The development of an RNAi therapy against acute respiratory viral infection

McCaskill, Jana (2014). The development of an RNAi therapy against acute respiratory viral infection PhD Thesis, UQ Diamantina Institute, The University of Queensland.

       
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Author McCaskill, Jana
Thesis Title The development of an RNAi therapy against acute respiratory viral infection
School, Centre or Institute UQ Diamantina Institute
Institution The University of Queensland
Publication date 2014
Thesis type PhD Thesis
Supervisor Nicholas Saunders
Nigel McMillan
Timothy Doran
Antje Blumenthal
Total pages 168
Language eng
Subjects 0605 Microbiology
0699 Other Biological Sciences
100401 Gene and Molecular Therapy
110804 Medical Virology
1004 Medical Biotechnology
Formatted abstract
The development of therapeutic agents to treat acute viral infections has historically resulted in a limited number of treatments progressing into the clinic. A new technology, RNA interference (RNAi), allows for the specific blockade of individual genes and offers a promising avenue for novel antiviral treatments. The use of RNAi for the treatment of acute respiratory viral infection was evaluated for its effectiveness against two human pathogens, Hendra virus (HeV) and Respiratory syncytial virus (RSV). HeV is a highly pathogenic zoonotic paramyxovirus that causes fatal disease in a wide range of species, and is of significant concern to human health due to its high mortality rate, increasing emergence, absence of vaccines and limited post exposure therapies. This thesis initially investigated the use of RNAi based therapeutics targeting HeV in conjunction with the TLR3 agonist Poly I:C and showed that they are potent inhibitors of HeV infection in vitro. Short interfering RNAs (siRNAs) targeting the abundantly expressed genes of HeV caused over 95% reduction of HeV virus titre, protein and mRNA. Furthermore, the combination of HeV targeting siRNA and Poly I:C had an additive effect in suppressing HeV infection. These results demonstrate for the first time that RNAi and type I interferon stimulation are effective inhibitors of HeV replication in vitro and may provide an effective therapy for this highly lethal, zoonotic pathogen.

RNAi may therefore provide a therapeutic solution to many pulmonary viral infections. However, the main barrier to the clinical use of RNAi remains the lack of efficient delivery vectors. Research has mainly concentrated on the intranasal route of delivery of siRNA effector molecules for the treatment of respiratory diseases. However, this may be complicated in a diseased state due to increased fluid production and tissue remodeling. Therefore, this thesis investigated hydration of a freeze-dried matrix (HFDM) formulated liposomes for systemic delivery to the lung epithelium. It was shown that 45 ± 2% of epithelial murine lung cells receive siRNA delivery upon intravenous (IV) liposomal administration. Furthermore, it was demonstrated that liposomal siRNA delivery resulted in targeted gene and protein knockdown throughout the lung, including lung epithelium. Taken together, this is the first description of lung epithelial delivery via cationic liposomes, and provides a proof of concept for the use of IV liposomal RNAi delivery to specifically knockdown targeted genes in the respiratory system. This approach may provide an attractive alternate therapeutic delivery strategy for the treatment of lung epithelium diseases such as viral infection.

The concepts of RNAi knockdown of acute respiratory viral infection and intravenous delivery to the lungs was subsequently evaluated for the treatment of RSV. RSV is a human specific paramyxovirus that is the leading cause of bronchiolitis in children, infecting almost 100% of children by 2-3 years of age. Initially, siRNAs were designed that targeted highly conserved regions of RSV. The effect of RSV knockdown by these siRNAs was examined in vitro and sequences that displayed potent gene silencing were identified. HFDM formulated liposomes were subsequently used to deliver RSV targeting siRNAs intravenously in a mouse model of RSV infection to investigate their potential in the treatment of RSV infection. The benefits of immune stimulation during RSV infection was also evaluated through IV delivery of type I interferon agonists. These experiments are the first description of intravenous liposomal delivery to the lungs for the treatment of acute respiratory viral infection and provide a proof of concept for development of a RSV therapeutic.
Keyword Virus
RNA interference
siRNA
Liposomes
Delivery
Respiratory
Immune Stimulation

 
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Created: Fri, 21 Feb 2014, 01:16:38 EST by Miss Jana Mccaskill on behalf of Scholarly Communication and Digitisation Service