Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice

Abstract

In this study, we investigated a series of cationic polyelectrolytes (PEs) with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that the poly(diallyldimethylammonium chloride) (PDDAC) polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro. The enhanced PMO delivery and targeting to dystrophin exon 23 was further observed in mdx mice, up to fourfold with the PE-4, compared with PMO alone. The cytotoxicity of the PEs was lower than that of Endoporter and polyethylenimine 25,000 Da in vitro, and was not clearly detected in muscle in vivo under the tested concentrations. Together, these results demonstrate that optimization of PE molecular size, composition, and distribution of cationic charge are key factors to achieve enhanced PMO exon-skipping efficiency. The increased efficiency and lower toxicity show this PDDAC series to be capable gene/antisense oligonucleotide delivery-enhancing agents for treating muscular dystrophy and other diseases.

Tween 85-Modified Low Molecular Weight PEI Enhances Exon-Skipping of Antisense Morpholino Oligomer In Vitro and in mdx Mice

“…Transfection efficiencies were recorded with the Olympus IX71 fluorescent microscope (Olympus America, Melville, NY, USA). TE was examined quantitatively using flow cytometry (BD, Franklin Lakes, NJ, USA), and the exon-skipping was also further examined by RT-PCR in the C2C12E23 cell line 31, 32, 33, 34, 35, 39. One hundred nanograms of template RNA was used for each 25 μL RT-PCR reaction.…”

Triazine-cored polymeric vectors for antisense oligonucleotide delivery in vitro and in vivo

“…Cytotoxicity was evaluated in C2C12E50 cell line using the MTS assay [30][31][32][33][34][35]. The relative cell viability was calculated by: A treated − A background × 100/ A control − A background , the absorbance (A) was measured at 570 nm using a Tecan 500 Plate reader (Tecan US, Inc, Morrisville, NC, USA) to obtain the metabolic activity of the cell.…”

“…The polymer/AO polyplex solution containing 1 µg of AO was prepared at a designated weight ratio in 100 µL 0.9% saline and analyzed using transmission electron microscopy (TEM; JEM-1400Plus TEM by JEOL USA, Inc.) with AMT-XR80S-B wide angle side Mount 8MPixel, CCD Camera. The samples were prepared using negative staining with 1% phosphotungstic acid [30][31][32][33][34][35].…”

“…The polymer-based drug/gene delivery is promising for the treatment of inherent or acquire disease, because of the polymer's structural flexibility, larger capacity for therapeutic agent, low host immunogenicity and less cost than viral one [21][22][23][24]. Amphiphilic polymers developed for gene delivery by our group and others have demonstrated enhanced gene transfection in skeletal muscles [25][26][27][28][29][30][31][32][33][34][35]. Recently, a set of triazine-cored amphiphilic polymers (TAPs) constructed from Jeffamine M series and low molecular weight polyethyleneimine (LPEI) were prepared by combinatorial chemistry and characterized for pDNA delivery in vitro and in vivo [34], which can condense DNA efficiently with nanosized particles below 200 nm at the weight ratio 5 of polymer/pDNA, being stable in the presence of serum.…”

Leveraging a Sturge-Weber Gene Discovery: An Agenda for Future Research

“…Both of these approaches can also be used in mice. 49;50 One of the goals of developing an animal model of SWS is to be able to screen potential compounds. Zebrafish models provide a robust drug screening platform to identify targets.…”