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

PURPOSE: This 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. METHODS: Formulations 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. RESULTS: The 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. CONCLUSIONS: These 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.

A simple protocol for transfecting human mesenchymal stem cells.

OBJECTIVES AND RESULTS: Mesenchymal stromal cells (MSCs) are potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and have significant expansion capability. These characteristics make them strong candidates for delivering genes and restoring organ systems function. However, as other primary cells, MSCs are difficult to transfect. In order to standardize a simple protocol for transfection of MSCs, we conducted a series of experiments and achieved a protocol that does not require the use of viral particles or specific expensive equipment. CONCLUSION: MSCs transfection at early passages using a ratio lipid/DNA of 3.0 µL/µg with Lipofectamine 3000® yields good transfection efficiencies for human MSCs (up to 26%) and is rapid, simple, and safe.

Laronidase-functionalized multiple-wall lipid-core nanocapsules: promising formulation for a more effective treatment of mucopolysaccharidosis type I.

PURPOSE: Mucopolysaccharidosis I is a genetic disorder caused by alpha-L-iduronidase deficiency. Its primary treatment is enzyme replacement therapy (ERT), which has limitations such as a high cost and a need for repeated infusions over the patient's lifetime. Considering that nanotechnological approaches may enhance enzyme delivery to organs and can reduce the dosage thereby enhancing ERT efficiency and/or reducing its cost, we synthesized laronidase surface-functionalized lipid-core nanocapsules (L-MLNC). METHODS: L-MLNCs were synthesized by using a metal complex. Size distributions were evaluated by laser diffraction and dynamic light scattering. The kinetic properties, cytotoxicity, cell uptake mechanisms, clearance profile and biodistribution were evaluated. RESULTS: Size distributions showed a D[4,3] of 134 nm and a z-average diameter of 71 nm. L-MLNC enhanced the Vmax and Kcat in comparison with laronidase. L-MLNC is not cytotoxic, and nanocapsule uptake by active transport is not only mediated by mannose-6-phosphate receptors. The clearance profile is better for L-MLNC than for laronidase. A biodistribution analysis showed enhanced enzyme activity in different organs within 4 h and 24 h for L-MLNC. CONCLUSIONS: The use of lipid-core nanocapsules as building blocks to synthesize surface-functionalized nanocapsules represents a new platform for producing decorated soft nanoparticles that are able to modify drug biodistribution.

Cationic Nanoemulsions as a Gene Delivery System: Proof of Concept in the Mucopolysaccharidosis I Murine Model.

Mucopolysaccharidosis I (MPS I) is an autosomal recessive lysosomal storage disease due to deficient a-L-iduronidase (IDUA) activity. It results in the accumulation of the glycosaminoglycans (GAGs) heparan and dermatan sulfate and leads to several clinical manifestations. This study describes the use of cationic nanoemulsions as a non-viral carrier for the plasmid named pIDUA, which has the gene that encodes for the IDUA enzyme. Cationic nanoemulsions, composed by a medium chain triglycerides oil core stabilized by DOTAP, DOPE and DSPE-PEG, were prepared by high pressure homogeneization. pIDUA was complexed with nanoemulsions in the end of manufacturing process. Physicochemical properties of complexes were influenced by the charge ratio used. From a charge ratio of +2/-, it was observed a total complexation of pIDUA with formulation as well as a protection of plasmid against DNAse I digestion. In vitro assay in fibroblasts of one MPS I patient presented greater and significant trasfection efficiency for pIDUA complexed to formulation in the +4/- charge ratio. This formulation was administered via the tail vein and the portal vein. Animals were compared to untreated MPS I mice. Transfection efficiency was measured as IDUA enzyme activity. After intravenous administration, IDUA activity was significantly higher in lungs and liver. The set of results shows the formulation obtained at the +4/- charge ratio as a promising non-viral gene delivery system, once showed increased enzyme activity both in vitro and in vivo.

Treatment of MPS I mice with microencapsulated cells overexpressing IDUA: effect of the prednisolone administration.

Cell encapsulation, although a promising strategy to deliver therapeutic products, is hampered by immune response against biomaterials. The aim of this article is to assess the effect of prednisolone on enzyme release by microencapsulated cells implanted in vivo. Recombinant cells encapsulated were implanted in the peritoneum of wild-type mice and mucopolysaccharidosis (MPS) I mice, with or without prednisolone. Later, microcapsules were recovered for histological and enzyme analysis. Blood was collected from MPS I mice. All animals receiving prednisolone had a smaller inflammatory infiltrate. In vitro, prednisolone increased the amount of enzyme released from the recovered capsules, but this was not accompanied by an increase in the amount of circulating enzyme in vivo after 15 days. However, in 7 days, prednisolone significantly increased the amount of enzyme detected in the serum. Although prednisolone improved enzyme release in vitro and in vivo after 7 days, it was unable to maintain this effect for a longer period.

