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.
Background
Mucopolysaccharidosis type I (MPS I) is an inherited disease caused by deficiency of the enzyme alpha‐l‐iduronidase (IDUA). MPS I affects several tissues, including the brain, leading to ...cognitive impairment in the severe form of the disease. Currently available treatments do not reach the brain. Therefore, in this study, we performed nasal administration (NA) of liposomal complexes carrying two plasmids encoding for the CRISPR/Cas9 system and for the IDUA gene targeting the ROSA26 locus, aiming at brain delivery in MPS I mice.
Methods
Liposomes were prepared by microfluidization, and the plasmids were complexed to the formulations by adsorption. Physicochemical characterization of the formulations and complexes, in vitro permeation, and mucoadhesion in porcine nasal mucosa (PNM) were assessed. We performed NA repeatedly for 30 days in young MPS I mice, which were euthanized at 6 months of age after performing behavioral tasks, and biochemical and molecular aspects were evaluated.
Results
Monodisperse mucoadhesive complexes around 110 nm, which are able to efficiently permeate the PNM. In animals, the treatment led to a modest increase in IDUA activity in the lung, heart, and brain areas, with reduction of glycosaminoglycan (GAG) levels in serum, urine, tissues, and brain cortex. Furthermore, treated mice showed improvement in behavioral tests, suggesting prevention of the cognitive damage.
Conclusion
Nonviral gene editing performed through nasal route represents a potential therapeutic alternative for the somatic and neurologic symptoms of MPS I and possibly for other neurological disorders.
Noninvasive nasal administration of liposomal vehicle for delivering CRISPR‐cas9/Donor plasmids showed an improvement in behavioral tasks and GAG reduction in MPS I mice due to 0.04–0.06% of gene edited cells, which led to a modest increase in IDUA enzyme activity in several tissues, including some brain areas.
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•Chitosan-coated RA nanoemulsion was successfully optimized using Box-Behnken design.•Mucoadhesive potential from chitosan-coated RA nanoemulsion was demonstrated.•Adequate ...physicochemical characteristics and prolonged RA release was also observed.•Long-lasting permeation and higher RA retention in porcine nasal mucosa was achieved.•Safety profile from chitosan-coated RA nanoemulsion was investigated in fibroblasts.
Mucoadhesive chitosan-coated nanoemulsions for rosmarinic acid (RA) nasal delivery were optimized. The optimum ratio between the formulation components that led to minimum droplet size and PDI, and maximal ζ-potential and RA content was obtained using Box-Behnken design (BBD). Optimized conditions were 8.5% oil phase (w/v), 3:10 lecithin to oil phase ratio (w/w), and 0.1% chitosan final concentration (w/v). Physicochemical characterization, mucoadhesion measurement, in vitro release and permeation/retention were performed. Optimized chitosan-coated RA nanoemulsions presented adequate physicochemical characteristics, high mucoadhesive potential, prolonged drug release, and long-lasting permeation time with a higher RA penetration/retention through porcine nasal mucosa. Cell viability and death by necrosis in fibroblasts cells were also evaluated to investigate the formulations safety. Formulations did not induce cytotoxicity following 24 h (3.125–50 μM) or 48 h (3.125–25 μM) of treatments. Overall results demonstrated that optimized chitosan-coated nanoemulsion showed to be a suitable carrier for RA nasal delivery aiming neuroprotective therapies.
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.
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•Liposomes 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.
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.
In the present study, we evaluated the transfection efficiency of repeated intravenous administrations of cationic nanoemulsions associated with the plasmid pIDUA (containing IDUA gene).
Cationic nanoemulsions were composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(aminopolyethylene 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.
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.
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.
Coumarins are benzopyrones found in several plant genera, including
(Asteraceae). These compounds represent an important source of new treatments, especially as antimicrobial and antifungal agents. ...In this study, two coumarin-rich extracts from
using green technologies were obtained through aqueous maceration (AE) and supercritical fluid extraction (SFE). Such extracts were incorporated into nanoemulsions (NAE and NSFE) composed of a medium-chain triglyceride oil core stabilized by phospholipids. The nanoemulsions exhibited droplet sizes between 127 and 162 nm, pH above 5.0, and viscosity of approximately 1.0 cP, properties compatible with the topical route. The coumarins permeation/retention from formulations through ear porcine skin using Franz-type diffusion cells were evaluated. Whatever the extract, coumarins were distributed in skin layers, especially in the dermis in both intact and impaired (tape stripping) skin. In addition, a significant increase in coumarins that reached up to the receptor fluid was observed for impaired skin, with increases of approximately threefold for NAE and fourfold for NSFE. Finally, antifungal activity of nanoemulsions was evaluated according to minimum inhibitory concentrations, and the values were 250 µg/mL for all strains tested. The overall results demonstrated the feasibility of incorporating
extracts into nanoemulsions and showed a potential alternative for the treatment of sporotrichosis.
