Affinity reagents for the sub-fractionation of specific exosomal populations

The goal of this Phase I SBIR project is to develop affinity reagents useful for the sub fractionation of specific exosomal populationsBecause exosomal makeup and cargo reflect the typedevelopmental stageand the disease state of the cells they are shed fromthey are attractive targets for non invasive diagnosticsExosomes are also being investigated for therapeutic applications such as vaccines and drug deliveryBecause exosomes from bodily fluids generally contain exosomes from many different cellstools are needed to isolate specific populationsCurrent exosome purification techniques like differential centrifugation and filtration techniques are effective for isolating exosomes from cell culture and biological fluidsbut do not discriminate between exosomes from different sourcesAffinity purification of exosomes based on unique surface markers is capable of separating subpopulationsbut is expensivecumbersomeand hard to scaleExosomes isolated by affinity purification using traditional antibodies are usually difficult to elutea property making sub fractionation of the primary elution infeasibleAffinity reagents that would allow the sequential isolation of exosomes based on different surface markers would enable unprecedented sorting of exosomal populationsnanoCLAMPs are a newly described class of single domain affinity reagents with high affinityhigh specificity and the rare property of polyol responsivenessnanoCLAMPs have the ability to release functionally and structurally intact targets upon exposure to buffer containing polyol and saltPanels of nanoCLAMPs to new protein targets are easily generated from our validated synthetic phage display library in a few weeksa process that eliminates the need for animal immunization and provides multiple binders per targetWe hypothesize that nanoCLAMP affinity resins to exosomal surface proteins will allow the sequential sub fractionation of exosomes based on surface proteins specific to those populationsWe propose to develop a panel of nanoCLAMP affinity resins to the extracellular domains of several common exosomal surface proteinsdemonstrate their utility in binding to and purifying exosomescompare their performance to current techniquescharacterize the eluted populations of exosomes for surface proteinsyieldand morphologyand finally utilize the nanoCLAMPs to purify and sub fractionate exosomes from bodily fluidsThe successful completion of this project will result in a platform for the facile production of novel tools for the isolation of exosomes based on the ever increasing identification of unique surface markers related to a wide field of cellular origins and conditions Project Narrative The development of novel tools for the specific isolation and sub fractionation of exosomal populations will enable investigators to precisely isolate and analyze exosomes from complex mixtures in bodily fluidsThese tools will support the development of innovative assays and diagnostic tests providing information about the specific cell typedevelopmental stagedisease stateand drug response of the cells under investigation