Cell microencapsulation: a potential tool for the treatment of neuronopathic lysosomal storage diseases.

Lysosomal storage disorders (LSD) are monogenic diseases caused by the deficiency of different lysosomal enzymes that degrade complex substrates such as glycosaminoglycans, sphingolipids, and others. As a consequence there is multisystemic storage of these substrates. Most treatments for these disorders are based in the fact that most of these enzymes are soluble and can be internalized by adjacent cells via mannose-6-phosphate receptor. In that sense, these disorders are good candidates to be treated by somatic gene therapy based on cell microencapsulation. Here, we review the existing data about this approach focused on the LSD treatments, the advantages and limitations faced by these studies.

Gene Therapy of Mucopolysaccharidosis Type I Mice: Repeated Administrations and Safety Assessment of pIDUA/Nanoemulsion Complexes.

BACKGROUND: Mucopolysaccharidosis type I (MPS I) is an inherited disorder caused by α-L-iduronidase (IDUA) deficiency. The available treatments are not effective in improving all signs and symptoms of the disease. OBJECTIVE: In the present study, we evaluated the transfection efficiency of repeated intravenous administrations of cationic nanoemulsions associated with the plasmid pIDUA (containing IDUA gene). METHODS: Cationic nanoemulsions were composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(amino[polyethylene glycol]- 2000) (DSPE-PEG), 1,2-dioleoyl-sn-glycero-3-trimethylammonium propane (DOTAP), medium- chain triglycerides, glycerol, and water and were prepared by high-pressure homogenization and were repeatedly administered to MPS I mice for IDUA production and gene expression. RESULTS: A significant increase in IDUA expression was observed in all organs analyzed, and IDUA activity tended to increase with repeated administrations when compared to our previous report when mice received a single administration of the same dose. In addition, GAGs were partially cleared from organs, as assessed through biochemical and histological analyzes. There was no presence of inflammatory infiltrate, necrosis, or signs of an increase in apoptosis. Furthermore, immunohistochemistry for CD68 showed a reduced presence of macrophage cells in treated than in untreated MPS I mice. CONCLUSION: These sets of results suggest that repeated administrations can improve transfection efficiency of cationic complexes without a significant increase in toxicity in the MPS I murine model.

Validation of keratan sulfate level in mucopolysaccharidosis type IVA by liquid chromatography-tandem mass spectrometry.

Mucopolysaccharidosis type IVA (MPS IVA, Morquio A disease), a progressive lysosomal storage disease, causes skeletal chondrodysplasia through excessive storage of keratan sulfate (KS). KS is synthesized mainly in cartilage and released to the circulation. The excess storage of KS disrupts cartilage, consequently releasing more KS into circulation, which is a critical biomarker for MPS IVA. Thus, assessment of KS level provides a potential screening strategy and determines clinical course and efficacy of therapies. We have recently developed a tandem mass spectrometry liquid chromatography [LC/MS/MS] method to assay KS levels in blood. Forty-nine blood specimens from patients with MPS IVA [severe (n = 33), attenuated (n = 11) and undefined (n = 5)] were analyzed for comparison of blood KS concentration with that of healthy subjects and for correlation with clinical severity. Plasma samples were digested by keratanase II to obtain disaccharides of KS. Digested samples were assayed by LC/MS/MS. We found that blood KS levels (0.4-26 µg/ml) in MPS IVA patients were significantly higher than those in age-matched controls (0.67-4.6 µg/ml; P < 0.0001). It was found that blood KS level varied with age and clinical severity in the patients. Blood KS levels in MPS IVA peaked between 2 years and 5 years of age (mean 11.4 µg/ml). Blood KS levels in severe MPS IVA (mean 7.3 µg/ml) were higher than in the attenuated form (mean 2.1 µg/ml) (P = 0.012). We also found elevated blood KS levels in other types of MPS. These findings indicate that the new KS assay for blood is suitable for early diagnosis and longitudinal assessment of disease severity in MPS IVA.

Dermatan sulfate and heparan sulfate as a biomarker for mucopolysaccharidosis I.

