in female mice were approximately 4.6-fold higher than in male mice, with GALNS enzyme activity levels approximately 6.8-fold lower. Overall, male mice treated with AAV-based gene therapy showed pathological improvement in the femur and tibial growth plates, ligaments, and articular cartilage as determined by contrasting differences in pathology scores compared to females. Cardiac histology revealed a failure to normalize vacuolation in females, in contrast, to complete correction in male mice. These findings promote the need for further determination of sex-based differences in response to AAV-mediated gene therapy related to developing treatments for MPS IVA.

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.

Cost-utility analysis of an integrated care program for children with asthma in a medium-income country.

OBJECTIVE: To evaluate the cost-utility of an integrated care program (ASMAIRE Infantil Program [PAI]) for children with asthma compared with standard of care. METHODS: A decision-analytic model was used to compare an integrated care program compared to the standard of care in children with asthma in Bogota, Colombia. Baseline characteristics of the patients were established according to the distribution of patients in the PAI database. Other inputs were obtained from published meta-analysis, local registries, medical bills, general mortality data, and expert opinion. Costs were presented in 2017 Colombian pesos. Outcomes included quality-adjusted life-years (QALYs). Costs and outcomes were discounted by 5% per year. Incremental cost-utility ratios were presented for PAI compared with standard of care. Univariate and multivariate probabilistic sensitivity analyses were conducted to assess model robustness to parameter uncertainty. RESULTS: The model predicted that patients that are part of the PAI would accrue more QALYs than patients on standard of care. The incremental results suggest that the PAI is a cost-effective treatment (incremental cost-utility ratio of Colombian pesos $33 753 817/QALY) compared with standard of care. Sensitivity analyses suggest that results are most sensitive to cost of care (with and without PAI) and costs of severe exacerbation. However, the PAI is cost-effective irrespective of variation in any of the input parameters. CONCLUSION: Our model predicted that an integrated intervention for the management of asthma in pediatric patients improves QALYs, reduces number of disease related exacerbations compared to standard therapy and is cost-effective for the long-term control of the disease in Colombia.