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

Abstract

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.

Introduction

Mucopolysaccharidosis type I (MPS I, OMIM N^o 607014) is an autosomal recessive lysosomal disorder characterized by a deficiency of alpha-l-iduronidase (IDUA, EC 3.2.1.76) which results in the lysosomal storage of partially degraded heparan sulphate and dermatan sulphate glycosaminoglycans (GAGs). The clinical manifestations of this disease include stiff joints, coarse facial features, visual impairment, deafness, cardiac valve disease, and intellectual disability [1]. Although treatments such as enzyme replacement therapy (ERT) and haematopoietic stem cell transplantation (HSCT) are often prescribed to patients with MPS I, their effects are limited by the fact that intravenous ERT does not cross the blood–brain barrier, and, therefore, does not address the neurological complications of the disease, while HSCT is only effective when initiated early in life, and is associated with high morbidity and mortality. Despite some advances, these treatments still have several limitations [2], [3].

Non-viral gene therapy can be considered as an interesting treatment option for patients with MPS I, given that non-viral vectors are safer and easier to produce than viral ones [4], [5], [6]. The design of non-viral vectors, based on cationic polymers (such as polyethylenimine) and phospholipids/cationic lipids (such as DOPE and DOTAP), is among the most widespread strategies for circumventing the poor cell uptake and instability of nucleic acids against enzymatic degradation. A well-documented literature has showed an improved transfection efficiency of polyplexes and lipoplexes, mainly in vitro [7], [8]. Despite extensive research in the field, limitations in gene therapy need to be overcome to design an optimal system that is able to protect and carry the nucleic acid to the site of action for a sufficient period of time.

In recent years, considerable efforts have been made to refine the properties of the cationic nanoemulsions/pDNA complexes used in gene therapy, especially since studies revealed interesting results regarding transfection efficiency after administration by different routes [9], [10]. Nanoemulsions are composed of a liquid lipid oil core stabilized by a binary mixture of phospholipids and a cationic lipid. We have recently described PEGylated nanoemulsions composed of medium-chain triglycerides, dioleoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-trimethylammonium propane (DOTAP), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) as a carrier for a plasmid containing the alpha-l-iduronidase (IDUA) gene [11]. Transfection efficiency was evaluated in an animal model of MPS I developed by Neufeld et al., which has proved to be a remarkable tool for the study of MPS I pathogenesis and treatment options [12], [13], [14], since it was created by the disruption of exon 6 of the mouse IDUA gene, resulting in mice with a severe form of the disease [14]. Preliminary results showed that our pIDUA/PEGylated nanoemulsion complexes were able to increase IDUA activity after intravenous administration in MPS I mice when compared with non-treated MPS I animals [11].

Therefore, the goal of the present study was to follow up on this finding by investigating the effect of pIDUA loading processes obtained by adsorption or encapsulation of preformed pIDUA-cationic lipid complexes in nanoemulsions on transfection efficiency in the MPS I murine model. Based on previous findings regarding the preparation of reconstituted chylomicron remnant particles [15], [16] we hypothesized that pIDUA would behave differently when encapsulated in oil droplets of PEGylated nanoemulsions than it would in a single adsorption at the o/w interface of nanoemulsions. The size, charge, structural organization, and decomplexation of pIDUA from formulations were also investigated.