Part II: Functional delivery of a neurotherapeutic gene to neural stem cells using minicircle DNA and nanoparticles: Translational advantages for regenerative neurology

Abstract

Both neurotrophin-based therapy and neural stem cell (NSC)-based strategies have progressed to clinical trials for treatment of neurological diseases and injuries. Brain-derived neurotrophic factor (BDNF) in particular can confer neuroprotective and neuro-regenerative effects in preclinical studies, complementing the cell replacement benefits of NSCs.Therefore, combining both approaches by genetically-engineering NSCs to express BDNF is an attractive approach to achieve combinatorial therapy for complex neural injuries. Current genetic engineering approaches almost exclusively employ viral vectors for gene delivery to NSCs though safety and scalability pose major concerns for clinical translation and applicability. Magnetofection, a non-viral gene transfer approach deploying magnetic nanoparticles and DNA with magnetic fields offers a safe alternative but significant improvements are required to enhance its clinical application for delivery of large sized therapeutic plasmids. Here, we demonstrate for the first time the feasibility of using minicircles with magnetofection technology to safely engineer NSCs to overexpress BDNF.Primary mouse NSCs overexpressing BDNF generated increased daughter neuronal cell numbers post-differentiation, with accelerated maturation over a four-week period. Based on our findings we highlight the clinical potential of minicircle/magnetofection technology for therapeutic delivery of key neurotrophic agents.

Electrophysiological assessment of primary cortical neurons genetically engineered using iron oxide nanoparticles

“…Comparison between studies in terms of metal or particle concentration is difficult in as much as uptake into the cells is highly dependent on particle size and cell type. The MNPs employed here, at ~160 nm diameter [20], and with an additional chemical envelope for plasmid attachment, are relatively large, but these and similar particles have a well-documented safety profile across a range of neural cell types [7,[15][16][17][18]20]. Further investigation should be directed towards more mature cells and to track changes post-transfection over longer time periods.…”

“…It has been used extensively for genetic modification of both neuronal cell lines and primary neurons, including for delivery of physiologically relevant biomolecules. For example, reporter genes (such as GFP) have been delivered in transfection studies of neurons derived from stem cells [7], as well as in embryonic primary motor neurons [8], in order to test the suitability of this method at different developmental stages. The approach has also been used to study the localization and axonal transport of the spinal muscular atrophy protein (SMN) in motor neurons [8].…”

“…75 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 µL DMEM: F12 medium free of complex were added to the controls. Plates were then incubated for 30 min on the magnefect-nano array system [24 magnets, matching the culture plate; frequency (F) = 4 Hz; amplitude = 200 nm], then incubated for 24-72 h in the absence of the magnetic field [7].…”

Magnetic Nanoparticle‐Mediated Gene Delivery to Two‐ and Three‐Dimensional Neural Stem Cell Cultures: Magnet‐Assisted Transfection and Multifection Approaches to Enhance Outcomes

“…These vectors are further suited to translation as they contain no bacterial sequence elements (e.g., antibiotic resistance genes and origin of replication)meaning they are safer than conventional plasmids and also demonstrate sustained gene expression due to reduction of mammalian transgene silencing mechanisms (Fernandes & Chari, 2016a). Minicircle DNA encoding brain-derived neurotrophic factor (BDNF) has been successfully delivered to NSCs using magnetofection technology, with functional in vitro effects observed from secreted BDNF, including enhanced NSC proliferation and differentiation into neurons (Fernandes & Chari, 2016b).…”

Cre‐Mediated Transgene Integration in Chinese Hamster Ovary Cells Using Minicircle DNA Vectors

“…Minicircle DNA vectors lacking plasmid backbone sequences can be generated by site‐specific recombination at the two joint regions between backbone sequences and eukaryotic expression cassettes . Minicircle vectors have been extensively used for gene delivery in immunotherapy, cancer‐related research, stem cell reprogramming, and as DNA vaccines . Compared with conventional plasmid vectors, minicircle DNA vectors demonstrate i) lower cell toxicity by avoiding the negative effects caused by plasmid backbone sequences, such as immunogenicity and gene silencing; ii) improved transfection efficiencies because of their small size; and iii) more stable transgene expression resulting from structural compactness in transient expression.…”