Lack of RNA-DNA oligonucleotide (chimeraplast) mutagenic activity in mouse embryos

There are numerous reports of the use of RNA-DNA oligonucleoticles (chimeraplasts) to correct point mutations in vitro and in vivo, including the human apolipoprotein E gene (ApoE). Despite the absence of selection for targeting, high efficiency conversion has been reported. Although mainly used to revert deleterious mutations for gene therapy applications, successful use of this approach would have the potential to greatly facilitate the production of defined mutations in mice and other species. We have attempted to create a point mutation in the mouse ApoE gene by microinjection of chimeraplast into the pronuclei of 1-cell mouse eggs. Following transfer of microinjected eggs we analysed 139 E12.5 embryos, but obtained no evidence for successful conversion. (c) 2005 Wiley-Liss, Inc

Correction of the neuropathogenic human apolipoprotein E4 (APOE4) gene to APOE3 in vitro using synthetic RNA/DNA oligonucleotides (chimeraplasts)

Apolipoprotein E (apoE) is a multifunctional circulating 34-kDa protein, whose gene encodes single-nucleotide polymorphisms linked to several neurodegenerative diseases. Here, we evaluate whether synthetic RNA/DNA oligonucleoticles (chimeraplasts) can convert a dysfunctional gene, APOE4 (C -> T, Cys112Arg), a risk factor for Alzheimer's disease and other neurological disorders, into wild-type APOE3. In preliminary experiments, we treated recombinant Chinese hamster ovary (CHO) cells stably secreting apoE4 and lymphocytes from a patient homozygous for the epsilon 4 allele with a 68-mer apoE4-to-apoE3 chimeraplast, complexed to the cationic delivery reagent, polyethyleneimine. Genotypes were analyzed after 48 h by routine polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and by genomic sequencing. Clear conversions of APOE4 to APOE3 were detected using either technique, although high concentrations of chimeraplast were needed (>= 800 nM). Spiking experiments of PCR reactions or CHO-K1 cells with the chimeraplast confirmed that the repair was not artifactual. However, when treated recombinant CHO cells were passaged for 10 d and then subcloned, no conversion could be detected when > 90 clones were analyzed by locus-specific PCR-RFLP. We conclude that the apparent efficient repair of the APOE4 gene in CHO cells or lymphocytes 48 h post-treatment is unstable, possibly because the high levels of chimeraplast and polyethyleneimine that were needed to induce nucleotide substitution are cytotoxic

Apolipoprotein E delivery by peritoneal implantation of encapsulated recombinant cells improves the hyperlipidaemic profile in apoE-deficient mice

Plasma apolipoprotein E (apoE) is a 34-kDa polymorphic protein which has atheroprotective actions by clearing remnant lipoproteins and sequestering excess cellular cholesterol. Low or dysfunctional apoE is a risk factor for hyperlipidaemia and atherosclerosis, and for restenosis after angioplasty. Here, in short-term studies designed to establish proof-of-principle, we investigate whether encapsulated recombinant Chinese hamster ovary (CHO) cells can secrete wild-type apoE3 protein in vitro and then determine whether peritoneal implantation of the microcapsules into apoE-deficient (apoE(-/-)) mice reduces their hypercholesterolaemia.Recombinant CHO-E3 cells were encapsulated into either alginate poly-L-lysine or alginate polyethyleneimine/polybrene microspheres. After verifying stability and apoE3 secretion, the beads were then implanted into the peritoneal cavity of apoE(-/-) mice; levels of plasma apoE3, cholesterol and lipoproteins were monitored for up to 14 days post-implantation.Encapsulated CHO-E3 cells continued to secrete apoE3 protein throughout a 60-day study period in vitro, though levels declined after 14 days. This cell-derived apoE3 was biologically active. When conditioned medium from encapsulated CHO-E3 cells was incubated with cultured cells pre-labelled with [H-3]-cholesterol, efflux of cholesterol was two to four times greater than with normal medium (at 8 h, for example, 7.4+/-0.3% vs. 2.4+/-0.2% of cellular cholesterol; P<0.001). Moreover, when secreted apoE3 was injected intraperitoneally into apoE(-/-) mice, apoE3 was detected in plasma and the hyperlipidaemia improved. Similarly, when alginate polyethyleneimine/polybrene capsules were implanted into the peritoneum of apoE(-/-) mice, apoE3 was secreted into plasma and at 7 days total cholesterol was reduced, while atheroprotective high-density lipoprotein (HDL) increased. In a second study, apoE was detectable in plasma of five mice treated with alginate poly-L-lysine beads, 4 and 7 days post-implantation, though not at day 14. Furthermore, their hypercholesterolaemia was reduced, while HDL was clearly elevated in all mice at days 4 and 7 (from 18.4+/-6.2% of total lipoproteins to 31.1+/-6.8% at 7 days; P<0.001); however, these had rebounded by day 14, possibly due to the emergence of anti-apoE antibodies.We conclude that microencapsulated apoE-secreting cells have the potential to ameliorate the hyperlipidaemia of apoE deficiency, but that the technology must be improved to become a feasible therapeutic to treat atherosclerosis. (C) 2004 Elsevier B.V. All rights reserved

