Abstract
Hepatitis B infection is a major causative factor for chronic liver disease and hepatocellular carcinoma. Recent studies have shown RNA interference (RNAi), the process of gene regulation via specific degradation of target mRNA, can be applied to inhibit hepatitis B replication, however administration to patients is a major issue. Short interfering RNA (siRNA) can be encoded as hairpins within DNA, however viral vectors commonly evoke an immune response, limiting the pool of vector available, whilst non-viral vectors are often poorly efficient due to barriers including the nuclear membrane. We were therefore interested to develop the use of polyelectrolyte complexes based on pH responsive cationic polymers containing reducible disulphides to condense siRNA. These vectors are designed to undergo reduction-triggered intracytoplasmic unpacking and release of biologically active siRNA in its final form. The advantage of such a system is the avoidance of the need to translocate the nuclear membrane in non-dividing cells.
Cationic oligopeptides of varying sequence (CK6C, CK8C, CK10C, CH3K3H3C, CH6K3H6C) were synthesised and subjected to oxidative polycondensation to produce reducible polycations (RPCs). Complexation of RPCs with siRNA form 100nm particles stable in the weakly reducing extracellular environment but designed to disintegrate in the intracellular reducing environment. Complexation completely retarded the mobility of siRNA on an agarose gel, with the amount of RPC required dependent on the lysine content. Treatment with 8mM glutathione restored siRNA mobility, indicating cleavage of disulphide bonds within the RPC, thus simulating the reducing conditions within the cytoplasm. Indeed a punctate pattern of glutathione localisation was demonstrated within liver cells using two-photon laser scanning microscopy.
A GFP-expressing stable liver cell line was generated to test the biological activity of siRNA delivered by various RPCs. siRNA delivered using pH-responsive histidine containing RPCs exhibited the greatest level of GFP repression consistent with improved endosomal escape and decomplexation of particles within the cytoplasm.
The intracellular fate of complexes is being assessed using Cy3 labelled siRNA. Furthermore RPCs terminated in hepatocyte-binding oligopeptides will be employed to improve cell uptake via receptor-mediated endocytosis. Ultimately it is hoped this vector system will provide a platform for delivery of siRNA targeting hepatitis B and C proteins for treatment of liver disease.
Cationic oligopeptides of varying sequence (CK6C, CK8C, CK10C, CH3K3H3C, CH6K3H6C) were synthesised and subjected to oxidative polycondensation to produce reducible polycations (RPCs). Complexation of RPCs with siRNA form 100nm particles stable in the weakly reducing extracellular environment but designed to disintegrate in the intracellular reducing environment. Complexation completely retarded the mobility of siRNA on an agarose gel, with the amount of RPC required dependent on the lysine content. Treatment with 8mM glutathione restored siRNA mobility, indicating cleavage of disulphide bonds within the RPC, thus simulating the reducing conditions within the cytoplasm. Indeed a punctate pattern of glutathione localisation was demonstrated within liver cells using two-photon laser scanning microscopy.
A GFP-expressing stable liver cell line was generated to test the biological activity of siRNA delivered by various RPCs. siRNA delivered using pH-responsive histidine containing RPCs exhibited the greatest level of GFP repression consistent with improved endosomal escape and decomplexation of particles within the cytoplasm.
The intracellular fate of complexes is being assessed using Cy3 labelled siRNA. Furthermore RPCs terminated in hepatocyte-binding oligopeptides will be employed to improve cell uptake via receptor-mediated endocytosis. Ultimately it is hoped this vector system will provide a platform for delivery of siRNA targeting hepatitis B and C proteins for treatment of liver disease.
Original language | English |
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Article number | P197 |
Journal | Molecular Therapy |
Volume | 11(1_Suppl) |
Publication status | Published - 1 Jun 2005 |
Event | American Society of Gene Therapy - Saint Louis, MO, United States Duration: 1 Jun 2005 → 5 Jun 2005 |