Mucopolysaccharidosis type I (MPS I) is caused by deficiency of alpha-L-iduronidase (IDUA), leading to multisystemic accumulation of glycosaminoglycans (GAG). Untreated MPS I patients may die in the ...first decades of life, mostly due to cardiovascular and respiratory complications. We previously reported that the treatment of newborn MPS I mice with intravenous administration of lipossomal CRISPR/Cas9 complexes carrying the murine Idua gene aiming at the ROSA26 locus resulted in long-lasting IDUA activity and GAG reduction in various tissues. Following this, the present study reports the effects of gene editing in cardiovascular, respiratory, bone, and neurologic functions in MPS I mice. Bone morphology, specifically the width of zygomatic and femoral bones, showed partial improvement. Although heart valves were still thickened, cardiac mass and aortic elastin breaks were reduced, with normalization of aortic diameter. Pulmonary resistance was normalized, suggesting improvement in respiratory function. In contrast, behavioral abnormalities and neuroinflammation still persisted, suggesting deterioration of the neurological functions. The set of results shows that gene editing performed in newborn animals improved some manifestations of the MPS I disorder in bone, respiratory, and cardiovascular systems. However, further studies will be imperative to find better delivery strategies to reach "hard-to-treat" tissues to ensure better systemic and neurological effects.
Mucopolysaccharidoses (MPS) are genetic metabolic diseases characterized by defects in the activity of lysosomal hydrolases. In MPS, secondary cell disturbance affects pathways related to ...cardiovascular disorders. Hence, the study aimed to identify MPS-related drugs targeting cardiovascular disease and select a list of drugs for repositioning. We obtained a list of differentially expressed genes and pathways. To identify drug perturbation-driven gene expression and drug pathways interactions, we used the CMAP and LINCS databases. For molecular docking, we used the DockThor web server. Our results suggest that pirfenidone and colchicine are promising drugs to treat cardiovascular disease in MPS patients. We also provide a brief description of good practices for the repositioning analysis. Furthermore, the list of drugs and related MPS-enriched genes could be helpful to new treatments and considered for pathophysiological studies.
Advanced Therapies are a class of innovative complex biological products used for therapeutic purposes, encompassing cell therapy, tissue engineering, and gene therapy products. These are promising ...therapeutic strategies for several complex diseases with low or non-existent therapeutic alternatives. The proper transposition of basic research in this area into medicinal products must comply with regulatory requirements. Here we review the main regulatory recommendations, emphasizing on the Brazilian regulation. The critical points are the manufacturing process, challenges in characterizing the product, development of non-clinical trials, lack of adequate animal models representative of the clinical situation, and absence of valid and measurable therapeutic endpoints. Based on that, we propose a framework for strategic planning of pre-clinical studies in this field. The detailed example involves producing a nonviral vector-based gene editing product, but the regulations and methods may be extrapolated for developing different types of advanced therapies.
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•Preparation methods have major influence in the physicochemical characteristics of cationic nanoemulsions and high-pressure homogenization leads to small monodisperse ...droplets.•Various physicochemical parameters may affect nucleic acids cell uptake and efficiency, but charge ratio seems to be the main important parameter.•Many factors can contribute to in vitro delivery and citotoxicity of nucleic acid/cationic nanoemulsions complexes, especially the composition and excess of positive charges.•Transfection efficiency after in vivo administration of nucleic acid/cationic nanoemulsions complexes can be measured qualitatively or quantitatively.
Since the first clinical studies, knowledge in the field of gene therapy has advanced significantly, and these advances led to the development and subsequent approval of the first gene medicines. Although viral vectors-based products offer efficient gene expression, problems related to their safety and immune response have limited their clinical use. Thus, design and optimization of nonviral vectors is presented as a promising strategy in this scenario. Nonviral systems are nanotechnology-based products composed of polymers or lipids, which are usually biodegradable and biocompatible. Cationic liposomes are the most studied nonviral carriers and knowledge about these systems has greatly evolved, especially in understanding the role of phospholipids and cationic lipids. However, the search for efficient delivery systems aiming at gene therapy remains a challenge. In this context, cationic nanoemulsions have proved to be an interesting approach, as their ability to protect and efficiently deliver nucleic acids for diverse therapeutic applications has been demonstrated. This review focused on cationic nanoemulsions designed for gene therapy, providing an overview on their composition, physicochemical properties, and their efficacy on biological response in vitro and in vivo.
