Efficient CRISPR/Cas9 nickase-mediated genome editing in an in vitro model of mucopolysaccharidosis IVA.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder (LSD) caused by mutations in gene encoding for GALNS enzyme. Lack of GALNS activity leads to the accumulation of glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate. Although enzyme replacement therapy has been approved since 2014 for MPS IVA, still there is an unmet medical need to have improved therapies for this disorder. CRISPR/Cas9-based gene therapy has been tested for several LSDs with encouraging findings, but to date it has not been assayed on MPS IVA. In this work, we validated for the first time the use of CRISPR/Cas9, using a Cas9 nickase, for the knock-in of an expression cassette containing GALNS cDNA in an in vitro model of MPS IVA. The results showed the successful homologous recombination of the expression cassette into the AAVS1 locus, as well as a long-term increase in GALNS activity reaching up to 40% of wild-type levels. We also observed normalization of lysosomal mass, total GAGs, and oxidative stress, which are some of the major findings regarding the pathophysiological events in MPS IVA. These results represent a proof-of-concept of the use of CRISPR/Cas9 nickase strategy for the development of a novel therapeutic alternative for MPS IVA.

Morphological damage in Sertoli, myoid and interstitial cells in a mouse model of mucopolysaccharidosis type I (MPS I).

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease caused by a mutation in the IDUA gene, which codes α-L-iduronidase (IDUA), a lysosomal hydrolase that degrades two glycosaminoglycans (GAGs): heparan sulfate (HS) and dermatan sulfate (DS). GAGs are macromolecules found mainly in the extracellular matrix and have important signaling and structural roles which are essential to the maintenance of cell and tissue physiology. Nondegraded GAGs accumulate in various cell types, which characterizes MPS I as a multisystemic progressive disease. Many tissues and vital organs have been described in MPS I models, but there is a lack of studies focused on their effects on the reproductive tract. Our previous studies indicated lower sperm production and morphological damage in the epididymis and accessory glands in male MPS I mice, despite their ability to copulate and to impregnate females. Our aim was to improve the testicular characterization of the MPS I model, with a specific focus on ultrastructural observation of the different cell types that compose the seminiferous tubules and interstitium. We investigated the testicular morphology of 6-month-old male C57BL/6 wild-type (Idua+/+) and MPS I (Idua-/-) mice. We found vacuolated cells widely present in the interstitium and important signs of damage in myoid, Sertoli and Leydig cells. In the cytoplasmic region of Sertoli cells, we found an increased number of vesicles with substrates under digestion and a decreased number of electron-dense vesicles similar to lysosomes, suggesting an impaired flux of substrate degradation. Conclusions: Idua exerts an important role in the morphological maintenance of the seminiferous tubules and the testicular interstitium, which may influence the quality of spermatogenesis, having a greater effect with the progression of the disease.

Co-culture of annulus fibrosus cells and bone marrow mesenchymal stem cells.

To investigate the effect on cell proliferation and extracellular matrix expression of annulus fibrosus (AF) cells when co-cultured with bone marrow mesenchymal stem cells (BMSCs). Primary isolated rabbit BMSCs and AF cells were cultured and harvested cells were placed into a 15-mL centrifugal tube co-culture system in a ratio of 2:1 (A group), 1:1 (B group), and 1:2 (C group). Cell proliferation was evaluated using cell counting kit-8, and mRNA of collagen II and mucopolysaccharide was quantified using real-time polymerase chain reaction (PCR) on Days 7, 14, and 21. The cell count, synthesized collagen II and mucopolysaccharide increased in a time-dependent manner, with a peak at Day 21. Cells in Group B proliferated faster and synthesized more collagen II and mucopolysaccharide than groups A and C, where the difference was significant. AF cells and BMSCs in the ratio of 1:1, cultured using the centrifugal tube three-dimensional co-culture system showed that BMSCs can promote AF cell proliferation and extracellular matrix synthesis.

Versican expression in myoepithelial cells from carcinomas in canine mixed mammary tumors.