Mucopolysaccharidosis I (MPS I) is an autosomal recessive disorder caused by deficiency of alpha-L-iduronidase leading to accumulation of its catabolic substrates, dermatan sulfate (DS) and heparan sulfate (HS), in lysosomes. This results in progressive multiorgan dysfunction and death in early childhood. The recent success of enzyme replacement therapy (ERT) for MPS I highlights the need for biomarkers that reflect response to such therapy. To determine which biochemical markers are better, we determined serum and urine DS and HS levels by liquid chromatography tandem mass spectrometry in ERT-treated MPS I patients. The group included one Hurler, 11 Hurler/Scheie, and two Scheie patients. Seven patients were treated from week 1, whereas the other seven were treated from week 26. Serum and urine DS (DeltaDi-4S/6S) and HS (DeltaDiHS-0S, DeltaDiHS-NS) were measured at baseline, week 26, and week 72. Serum DeltaDi-4S/6S, DeltaDiHS-0S, and DeltaDiHS-NS levels decreased by 72%, 56%, and 56%, respectively, from baseline at week 72. Urinary glycosaminoglycan level decreased by 61.2%, whereas urine DeltaDi-4S/6S, DeltaDiHS-0S, and DeltaDiHS-NS decreased by 66.8%, 71.8%, and 71%, respectively. Regardless of age and clinical severity, all patients showed marked decrease of DS and HS in blood and urine samples. We also evaluated serum DS and HS from dried blood-spot samples of three MPS I newborn patients, showing marked elevation of DS and HS levels compared with those in control newborns. In conclusion, blood and urine levels of DS and HS provide an intrinsic monitoring and screening tool for MPS I patients.

Effects of cryopreservation and hypothermic storage on cell viability and enzyme activity in recombinant encapsulated cells overexpressing alpha-L-iduronidase.

Here, we show the effects of cryopreservation and hypothermic storage upon cell viability and enzyme release in alginate beads containing baby hamster kidney cells overexpressing alpha-L-iduronidase (IDUA), the enzyme deficient in mucopolysaccharidosis type I. In addition, we compared two different concentrations of alginate gel (1% and 1.5%) in respect to enzyme release from the beads and their shape and integrity. Our results indicate that in both alginate concentrations, the enzyme is released in lower amounts compared with nonencapsulated cells. Alginate 1% beads presented increased levels of IDUA release, although this group presented more deformities when compared with alginate 1.5% beads. Importantly, both encapsulated groups presented higher cell viability after long cryopreservation period and hypothermic storage. In addition, alginate 1.5% beads presented higher enzyme release after freezing protocols. Taken together, our findings suggest a benefic effect of alginate upon cell viability and functionality. These results may have important application for treatment of both genetic and nongenetic diseases using microencapsulation-based artificial organs.

Gene editing of MPS I human fibroblasts by co-delivery of a CRISPR/Cas9 plasmid and a donor oligonucleotide using nanoemulsions as nonviral carriers.

Mucopolysaccharidosis type I (MPS I) is an inherited disease caused by the deficiency of alpha-L-iduronidase (IDUA). This study shows the use of nanoemulsions co-complexed with the plasmid of CRISPR/Cas9 system and a donor oligonucleotide aiming at MPS I gene editing in vitro. Nanoemulsions composed of MCT, DOPE, DOTAP, DSPE-PEG, and water were prepared by high-pressure homogenization. The DNA was complexed by adsorption (NA) or encapsulation (NE) of preformed DNA/DOTAP complexes with nanoemulsions at +4/-1 charge ratio. The incubation in pure DMEM or supplemented with serum showed that the complexation with DNA was stable after 1 h of incubation, but the complexes tended to release the adsorbed DNA after 24 h of incubation, while the encapsulated DNA remained complexed in the oil core of the nanoemulsions even 48 h after incubation with DMEM. The treatment of MPS I patient's fibroblasts homozygous for the p.Trp402∗ mutation led to a significant increase in IDUA activity at 2, 15, and 30 days when compared to MPS I untreated fibroblasts. Flow cytometry and confocal microscopy demonstrated that there was a reduction in the area of lysosomes to values similar to normal, an indicator of correction of the cellular phenotype. These results show that the nanoemulsions co-complexed with the CRISPR/Cas9 system and a donor oligonucleotide could effectively transfect MPS I p.Trp402∗ patient's fibroblasts, as well as enable the production of IDUA, and represent a potential new treatment option for MPS I.

