Édina Poletto

Physicochemical properties of cationic nanoemulsions and liposomes obtained by microfluidization complexed with a single plasmid or along with an oligonucleotide: Implications for CRISPR/Cas technology

In this study, we investigated the effects of the association of a single plasmid or its co-complexation along with an oligonucleotide on the physicochemical properties of cationic nanoemulsions and liposomes intended for gene editing. Formulations composed of DOPE, DOTAP, DSPE-PEG (liposomes), MCT (nanoemulsions), and water were obtained by microfluidization. DSPE-PEG was found to play a crucial role on the size and polydispersity index of nanocarriers. Nucleic acids were complexated by adsorption at different charge ratios. No significant differences were noticed in the physicochemical properties of nanocarriers (i.e. droplet size, polydispersity index, or zeta potential) when a single plasmid or both plasmid and oligonucleotide were adsorbed to the formulations. Transmission electron microscopy photomicrographs suggested round nanostructures with the nucleic acids and DSPE-PEG enfolding the surface. Complexes at +4/-1 charge ratio protected nucleic acids against DNase I degradation. The oligonucleotide seemed to be released from the liposomal complexes, while nanoemulsions only released the plasmid after 24 and 48 h of incubation in DMEM supplemented or not. In vitro experiments demonstrated that complexes were highly tolerable to human fibroblasts, Hep-G2, and HEK-293 cells, demonstrating also an uptake ability of about 30%, 30%, and 90%, respectively, no matter what the formulation or the combination of nucleic acids used. Transfection efficiency of the formulations was around 25% in human fibroblasts, 32% in HEK-293, and 15% in Hep-G2 cells. The overall results demonstrated the behavior of liposomes and nanoemulsions complexed with a plasmid or a mixture of a plasmid and an oligonucleotide, and demonstrated that the association with one or two nucleic acids sequences of different length does not seem to interfere in the physicochemical characteristics of complexes or in the uptake capacity by three different types of cells.

Progressive heart disease in mucopolysaccharidosis type I mice may be mediated by increased cathepsin B activity

Mucopolysaccharidosis type I (MPS I) is a lysosomal disorder characterized by a deficiency of alpha-L-iduronidase and storage of undegraded glycosaminoglycans (GAGs). Clinical findings of the disease include heart failure, and patients often need valve replacement. It has been shown that, later in life, MPS I mice develop those abnormalities, but to date, there have not been studies on the progression and pathogenesis of the disease. Therefore, in the present study, we evaluated heart function in normal and MPS I male mice from 2 to 8 months of age. Echocardiographic analysis showed left ventricular enlargement with progressive reduction in ejection fraction, fractional area change, and left ventricular fractional shortening in the MPS I hearts at 6 and 8 months of age and a reduction in acceleration time/ejection time ratio of the pulmonary artery starting at 6 months of age, which suggests pulmonary vascular resistance. Histological and biochemical analysis confirmed progressive GAG storage from 2 months of age and onwards in the myocardium and heart valves, which had also increased in thickness. Additionally, macrophages were present in the MPS I heart tissue. Collagen content was reduced in the MPS I mouse valves. Cathepsin B, an enzyme that is known to be able to degrade collagen and is involved in heart dilatation, displayed a marked elevation in activity in the MPS I mice and could be responsible for the heart dilatation and valves alterations observed. Our results suggest that the MPS I mice have progressive heart failure and valve disease, which may be caused by cathepsin B overexpression.

In vivo genome editing of mucopolysaccharidosis I mice using the CRISPR/Cas9 system

ABSTRACT Mucopolysaccharidosis type I (MPS I) is a multisystemic disorder caused by the deficiency of alpha‐L‐iduronidase (IDUA) that leads to intracellular accumulation of glycosaminoglycans (GAG). In the present study we aimed to use cationic liposomes carrying the CRISPR/Cas9 plasmid and a donor vector for in vitro and in vivo MPS I gene editing, and compare to treatment with naked plasmids. The liposomal formulation was prepared by microfluidization. Complexes were obtained by the addition of DNA at +4/−1 charge ratio. The overall results showed complexes of about 110 nm, with positive zeta potential of +30 mV. The incubation of the complexes with fibroblasts from MPS I patients led to a significant increase in IDUA activity and reduction of lysosomal abnormalities. Hydrodynamic injection of the liposomal complex in newborn MPS I mice led to a significant increase in serum IDUA levels for up to six months. The biodistribution of complexes after hydrodynamic injection was markedly detected in the lungs and heart, corroborating the results of increased IDUA activity and decreased GAG storage especially in these tissues, while the group that received the naked plasmids presented increased enzyme activity especially in the liver. Furthermore, animals treated with the liposomal formulation presented improvement in cardiovascular parameters, one of the main causes of death observed in MPS I patients. We conclude that the IDUA production in multiple organs had a significant beneficial effect on the characteristics of MPS I disease, which may bring hope to gene therapy of Hurler patients. Graphical abstract Figure. No Caption available. HighlightsLiposomes are efficient carriers for CRISPR/Cas9 system.Liposomal CRISPR/Cas9 complexes showed efficient gene editing of MPS I fibroblasts.Treatment of newborn MPS I mice promoted high serum IDUA levels for up to 6 months.Treatment with liposomal complexes was more efficient than Naked CRISPR/Cas9.Liposomal CRISPR/Cas9‐treated mice improved GAG accumulation and cardiac function.