The matrix of canine mixed mammary tumors (CMMTs) consists of proliferating spindle cells of possible myoepithelial origin, as well as myxomatous tissue, cartilage matrix and/or bone. Among the multiple components of this tumor extracellular matrix, versican probably plays a prominent role due to its importance in tumor progression, cell proliferation and differentiation. However, there are few data related to a possible association between versican expression and the state of myoepithelial cell differentiation in CMMTs. Using immunohistochemistry and histochemistry, the objective of this study was to evaluate the expression of versican, sulfated proteoglycans and mucopolysaccharides in myoepithelial cells at different stages of differentiation and to explore a potential relationship with p63 and α-smooth muscle actin (SMA) expression. A significant difference in versican expression was observed among the different stages of myoepithelial cell differentiation with an inverse correlation between versican and p63/SMA expression. These results suggest that at an early stage of proliferation, myoepithelial cells acquire a phenotype consistent with a role in chondrogenesis. Moreover, myoepithelial cells showed an affinity for safranin and periodic acid-Schiff staining at different stages of proliferation supporting the myoepithelial origin of spindle cells from CMMTs.

Enzyme replacement therapy started at birth improves outcome in difficult-to-treat organs in mucopolysaccharidosis I mice.

Since we previously observed that in patients with mucopolysaccharidosis (MPS) the storage of undegraded glycosaminoglycans (GAG) occurs from birth, in the present study we aimed to compare normal, untreated MPS I mice (knockout for alpha-l-iduronidase-IDUA), and MPS I mice treated with enzyme replacement therapy (ERT, Laronidase, 1.2mg/kg every 2 weeks) started from birth (ERT-neo) or from 2 months of age (ERT-ad). All mice were sacrificed at 6 months. Both treatments were equally effective in normalizing GAG levels in the viscera but had no detectable effect on the joint. Heart function was also improved with both treatments. On the other hand, mice treated from birth presented better outcomes in the difficult-to-treat aortas and heart valves. Surprisingly, both groups had improvements in behavior tests, and normalization of GAG levels in the brain and IDUA injection resulted in detectable levels of enzyme in the brain tissue 1h after administration. ERT-ad mice developed significantly more anti-IDUA-IgG antibodies, and mice that didn't develop antibodies had better performances in behavior tests, indicating that development of antibodies may reduce enzyme bioavailability. Our results suggest that ERT started from birth leads to better outcomes in the aorta and heart valves, as well as a reduction in antibody levels. Some poor vascularized organs, such as the joints, had partial or no benefit and ancillary therapies might be needed for patients. The results presented here support the idea that ERT started from birth leads to better treatment outcomes and should be considered whenever possible, a observation that gains relevance as newborn screening programs are being considered for MPS and other treatable lysosomal storage disorders.

Intraperitoneal implant of recombinant encapsulated cells overexpressing alpha-L-iduronidase partially corrects visceral pathology in mucopolysaccharidosis type I mice.

BACKGROUND AIMS: Mucopolysaccharidosis type I (MPS I) is characterized by deficiency of the enzyme alpha-L-iduronidase (IDUA) and storage of glycosaminoglycans (GAG) in several tissues. Current available treatments present limitations, thus the search for new therapies. Encapsulation of recombinant cells within polymeric structures combines gene and cell therapy and is a promising approach for treating MPS I. METHODS: We produced alginate microcapsules containing baby hamster kidney (BHK) cells overexpressing IDUA and implanted these capsules in the peritoneum of MPS I mice. RESULTS: An increase in serum and tissue IDUA activity was observed at early time-points, as well as a reduction in GAG storage; however, correction in the long term was only partially achieved, with a drop in the IDUA activity being observed a few weeks after the implant. Analysis of the capsules obtained from the peritoneum revealed inflammation and a pericapsular fibrotic process, which could be responsible for the reduction in IDUA levels observed in the long term. In addition, treated mice developed antibodies against the enzyme. CONCLUSIONS: The results suggest that the encapsulation process is effective in the short term but improvements must be achieved in order to reduce the immune response and reach a stable correction.

Genomic instability in blood cells from murine model of mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of alfa-iduronidase (IDUA), which leads to intralysosomal accumulation of glycosaminoglycans. Some studies have revealed that oxidative stress plays an important role in MPS I. However, the mechanisms by which these alterations occur are still not fully understood. The aim of this study was to analyze genomic instability in blood cells from murine model of MPS I by single cell gel (comet) assay and micronucleus test. The results pointed out genetic damage in blood cells as depicted by the single cell gel (comet) assay results. By contrast, no increase of micronucleated cells were found in mouse blood cells when compared to negative control. Taken together, our results suggest that IDUA deficiency induces genomic damage in blood cells. Certainly, this finding offers new insights into the mechanisms underlying the relation between IDUA deficiency and clinical manifestations that can occur in MPS I patients.