| 1. |
- Dash-Wagh, Suvarna, et al.
(författare)
-
Intracellular Delivery of Short Interfering RNA in Rat Organ of Corti Using a Cell-penetrating Peptide PepFect6
- 2012
-
Ingår i: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 1:e61
-
Tidskriftsartikel (refereegranskat)abstract
- RNA interference (RNAi) using short interfering RNA (siRNA) is an attractive therapeutic approach for treatment of dominant-negative mutations. Some rare missense dominant-negative mutations lead to congenital-hearing impairments. A variety of viral vectors have been tested with variable efficacy for modulating gene expression in inner ear. However, there is concern regarding their safety for clinical use. Here, we report a novel cell-penetrating peptide (CPP)-based nonviral approach for delivering siRNA into inner ear tissue using organotypic cultures as model system. PepFect6 (PF6), a variant of stearyl-TP10, was specially designed for improved delivery of siRNA by facilitating endosomal release. We show that PF6 was internalized by all cells without inducing cytotoxicity in cochlear cultures. PF6/siRNA nanoparticles lead to knockdown of target genes, a housekeeping gene and supporting cell-specific connexin 26. Interestingly, application of PF6/connexin 26 siRNA exhibited knockdown of both connexin 26 and 30 mRNA and their absence led to impaired intercellular communication as demonstrated by reduced transfer of calcein among the PF6/connexin 26-siRNA-treated cells. Thus, we conclude that PF6 is an efficient nonviral vector for delivery of siRNA, which can be applied as a tool for the development of siRNA-based therapeutic applications for hearing impairments.
|
|
| 2. |
|
|
| 3. |
|
|
| 4. |
|
|
| 5. |
|
|
| 6. |
|
|
| 7. |
|
|
| 8. |
- Stenler, S., et al.
(författare)
-
Micro-minicircle gene therapy : Implications of size on fermentation, complexation, shearing resistance, and expression
- 2014
-
Ingår i: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 3, s. e140-
-
Tidskriftsartikel (refereegranskat)abstract
- The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting U7 small nuclear RNA, which results in a vector of 650 base pairs (bp), as compared to a conventional 3600 bp plasmid carrying the same expression cassette. Furthermore, we construct miMCs of varying sizes carrying different number of these cassettes. This allows us to evaluate how size influences production, supercoiling, stability and efficiency of the vector. We characterize coiling morphology by atomic force microscopy and measure the resistance to shearing forces caused by an injector device, the Biojector. We compare the behavior of miMCs and plasmids in vitro using lipofection and electroporation, as well as in vivo in mice. We here show that when the size of the miMC is reduced, the formation of dimers and trimers increases. There seems to be a lower size limit for efficient expression. We demonstrate that miMCs are more robust than plasmids when exposed to shearing forces, and that they show extended expression in vivo.
|
|
| 9. |
|
|
