Highly efficient delivery of siRNA to a heart transplant model by a novel cell penetrating peptide-dsRNA binding domain.
Small interfering RNAs (siRNAs) delivery remains a bottleneck for RNA interference (RNAi) - based therapies in the clinic. In the present study, a fusion protein with two cell-penetrating peptides (CPP), Hph1-Hph1, and a double-stranded RNA binding domain (dsRBD), was constructed for the siRNA delivery: dsRBD was designed to bind siRNA, and CPP would subsequently transport the dsRBD/siRNA complex into cells. We assessed the efficiency of the fusion protein, Hph1-Hph1-dsRBD, as a siRNA carrier. Calcium-condensed effects were assessed on GAPDH and green fluorescent protein (GFP) genes by western blot, real time polymerase chain reaction (RT-PCR), and flow cytometry analysis in vitro. Evaluations were also made in an in vivo heart transplantation model. The results demonstrated that the fusion protein, Hph1-Hph1-dsRBD, is highly efficient at delivering siRNA in vitro, and exhibits efficiency on GAPDH and GFP genes similar to or greater than lipofectamine. Interestingly, the calcium-condensed effects dramatically enhanced cellular uptake of the protein-siRNA complex. In vivo, Hph1-Hph1-dsRBD transferred and distributed ^ targeted siRNA throughout the whole mouse heart graft. Together, these results indicate that Hph1-Hph1-dsRBD has potential as an siRNA carrier for applications in the clinic or in biomedical research.
The Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Institute for Regenerative Medicine and Cancer, Huzhou University School of Medicine, Huzhou, China.
Hua
H
Li
The Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Xiangtao
X
Zheng
The Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Viktoria
V
Koren
The Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Yogesh Kumar
YK
Vashist
The Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Electronic address: tytsui@gmail.com.
Tung Yu
TY
Tsui
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000378:metabolism*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006367:HeLa Cells; D016027:Heart Transplantation; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D057846:Cell-Penetrating Peptides; D010446:Peptide Fragments; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D002118:Calcium
Cell penetrating peptide; Double-stranded RNA binding domain; Gene delivery; RNA interference