Peptide-modified vectors 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.
Section snippets
PC-12 cell culture
PC-12 cells were obtained from ATCC (CRL-1721) and were maintained in growth medium (F-12K medium supplemented with 15% horse serum, 2.5% fetal bovine serum, and antibiotics) in a 37 °C, 5% CO2 environment. Medium was replaced every 2–3 days and cells were passaged when 60–80% confluent. Cells were detached by incubation with Trypsin–EDTA and resuspended in 1 mL of F-12 K medium. In order to obtain a single cell suspension, cells were passed through a fire-polished glass pipette. For
Neuron targeting
For neuron-specific delivery, it is desirable to target vehicles using ligand–receptor interactions in order to minimize secondary effects due to off-target delivery. To this end, several classes of neuron-specific ligands have been employed: neuropeptides, neurotrophins, and neurotoxins [9]. Of particular note is tetanus toxin (TeNT), which binds to peripheral neurons at their presynaptic terminals and is transported retrograde in motor neurons. The heavy chain of TeNT (TeNT Hc), which is
Conclusions and future directions
We have summarized our recent work identifying and applying bioactive peptides for enhancing non-viral gene delivery and have demonstrated the potential of multicomponent, peptide-modified vectors for neuron-targeted delivery. Synthetic peptides are promising materials because they can be economically produced [49] and modularly incorporated into existing vectors. In this work, we have demonstrated that multiple peptides can be combined to synergistically improve gene transfection efficiencies.
Acknowledgments
This work was funded by the NIH/NINDS (R21NS052030) and an NSF CAREER Award (CBET 0448547). EJK and JMB acknowledge the University of Washington's Engineered Biomaterials Training Program for graduate fellowship support (T32GM065098). We are grateful to Patrick Stayton (UW Bioengineering) for the use of his ZetaPALS dynamic light scattering analyzer.
References (49)
- et al.
Gene delivery to the nervous system
Molecular Therapy
(2008) - et al.
Gene delivery to differentiated neurotypic cells with RGD and HIV Tat peptide functionalized polymeric nanoparticles
Biomaterials
(2006) - et al.
Protein diffusion in living skeletal muscle fibers: dependence on protein size, fiber type, and contraction
Biophysical Journal
(2000) Solute and macromolecule diffusion in cellular aqueous compartments
Trends in Biochemical Sciences
(2002)- et al.
Characterization of commercially available and synthesized polyethylenimines for gene delivery
Journal of Controlled Release
(2000) - et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method
Methods (San Diego, Calif)
(2001) - et al.
Retrograde trans-neuronal transfer of the C-fragment of tetanus toxin
Brain Research
(1987) - et al.
A novel peptide defined through phage display for therapeutic protein and vector neuronal targeting
Neurobiology of Disease
(2005) - et al.
Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes
Journal of biological chemistry
(2003) - et al.
Membrane-active peptides for non-viral gene therapy: making the safest easier
Trends in Biotechnology
(2008)
The membranotropic regions of the endo and ecto domains of HIV gp41 envelope glycoprotein
Biochimica et Biophysica Acta
The molecular motor toolbox for intracellular transport
Cell
Structure and dynamics of LC8 complexes with KXTOT-motif peptides: swallow and dynein intermediate chain compete for a common site
Journal of Molecular Biology
Solution structure of the Tctex1 dimer reveals a mechanism for dynein–cargo interactions
Structure
Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model
Science
Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model
Natural Medicines
Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson's disease in rats
Proceedings of the National Academy of Sciences of the United States of America
RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model
Proceedings of the National Academy of Sciences of the United States of America
RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia
Natural Medicines
Induction of antigen-specific tolerance in multiple sclerosis after immunization with DNA encoding myelin basic protein in a randomized, placebo-controlled phase 1/2 trial
Archives of Neurology
Nonviral approaches for neuronal delivery of nucleic acids
Pharmaceutical Research
Analysis of the intracellular barriers encountered by nonviral gene carriers in a model of spatially controlled delivery to neurons
Journal of Gene Medicine
Quantifying the intracellular transport of viral and nonviral gene vectors in primary neurons
Experimental biology and medicine (Maywood)
Cited by (31)
Virus-inspired nucleic acid delivery system: Linking virus and viral mimicry
2016, Advanced Drug Delivery ReviewsNon-Viral nanosystems for gene and small interfering RNA delivery to the central nervous system: Formulating the solution
2013, Journal of Pharmaceutical SciencesCitation Excerpt :Tet1 is a synthetic peptide which displays high affinity for the triasialoganglioside receptor on motor neurons.138 Tet1 undergoes retrograde transport from the pre-synaptic axon terminus and has been conjugated to PEI to facilitate internalisation of pDNA and siRNA polyplexes.133,138 Other receptor–ligand interactions have also been exploited to facilitate uptake.
Non-viral gene therapy for neurological diseases, with an emphasis on targeted gene delivery
2012, Journal of Controlled ReleaseCitation Excerpt :siRNA has the advantage of not having to enter the nucleus and this may one of the reasons siRNA is more efficient in transfecting cells than DNA [44]. Recent research has revealed that movement of non-viral vectors complexed to pDNA across the cytoplasm to the nucleus can be enhanced by conjugation to dynein-binding peptides [63]. If it is necessary for pDNA to be released from binding before it enters the nucleus is uncertain.
HPMA-oligolysine copolymers for gene delivery: Optimization of peptide length and polymer molecular weight
2011, Journal of Controlled ReleaseCitation Excerpt :We further demonstrated that HPMA-oligolysine copolymers can be synthesized using reversible addition-fragmentation transfer (RAFT) copolymerization [14]. The efficient and statistical incorporation of peptides in copolymers by RAFT polymerization raises the possibility of efficient and well-defined synthesis of multifunctional peptide-based polymers that incorporate various monomers to facilitate delivery [15–17]. However, the properties of the base material, HPMA-oligolysine copolymer, should first be optimized before including peptides for cell targeting [18,19] or endosomal escape [20–23].
Targeted nonviral delivery vehicles to neural progenitor cells in the mouse subventricular zone
2010, BiomaterialsCitation Excerpt :The use of small peptides over full-length polypeptides is advantageous for nanoparticulate delivery systems because they can be easily synthesized and incorporated while having minimal effects on the physicochemical properties of the particle. The Tet1 peptide has been successfully used in our group to target polyethylenimine (PEI)-based gene delivery vehicles in vitro to cultured neuronal cells [20,21]. The goal of this work is to evaluate Tet1-modified vehicles for in vivo targeting to neuronal populations in the CNS.
- 1
Current address: Department of Biomedical Sciences, Chonnam National University Medical School, South Korea.