Peptide-modified vectors for nucleic acid delivery to neurons

Abstract

Neuron-targeted nucleic acid delivery systems are important technologies for realizing the potential of gene therapy for nervous system disorders. However, neurons are difficult cells to transfect using non-viral vectors due in part to the specific and unique delivery challenges present in these cells. We have investigated several bioactive peptides for their ability to assist in overcoming delivery barriers in mammalian cells. We summarize here our recent progress in developing and applying peptide-modified polycations for nucleic acid delivery. In addition, we present data demonstrating the potential of using multicomponent, peptide-modified polycations for nucleic acid delivery to neurons.

Introduction

Neurological disorders affect up to one billion people worldwide and are the cause of 12% of total global deaths [1]. Despite significant advances in understanding the molecular basis of these diseases, many prevalent neurological disorders have no effective cure due to the unavailability of effective drugs and/or delivery systems. Nucleic acid-based therapies are a relatively new class of drugs that can potentially treat neurological disease. Indeed, nucleic acid-based therapies have yielded promising disease reduction in animal models of amyotrophic lateral sclerosis [2], [3], Parkinson's disease [4], Huntington's disease [5], and spinocerebellar ataxia [6], to name a few. In addition, gene therapies are currently being evaluated in clinical trials for Parkinson's disease and multiple sclerosis [7], [8].

Effective and specific gene delivery to target cells in the nervous system remains a major challenge in translating these technologies for clinical application. Most animal studies and clinical trials have utilized viral vectors due to their advantageous in vivo delivery efficiencies. However, synthetic (non-viral) vectors offer several merits compared with viral systems, including improved safety profiles, versatility in application, and relative ease in production. A recent review covers the development of synthetic systems for gene delivery to neurons, the key information-transmitting cells in the nervous system [9].

Non-viral gene delivery to neurons is particularly challenging, especially when vectors are administered to the neural projections, such as via intramuscular injection for motor neuron delivery. For successful nucleic acid delivery to the cell body, vectors must traverse a series of cellular barriers (Fig. 1). Vectors must interact with the neuronal membrane, become internalized (typically into endocytic vesicles), undergo retrograde transport toward the cell nucleus, escape from vesicular compartments, and deposit the therapeutic in the desirable subcellular location (the nucleus for plasmids and the perinuclear cytoplasm for oligonucleotides and small, interfering RNA (siRNA)).

Several intracellular trafficking studies conducted in neurons or neuron-like cells have highlighted unique delivery challenges presented by neurons that are not encountered in other mammalian cell types. Non-specific, electrostatic uptake of synthetic vectors is significantly reduced in differentiated, neuron-like cells compared to undifferentiated cells [10]. Furthermore, binding and internalization efficiencies of synthetic vectors are depressed at neurites compared with neuronal soma [11]. Vesicular escape by non-viral vectors in neurons is also extremely limited. Two hours after internalization into neurons, the release efficiency of polycation-based synthetic vectors from endocytic vesicles is low compared with adenoviral vectors (~ 15% compared with ~ 95%, respectively, as assessed using colocalization analysis of confocal microscopy images) [12]. Although retrograde transport of non-viral vectors residing within neuronal endocytic vesicles occurs [11], [12], any released vectors are likely to experience minimal motility in the cytoplasm due to limited diffusion of particles of this size range (~ 80–150 nm) [13], [14]. Viruses have been shown to overcome this limited cytoplasmic diffusion by hijacking the retrograde-biased motor protein dynein [15], [16], [17], [18], [19], [39], [40], [41].

We hypothesize that bioactive peptides can be integrated with synthetic delivery systems to result in neuron-targeted delivery vectors with high delivery efficiencies. Synthetic peptides have been successfully applied to enhance the efficiencies of several steps in the gene delivery pathway, including DNA condensation, cell binding, and endosomal escape, as reviewed elsewhere [20], [21]. In our laboratory, we have demonstrated the advantage of incorporating bioactive peptides into polycations for improved transfection to mammalian cells. Here, we review the application of various bioactive peptides for assisting in targeting, endosomal escape, and dynein-binding of polycation vectors. In addition, we present new data demonstrating the synergistic effects of conjugating neuron-targeting and endosomal escape peptides to polycation vectors for plasmid delivery to neuron-like cells.