Intra-articular nonviral gene therapy in mucopolysaccharidosis I mice

ABSTRACT Mucopolysaccharidosis type I (MPS I) is caused by the lysosomal accumulation of glycosaminoglycans (GAGs) due to the deficiency of the enzyme alpha‐L‐iduronidase (IDUA). Currently available treatments may improve several clinical manifestations, but they have limited effects on joint disease, resulting in persistent orthopedic complications and impaired mobility. Thus, this study aimed to perform an intra‐articular administration of cationic nanoemulsions complexed with the plasmid encoding for the IDUA protein (pIDUA) targeting MPS I gene therapy for the synovial joints. Formulations composed of DOPE, DOTAP, MCT (NE), and DSPE‐PEG (NE‐PEG) were prepared by high‐pressure homogenization, and the pIDUA plasmid was associated by adsorption onto the surface of nanoemulsions (pIDUA/NE or pIDUA/NE‐PEG). The physicochemical characterization showed that the presence of DSPE‐PEG in pIDUA/NE‐PEG formulations led to small and highly stable droplets even when incubated with simulated synovial fluid (SSF), when compared to the non‐pegylated complexes (pIDUA/NE). Uptake by fibroblast‐like synoviocytes (FLS) was demonstrated, and high cell viability (70%) in addition with increased IDUA activity (2.5% of normal) were observed after incubation with pIDUA/NE‐PEG. The intra‐articular injection of pIDUA/NE‐PEG complexes in MPS I mice showed that the complexes were localized in the joints, were able to transfect synovial cells, and thus promoted an increase in IDUA activity and expression in the synovial fluid, with no significant activity in other tissues (kidney, liver, lung, and spleen). The overall results demonstrated a contained, safe, tolerable, and effective in situ approach of nonviral intra‐articular gene therapy targeting the reduction or prevention of the debilitating orthopedic complications of MPS I disorder.

Losartan improves aortic dilatation and cardiovascular disease in mucopolysaccharidosis I

To the Editor, We read with great interest the manuscript titled BAngiotensin receptor blockade mediated amelioration of mucopoly saccharidosis type I cardiac and craniofacial pathology^ recently published in the Journal of Inherited Metabolic Disorders (Osborn et al., 2016). Simultaneously, we have been conducting a similar study, with results that are partially in accordance, and we thus have some additional important considerations concerning the content of their paper. We treated juvenile (8-week-old) Idua mice with losartan or other antihypertensive drug (propranolol) as an additional control (Suppl. Material andMethods) to determine the benefit of angiotensin receptor blockade (ARB) in mucopolysaccharidosis I (MPS I) and if this effect was specific to losartan. Mice were sacrificed at 6 months of age. Our data showed an increase in aortic diameter measured by a digital caliper in untreated Idua mice without significant gender differences (Suppl. Fig. 1). Treatment analysis was performed according to gender and without differentiation (Suppl. Fig. 2). Unlike Osborn et al.’s results, our data indicate that losartan is effective in decreasing aortic dilatation in MPS I at a similar rate for both genders (∼20%). As a comparison, we also treated female mice with propranolol (a betablocker), which was not able to reduce aortic dilatation. In agreement with Osborn et al., our echocardiographic analysis showed that losartan also improves ventricular contraction, expressed through left ventricular fraction shortening (LVSF) and suggesting an improved heartpumping ability. It also prevented enlargement of left ventricular chamber dimensions (Suppl. Table 1). However, it is important to point out that propranolol also improved cardiac function, which suggests that heart dysfunction may be independent from angiotensin receptor (AT-1R) activation. Propranolol might have its beneficial effect by reducing hemodynamic stress on the aortic vasculature. In contrast, we believe that losartan targets the underlying pathophysiology in MPS possibly by antagonism of transforming growth factor (TGF)-β or other pathways, which will be investigated. Aortic diameter was also measured by echo, which confirmed that only losartan prevented abnormalities in the aorta. The authors highlight the possibility that matrix metalloproteinase-12 (MMP-12) increase via AT-1R activation is one of the mechanisms involved in the pathogenesis of cardiovascular disease in MPS. Nonetheless, in a previous study, MPS I and VII mice developed aortic dilatation by similar mechanisms, and knocking out MMP-12 in MPS VII mice did not prevent aortic abnormalities (Baldo et al. 2011). This probably suggestst that multiple mechanisms and genes are responsible for this effect. Communicated by: Carla E. Hollak