CRISPR-Cas9-mediated gene editing in human MPS I fibroblasts.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder (LSD). It is caused by mutations in the IDUA gene, which lead to the accumulation of the glycosaminoglycans dermatan and heparan sulfate. The CRISPR-Cas9 system is a new and powerful tool that allows gene editing at precise points of the genome, resulting in gene correction through the introduction and genomic integration of a wildtype sequence. In this study, we used the CRISPR-Cas9 genome editing technology to correct in vitro the most common mutation causing MPS I. Human fibroblasts homozygous for p.Trp402* (legacy name W402X) were transfected and analyzed for up to one month after treatment. IDUA activity was significantly increased, lysosomal mass was decreased, and next generation sequencing confirmed that a percentage of cells carried the wildtype sequence. As a proof of concept, this study demonstrates that CRISPR-Cas9 genome editing may be used to correct causative mutations in MPS I. LIST OF ABBREVIATIONS.

Factors influencing transfection efficiency of pIDUA/nanoemulsion complexes in a mucopolysaccharidosis type I murine model.

Mucopolysaccharidosis type I (MPS I) is an autosomal disease caused by alpha-l-iduronidase (IDUA) deficiency. This study used IDUA knockout mice as a model to evaluate whether parameters such as dose of plasmid and time of treatment could influence the transfection efficiency of complexes formed with PEGylated cationic nanoemulsions and plasmid (pIDUA), which contains the gene that encodes for IDUA. Formulations were composed of medium chain triglycerides, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(amino[polyethylene glycol]-2000), 1,2-dioleoyl-sn-glycero-3-trimethylammonium propane (DOTAP), glycerol, and water and were prepared by the adsorption or encapsulation of preformed pIDUA-DOTAP complexes by high-pressure homogenization. A progressive increase in IDUA expression was observed with an increase in the dose and time of transfection for mice treated with both complexes (adsorbed and encapsulated), especially in the liver. Regardless of the complex administered, a significant increase in IDUA activity was detected in lungs and liver compared with nontreated MPS I when a dose of 60 µg was administered and IDUA activity was measured 7 days postadministration. Tissue sections of major organs showed no presence of cell necrosis, inflammatory infiltrate, or an increase in apoptosis. Furthermore, immunohistochemistry for CD68 showed no difference in the number of macrophage cells in treated and nontreated animals, indicating the absence of inflammatory reaction caused by the treatment. The data set obtained in this study allowed establishing that factors such as dose and time can influence transfection efficiency in different degrees and that these complexes did not lead to any lethal effect in the MPS I murine model used.

Effects of enzyme replacement therapy started late in a murine model of mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is a progressive disorder caused by deficiency of α-L-iduronidase (IDUA), which leads to storage of heparan and dermatan sulphate. It is suggested that early enzyme replacement therapy (ERT) leads to better outcomes, although many patients are diagnosed late and don't receive immediate treatment. This study aims to evaluate the effects of late onset ERT in a MPS I murine model. MPS I mice received treatment from 6 to 8 months of age (ERT 6-8mo) with 1.2mg laronidase/kg every 2 weeks and were compared to 8 months-old wild-type (Normal) and untreated animals (MPS I). ERT was effective in reducing urinary and visceral GAG to normal levels. Heart GAG levels and left ventricular (LV) shortening fraction were normalized but cardiac function was not completely improved. While no significant improvements were found on aortic wall width, treatment was able to significantly reduce heart valves thickening. High variability was found in behavior tests, with treated animals presenting intermediate results between normal and affected mice, without correlation with cerebral cortex GAG levels. Cathepsin D activity in cerebral cortex also did not correlate with behavior heterogeneity. All treated animals developed anti-laronidase antibodies but no correlation was found with any parameters analyzed. However, intermediary results from locomotion parameters analyzed are in accordance with intermediary levels of heart function, cathepsin D, activated glia and reduction of TNF-α expression in the cerebral cortex. In conclusion, even if started late, ERT can have beneficial effects on many aspects of the disease and should be considered whenever possible.

PEGylated cationic nanoemulsions can efficiently bind and transfect pIDUA in a mucopolysaccharidosis type I murine model.

