PEPFECT
Transfection by PepFect of siRNA, plasmids and splice correcting oligonucleotides into different cell types
PepFect, a novel transfection reagent for oligonucleotide delivery in solution and as solid formulation
Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform splice-correcting oligonucleotides into usable drug products.
A new transfection reagent, PepFect, efficiently delivers splice-correcting oligonucleotides to different cell models including HeLa pLuc705 and mdx mouse myotubes, a cell culture model of Duchenne’s muscular dystrophy (DMD). Non-covalent PepFect - splice-correcting oligonucleotide nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine™ 2000 (LF2000) and remain active in the presence of serum.
Furthermore, the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications has been demonstrated. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to the same procedure.
This shows that using PepFect is a very promising translational approach for the delivery of splice-correcting oligonucleotides in different pharmaceutical forms.
PepFect forms highly active and stable nanocomplexes with siRNA, which are also durable in simulated gastric conditions
Cell-penetrating peptides (CPPs) are short cationic peptides that have been extensively studied as drug delivery vehicles for proteins, nucleic acids and nanoparticles. However, the formulation of CPP-based therapeutics into different pharmaceutical formulations and their stability in relevant biological environments have not been given the same attention.
A newly developed transfection reagent, PepFect, forms non-covalent nanocomplexes with short interfering RNA (siRNA), which are able to elicit efficient RNA-interference (RNAi) response in different cell-lines. RNAi effect is obtained at low siRNA doses with a unique kinetic profile.
Solid dispersion technique is utilized to formulate PepFect/siRNA nanocomplexes into solid formulations that are as active as the freshly prepared nanocomplexes in solution. Importantly, the nanocomplexes are stable and active in mediating RNAi response after incubation with simulated gastric fluid that is highly acidic.
These results demonstrate the activity of PepFect in delivering and protecting siRNA in different pharmaceutical forms and biological environments.
Highlights
PepFect mediated knockdown of an endogenous gene - hypoxanthine phosphoribosyltransferase 1 (HPRT1).
HUH7 cells were transfected by siRNA with PepFect (in media with serum or without) or by siRNA only without PepFect, or with an unrelated siRNA together with PepFect.
Downregulation of HPRT1 gene expression by PepFect - siRNA complexes.
HUH7 cells were transfected with PepFect -siRNA complexes in FBS-containing and FBS-free media.
Size distribution of PepFect - siRNA nanocomplexes studied with Nanoparticle Tracking Analysis (NTA) system.
Black curve - Nanocomplexes formed in water. Red curve - Nanocomplexes formed in OptiMEM with 10% FBS.
A fairly uniform size distribution peaked at 106 nm is observed in both media.
Advantages of PepFect
- Transfection efficiency using PepFect is nearly 100%.
- PepFect facilitates efficient endosomal release of oligo-nucleotides.
- PepFect forms non-covalent nanocomplexes with nucleotides of 100 nm in size.
- PepFect delivers highly active oligonucleotides to their targets.
- PepFect is stable and highly active in different cell culture conditions.
- PepFect works well with siRNA, splice-correcting oligonucleotides, antisense, plasmid DNA and other types of nucleotides.
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