Nasal Administration of Cationic Nanoemulsions as Nucleic Acids Delivery Systems Aiming at Mucopolysaccharidosis Type I Gene Therapy

PurposeThis study demonstrates the nasal administration (NA) of nanoemulsions complexed with the plasmid encoding for IDUA protein (pIDUA) as an attempt to reach the brain aiming at MPS I gene therapy.MethodsFormulations composed of DOPE, DOTAP, MCT (NE), and DSPE-PEG (NE-PEG) were prepared by high-pressure homogenization, and assessed in vitro on human fibroblasts from MPS I patients and in vivo on MPS I mice for IDUA production and gene expression.ResultsThe physicochemical results showed that the presence of DSPE-PEG in the formulations led to smaller and more stable droplets even when submitted to dilution in simulated nasal medium (SNM). In vitro assays showed that pIDUA/NE-PEG complexes were internalized by cells, and led to a 5% significant increase in IDUA activity, besides promoting a two-fold increase in IDUA expression. The NA of pIDUA/NE-PEG complexes to MPS I mice demonstrated the ability to reach the brain, promoting increased IDUA activity and expression in this tissue, as well as in kidney and spleen tissues after treatment. An increase in serum IL-6 was observed after treatment, although with no signs of tissue inflammatory infiltrate according to histopathology and CD68 assessments.ConclusionsThese findings demonstrated that pIDUA/NE-PEG complexes could efficiently increase IDUA activity in vitro and in vivo after NA, and represent a potential treatment for the neurological impairment present in MPS I patients.

Cathepsin B inhibition attenuates cardiovascular pathology in mucopolysaccharidosis I mice.

ABSTRACT Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder with multisystemic features, including heart enlargement, heart valve dysfunction, and aortic stiffness and dilatation. Previous studies have shown that MPS I mice overexpress cathepsin B (CtsB) in multiple tissues, including those from the cardiovascular system. Here, we hypothesized that inhibition of CtsB could ameliorate cardiac function parameters, as well as aorta and valve abnormalities found in MPS I. First, we found that total elastase activity in an MPS I aorta is elevated. Following that, we demonstrated that CtsB leaks from the lysosome in MPS I human fibroblasts, possibly acting as a degradative agent of extracellular matrix components from the aorta, cardiac muscle, and heart valves. We then used a CtsB inhibitor in vivo in the MPS I mouse model. After 4 months of treatment, partial inhibition of CtsB activity in treated mice reduced aortic dilatation, as well as heart valve thickening, and led to improvements in cardiac function parameters, although none of these were completely normalized. Based on these results, we conclude that lysosomal alterations in this disease promote leakage of CtsB to outside the organelle, where this protein can have multiple pathological roles. CtsB inhibition improved cardiovascular parameters in MPS I mice and can have a potential benefit in this disease.

Worldwide distribution of common IDUA pathogenic variants

Mucopolysaccharidosis type I (MPS I) is a rare disorder caused by deleterious sequence variants in the α‐L‐iduronidase (IDUA) gene. More than 200 pathogenic variants have been described so far, but their frequencies have not yet been analyzed on a worldwide scale. To address this, we analyzed the genotypes of MPS I patients from 35 published studies papers. The most common pathogenic variant observed was p.Trp402Ter. With frequencies of up to 63%, it was the major allele in most European countries, America and Australia. The variant p.Gln70Ter was also frequent; it was found mainly in Northern and Eastern Europe. The most frequent variant in North African countries was p.Pro533Arg; in Morocco, it represented more than 90% of mutant alleles. Variants observed in East Asians were not found in Western populations, including c.1190‐1G>A, p.Ala79Val, p.Leu346Arg and c.613_617dupTGCTC. Conversely, p.Trp402Ter and p.Pro533Arg were not found in patients from East Asia. In conclusion, the most common pathogenic IDUA variant in MPS I patients are p.Trp402Ter, p.Gln70Ter and p.Pro533Arg. Knowledge about the genetic background of MPS I for each population is essential when developing new genotype‐targeted therapies, as well as to enable faster genetic analysis and improve patient management.