Improved in vivo delivery of siRNA

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop novel reagents to efficiently deliver functional siRNA into animals for gene silencing experiments. Pharmaceutical and biotechnology companies are actively testing siRNAs
as therapeutic agents and to validate drug targets. Unfortunately siRNA delivery in animals is usually not efficient and it has proven difficult to deliver siRNA to some therapeutically important cell types and tissues. The commercial success of in vivo ap
plications of siRNA hinges on the development of new delivery methods. The proposed research will result in the development of improved antibody-based conjugates to target siRNAs to cells overexpressing the appropriate cell surface antigen. Two different t
ypes of immunoconjugates will be developed. The first immunoconjugate type is produced by chemically crosslinking large recombinant RNA binding proteins to antibodies. The siRNA noncovalently associates with the targeting antibody via the RNA binding prote
in. The second type is produced by directly attaching functional modified siRNAs to antibodies using heterobifunctional crosslinking agents containing internal cell labile structures to promote release of the siRNA after internalization. Specific aims for
Phase I: 1. Develop novel immunoconjugates to significantly increase the amount of siRNA loaded per antibody molecule in the antibody/siRNA complex. 2: Covalently conjugate siRNA to antibodies using cleavable linkers to produce immunoconjugates which are s
table in plasma but efficiently release functional siRNA in cells. 3. Use the novel immunoconjugates produced in Aims 1 and 2 to specifically deliver functional siRNA into cultured cells overexpressing the appropriate cell surface antigen and into tumor ce
lls in mouse xenografts. The siRNA binding capacity and stability of each of immunoconjugates will be directly compared using fluorescence measurements. The ability of the immunoconjugates to deliver functional siRNA into targeted cells to silence specifi
c genes will also be tested using qRT-PCR. In Phase II, we will expand and adapt the technique to work with chemically modified siRNAs designed for in vivo applications. Narrative: The versatility and potency of siRNA in downregulating target gene
expression has made it a preferred tool to study gene function in mammalian cell culture systems. As a result of this success, the therapeutic potential of siRNA is being actively investigated in animal disease models and in humans. Unfortunately, the cur
rent methods to deliver siRNA in animals are very inefficient and difficult to control. The promise of therapeutic siRNA will not be realized until more efficient modes of delivery are developed. Therefore, we outline an efficient research plan to develop
improved methods of in vivo siRNA delivery. Our strategy involves the use of antibody-based delivery agents to target and deliver siRNA into specific cell types and if successful will greatly advance the delivery of siRNA in vivo for understanding and trea
ting cancer.