Role of liposome and peptide in the synergistic enhancement of transfection with a lipopolyplex vector

Lipopolyplexes are of widespread interest for gene therapy due to their multifunctionality and high transfection efficiencies. Here we compared the biological and biophysical properties of a lipopolyplex formulation with its lipoplex and polyplex equivalents to assess the role of the lipid and peptide components in the formation and function of the lipopolyplex formulation. We show that peptide efficiently packaged plasmid DNA forming spherical, highly cationic nanocomplexes that are taken up efficiently by cells. However, transgene expression was poor, most likely due to endosomal degradation since the polyplex lacks membrane trafficking properties. In addition the strong peptide-DNA interaction may prevent plasmid release from the complex and so limit plasmid DNA availability. Lipid/DNA lipoplexes, on the other hand, produced aggregated masses that showed poorer cellular uptake than the polyplex but contrastingly greater levels of transgene expression. This may be due to the greater ability of lipoplexes relative to polyplexes to promote endosomal escape. Lipopolyplex formulations formed spherical, cationic nanocomplexes with efficient cellular uptake and significantly enhanced transfection efficiency. The lipopolyplexes combined the optimal features of lipoplexes and polyplexes showing optimal cell uptake, endosomal escape and availability of plasmid for transcription, thus explaining the synergistic increase in transfection efficiency

Receptor-targeted liposome-peptide-siRNA nanoparticles represent a novel and efficient therapeutic approach to prevent conjunctival fibrosis.

There is increasing evidence that the Myocardin-related transcription factor/Serum response factor (MRTF/SRF) pathway plays a key role in fibroblast activation and that knocking down MRTF can lead to reduced scarring and fibrosis. Here, we have developed a receptor-targeted liposome-peptide-siRNA nanoparticle as a non-viral delivery system for MRTF-B siRNA in conjunctival fibrosis. Using 50 nM siRNA, the MRTF-B gene was efficiently silenced by 76% and 72% with LYR and LER nanoparticles, respectively. The silencing efficiency was low when non-targeting peptides or siRNA alone or liposome-siRNA alone were used. LYR and LER nanoparticles also showed higher silencing efficiency than PEGylated LYR-P and LER-P nanoparticles. The nanoparticles were not cytotoxic using different liposomes, targeting peptides, and 50 nM siRNA. Three-dimensional fibroblast-populated collagen matrices were also used as a functional assay to measure contraction in vitro, and showed that MRTF-B LYR nanoparticles completely blocked matrix contraction after a single transfection treatment. In conclusion, this is the first study to develop and show that receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient and safe non-viral siRNA delivery system that could be used to prevent fibrosis after glaucoma filtration surgery and other contractile scarring conditions in the eye

The role of the helper lipid on the DNA transfection efficiency of lipopolyplex formulations.

