This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
This Small Business Innovation Research (SBIR) Phase II project will develop technologies that optimize the use of RNA interference (RNAi) in animals. RNAi is an invaluable tool for characterizing gene function and is a promising candidate for gene therapy. The use of RNAi in tissue culture is well developed but is of limited use in experimental animals. RNAi agents must enter cells to exert their effects but this has proven to be challenging in animals. The current lack of such technologies is holding back the majority of important RNAi animal experiments. To open this bottleneck, kits and reagents will be developed based on Bioo Scientific?s Targeted Transport Technology (T3). Easy-to-use RNAi delivery products will be manufactured, validated and commercialized for use in animal experiments.
The broader impacts of this research are twofold. First, researchers will gain ready access to products that greatly simplify the use of RNAi in animals, thereby, stimulating a burst of validation experiments in animals to try to replicate prior results derived from tissue culture experiments. Animals are more complex than their tissue culture counterparts and it is uncertain that results can be duplicated in an animal. Second, T3 has the potential to be used for the therapeutic delivery of RNAi agents. In sum, this project will propel the validation of tissue culture results via T3 enabled animal experimentation, leading to a better understanding of cellular pathways, the identification of novel drug targets, and the potential to deliver RNAi agents as drugs.
Finding from Phase II NSF 0923854 (06/1/2011-12/31/2011) We have analyzed biodistribution of small RNA following administration with a carrier conjugate, to evaluate the ability of the HER2 carrier conjugate delivered miRNA-34 to inhibit tumor growth during an extended treatment schedule enabling Mirna Therapeutics to make informed decisions regarding this drug delivery technology. During this period we manufactured over 20mg of material for these experiments. The first experiments investigating loss of antibody during cleanup were done using different brands of concentrator columns, and different sizes molecular weight cutoff (MWC) filters, to see if the loss of antibody was due to the type or size of filter. In doing this we discovered that antibody loss was happening because the conjugated antibodies were essentially ‘cloaking’ themselves from the spectrophotometer because they attached to several protamine molecules. Pratamine inhibits proper spectrophotometer readings due to lack of readable amino acids. Before this work was initiated, we had no reason to quantify the conjugates before placing them into the concentrator column apparatus. If done, we would have seen that the concentration was already zero due to the cloaking effect. Also, samples with zero spectrophotometer readings (based on an optical density reading at 280nm) weren’t run on polyacrylamide gels containing SDS as we thought the antibody was lost before we understood of the cloaking effect. As more experiments were performed it was determined that the antibody was sticking to the filter because it was not recoverable from either flow-through or retentate but was present as a protamine conjugate (which reads zero using the spectrophotometer) before it was ever placed onto a concentrator column. This was confusing because all readings before column use, during column use, and after column use were zero. It was only by gel analysis that we could determine when the antibody was present and when it had disappeared. While with some antibody we found that the more clean up spins that were performed the more it was sticking to the filter and the less you that was observed on the gel. While with other batches of antibody the antibody the sticking was entirely taken place in the first spin. While still with other antibody, we were able to do 6 clean up spins and recover 90% or more of antibody that reads beautifully by spectrophotometry. We propose that this effect has something to do with amount of protamine binding to the antibody and the placement on the molecule. To avoid loss of the antibody but keep the cleanup step we started trying the methods listed above to clean the excess protamine from the conjugation reaction. In addition we also changed the buffer reaction solution by adding potassium salt after 16 hours. The common conjugation and modification buffers in the kit contain only sodium salts and do not contain potassium salts which inhibit conjugation. By adding KCl and KH2PO4 to the conjugates you essentially halt conjugation and prevent over conjugation. We added the potassium salts to the conjugates prior to trying the alternative clean-up methods. It was our prediction that adding these salts might help make the antibodies more easily recoverable during the clean up process. This however has not helped significantly. We have however enlisted this salt addition step (addition of injection buffer) in the kit to halt conjugation instead of cleaning out the excess protamine, which was only a problem when left in a solution that still had possible reactivity. If a batch of antibody sticks to the corning concentrator membrane previously included in the first version of the kit it will stick to dialysis membranes and all other apparatus. When using columns of any size (G-25,G-75,G-200) it has shown to be practically impossible to elute the antibody from the column. Standard PD-10 desalting columns are too short and the antibody is not able to separate from the excess protamine, so while the antibody is not lost using this process it is also not cleaned. We concluded that there is not a cost and time effective method for clearing the excess protamine away from the conjugated antibody for these ‘sticky’ conjugates so we decided to try another route and titrate down the amount of protamine used in the starting reaction. The T3 Conjugation Kit still contains the T3 carrier, cross-linking agents, a control antibody/carrier, modification buffer, conjugation buffer and desalting columns. The kit no longer contains the centriprep concentrator column. The pore size of the centriprep column was the main physical difference between the kit for larger than 75 kDa antibodies and the kit for smaller than 75 kDa antibodies. The manual and amounts used of each reagent are now the main difference between the two kits. The second version of the kit contains less siRNA carrier than the first version.