Mucopolysaccharidosis type I (MPS I) is an autosomal disease caused by alpha-L-iduronidase deficiency. This study proposed the use of cationic nanoemulsions as non-viral vectors for a plasmid (pIDUA) containing the gene that codes for alpha-L-iduronidase. Nanoemulsions composed of medium chain triglycerides (MCT)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/1,2-dioleoyl-sn-glycero-3-trimethylammonium propane (DOTAP)/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) were prepared by high pressure homogenization. Formulations were prepared by the adsorption or encapsulation of preformed pIDUA-DOTAP complexes into the oil core of nanoemulsions at different charge ratios. pIDUA complexed was protected from enzymatic degradation by DNase I. The physicochemical characteristics of complexes in protein-containing medium were mainly influenced by the presence of DSPE-PEG. Bragg reflections corresponding to a lamellar organization were identified for blank formulations by energy dispersive X-ray diffraction, which could not be detected after pIDUA complexation. The intravenous injection of these formulations in MPS I knockout mice led to a significant increase in IDUA activity (fluorescence assay) and expression (RT-qPCR) in different organs, especially the lungs and liver. These findings were more significant for formulations prepared at higher charge ratios (+4/-), suggesting a correlation between charge ratio and transfection efficiency. The present preclinical results demonstrated that these nanocomplexes represent a potential therapeutic option for the treatment of MPS I.

Increased glutamate receptor and transporter expression in the cerebral cortex and striatum of gcdh-/- mice: possible implications for the neuropathology of glutaric acidemia type I.

We determined mRNA expression of the ionotropic glutamate receptors NMDA (NR1, NR2A and NR2B subunits), AMPA (GluR2 subunit) and kainate (GluR6 subunit), as well as of the glutamate transporters GLAST and GLT1 in cerebral cortex and striatum of wild type (WT) and glutaryl-CoA dehydrogenase deficient (Gchh-/-) mice aged 7, 30 and 60 days. The protein expression levels of some of these membrane proteins were also measured. Overexpression of NR2A and NR2B in striatum and of GluR2 and GluR6 in cerebral cortex was observed in 7-day-old Gcdh-/-. There was also an increase of mRNA expression of all NMDA subunits in cerebral cortex and of NR2A and NR2B in striatum of 30-day-old Gcdh-/- mice. At 60 days of life, all ionotropic receptors were overexpressed in cerebral cortex and striatum of Gcdh-/- mice. Higher expression of GLAST and GLT1 transporters was also verified in cerebral cortex and striatum of Gcdh-/- mice aged 30 and 60 days, whereas at 7 days of life GLAST was overexpressed only in striatum from this mutant mice. Furthermore, high lysine intake induced mRNA overexpression of NR2A, NR2B and GLAST transcripts in striatum, as well as of GluR2 and GluR6 in both striatum and cerebral cortex of Gcdh-/- mice. Finally, we found that the protein expression of NR2A, NR2B, GLT1 and GLAST were significantly greater in cerebral cortex of Gcdh-/- mice, whereas NR2B and GLT1 was similarly enhanced in striatum, implying that these transcripts were translated into their products. These results provide evidence that glutamate receptor and transporter expression is higher in Gcdh-/- mice and that these alterations may be involved in the pathophysiology of GA I and possibly explain, at least in part, the vulnerability of striatum and cerebral cortex to injury in patients affected by GA I.

In vitro correction of ARSA deficiency in human skin fibroblasts from metachromatic leukodystrophy patients after treatment with microencapsulated recombinant cells.

Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder due to arylsulfatase A (ARSA) deficiency that affects primarily the central nervous system. Ongoing treatments include enzyme replacement therapy and bone marrow transplantation, both limited in their effects due to the blood-brain barrier. An alternative approach would be the in situ implantation of encapsulated cells over expressing ARSA. Based on that, we tested the ability of encapsulated BHK cells over expressing ARSA to correct the enzyme deficiency in MLD patients' fibroblasts. Three groups were analyzed: fibroblasts treated with ARSA-over expressing BHK cells (rBHK) trapped in alginate capsules (capsules group), fibroblasts treated with supernatant of non-encapsulated rBHK (uptake control) and fibroblasts treated with empty capsules (empty group). Untreated and normal fibroblasts were used as controls. rBHK obtained by clone selection after non-viral transfection with pTARSA-CMV2. ARSA activity was measured after 1, 2, 3 and 4 weeks of treatment and beta-gal was used as reference enzyme. Statistical analysis was performed using ANOVA and Tukey's test. Normal fibroblasts showed ARSA activity of 23.9 + /- 2.01 nmol/h/mg of protein, whereas untreated MLD fibroblasts had the low ARSA activity (2.22 + /- 0.17). In the empty group, ARSA activity was equal to that of untreated fibroblasts (2.71 + /- 0.34). Capsules and uptake control groups showed higher enzymatic activity levels, compared to MLD untreated, 23.42 + /- 6.39 and 42.35 + /- 5.20, respectively (p < 0.01 for all groups). Encapsulated rBHK clones show potential as a new therapeutic strategy for the treatment of MLD, reaching normal enzyme levels in human MLD fibroblasts.