Multifunctional, lipopolyplex formulations comprising a mixture of cationic liposomes and cationic, receptor-targeting peptides have potential use in gene therapy applications. Lipopolyplex formulations described here are typically far more efficient transfection agents than binary lipoplex or polyplex formulations. It has been shown previously that the peptide component mediates both DNA packaging and targeting of the nanoparticle while in this report we investigate the contribution of the lipid component. We hypothesised that the lipid components synergise with the peptides in the transfection process by promoting endosomal escape after lipid bilayer fusion. Lipopolyplexes were prepared with cationic liposomes comprising DOTAP with either neutral lipid DOPE or DOPC. DOPE promotes fusogenic, inverted hexagonal lipid structures while DOPC promotes more stable laminar structures. Lipopolyplexes containing DOPE showed substantially higher transfection efficiency than those formulated with DOPC, both in vitro and in vivo. DOPE-containing lipopolyplexes showed rapid endosomal trafficking and nuclear accumulation of DNA while DOPC-containing formulations remained within the late endo-lysosomal compartments. These findings are consistent with previous finding for the role of DOPE in lipoplexes and support the hypothesis regarding the function of the lipid components in lipopolyplexes. These findings will help to inform future lipopolyplex design, strategies and clinical development processes

Delivery of ENaC siRNA to epithelial cells mediated by a targeted nanocomplex: a therapeutic strategy for cystic fibrosis

The inhibition of ENaC may have therapeutic potential in CF airways by reducing sodium hyperabsorption, restoring lung epithelial surface fluid levels, airway hydration and mucociliary function. The challenge has been to deliver siRNA to the lung with sufficient efficacy for a sustained therapeutic effect. We have developed a self-assembling nanocomplex formulation for siRNA delivery to the airways that consists of a liposome (DOTMA/DOPE; L), an epithelial targeting peptide (P) and siRNA (R). LPR formulations were assessed for their ability to silence expression of the transcript of the gene encoding the α-subunit of the sodium channel ENaC in cell lines and primary epithelial cells, in submerged cultures or grown in air-liquid interface conditions. LPRs, containing 50 nM or 100 nM siRNA, showed high levels of silencing, particularly in primary airway epithelial cells. When nebulised these nanocomplexes still retained their biophysical properties and transfection efficiencies. The silencing ability was determined at protein level by confocal microscopy and western blotting. In vivo data demonstrated that these nanoparticles had the ability to silence expression of the α-ENaC subunit gene. In conclusion, these findings show that LPRs can modulate the activity of ENaC and this approach might be promising as co-adjuvant therapy for cystic fibrosis

Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung

Introduction Loss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport. Methods We investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air–liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (Vt), short circuit current (Isc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections. Results Nanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and βENaC mRNA by 30%. Transfections reduced Vt, the amiloride-sensitive Isc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%. Conclusion Nanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo

In vitro and in vivo delivery of a sustained release nanocarrier-based formulation of an MRTF/SRF inhibitor in conjunctival fibrosis

BACKGROUND: Sustained drug delivery is a large unmet clinical need in glaucoma. Here, we incorporated a Myocardin-Related Transcription Factor/Serum Response Factor inhibitor, CCG-222740, into slow release large unilamellar vesicles derived from the liposomes DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane) and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and tested their effects in vitro and in vivo. RESULTS: The vesicles were spherical particles of around 130 nm and were strongly cationic. A large amount of inhibitor could be incorporated into the vesicles. We showed that the nanocarrier CCG-222740 formulation gradually released the inhibitor over 14 days using high performance liquid chromatography. Nanocarrier CCG-222740 significantly decreased ACTA2 gene expression and was not cytotoxic in human conjunctival fibroblasts. In vivo, nanocarrier CCG-222740 doubled the bleb survival from 11.0 ± 0.6 days to 22.0 ± 1.3 days (p = 0.001), decreased conjunctival scarring and did not have any local or systemic adverse effects in a rabbit model of glaucoma filtration surgery. CONCLUSIONS: Our study demonstrates proof-of-concept that a nanocarrier-based formulation efficiently achieves a sustained release of a Myocardin-Related Transcription Factor/Serum Response Factor inhibitor and prevents conjunctival fibrosis in an established rabbit model of glaucoma filtration surgery