The Development of Indolicidin-Based Carrier to Deliver Antisense Oligonucleotide against Tnf-Α for the Treatment of Rheumatoid Arthritis

Silence of tumor necrosis factor-α (TNF-α) gene has been proved to effectively protect against therheumatoid arthritis (RA) development. So far, the lack of suitable delivering carriers continues to be amajor obstacle to not only the cellular effect in vitro but also practical therapy in vivo. Indolicidin (IL) is awell-known cationic antimicrobial peptide derived from bovine neutrophils. Our previous project (MOST104-2221-E-008-105-) demonstrated that IL can be applied to facilitate plasmid DNA delivery. Therefore, inthis project, we propose IL as novel candidates of carriers to deliver antisense oligonucleotides (ASOs)against TNF-α mRNA sequence. IL will be either directly linked to ASOs as a conjugate or applied asdimmer-form or bolaamphiphile-form carriers to complex with ASOs as nanoparticles. In addition, a cysteinwill be added to N or C teminals of IL to specifically control the conjugation sites, by which the interactionof IL domains and cell membrane can be manipulated. The differences of delivery performances in thesecases will elucidate the effects of conjugation form and the roles of hydrophobic and cationic domains in ILon ASO delivery. The TNF-α knockdown effect of the nanoparticles will be examined in vitro using mouseRaw 264.7 macrophage cell line treated with lipopolysaccharide (LPS) to stimulate TNF-α expression.Through real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA)assays, the time profile of mRNA degradation and the following attenuation of TNF-α expression will bedescribed. These results will be helpful to determine pharmacokinetics of ASO delivery. Furthermore, wewill optimize the administration protocol through the temporal effects and dosage-response experiments,and the biocompatibility of our developed IL-derived carriers will also be evaluated. Once we demonstratethat TNF-α expression in macrophages can be attenuated through IL-derived carrier delivery, we will furtherapply them to whole blood sample to investigate the feasibility of these systems to treat circulatingleukocytes. Finally, TNF-α ASOs will be delivered to RA disease model. The collagen-induced arthritis(CIA) model will be established in DBA/1j male mice by immunization of the mice with bovine collagen IIin adjuvant. The antisense effect of the nanoparticles will be analyzed by evaluating the incidence andseverity of arthritis induced in immunized mice. This comprehensive study provides an ideal solution todesign peptide-derived ASO carriers. Although our study is to develop an ASO therapy against RA, itsapplication should not be restricted. If we can determine an effective form of IL-derived ASO vehiclethrough this study, it will also be capable of treating other diseases which need to silence pathologic genesfor therapeutic purpose.