2013 - CTS 2013 Congress

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Oral Communications 3

8.4 - Imaging and targeting pancreatic islets with Aptamers

Presenter: Giacomo, Lanzoni, Miami, United States
Authors: Giacomo Lanzoni1,2,3, Adriana De La Fuente1,2,3, Alessia Zoso1,2,3, Silvio Bicciato1,2,3, Cristian Taccioli1,2,3, Luca Inverardi1,2,3, Paolo Serafini1,2,3

Imaging and targeting pancreatic islets with Aptamers

Giacomo Lanzoni1,2,3, Adriana De La Fuente1,2,3, Alessia Zoso1,2,3, Silvio Bicciato1,2,3, Cristian Taccioli1,2,3, Luca Inverardi1,2,3, Paolo Serafini1,2,3

1Diabetes Research Institute, University of Miami, Miami, FL, United States; 2Department of Microbiology and Immunology, Sylvester Cancer Center, University of Miami, Miami, FL, United States; 3Department of Biomedical Sciences, University of Modena, Modena, Italy

The development of technologies for pancreatic islet imaging has become a field of intense study to respond to the high demands in diabetes research and clinical practice. In Type 1 Diabetes (T1D) a selective destruction of islet β-cells precedes clinical manifestation and ultimately leads to the disease. The β-cells mass may change in relation to therapeutic treatments and during the course of the disease. Currently, β-cells mass can be measured only indirectly with functional tests, andimaging of native or transplanted islets remains challenging.Antibodies, peptides and other forms of nanoparticles have been explored as imaging reagents, but they present several limitations: non-penetrating structures, non specific binding, toxicity, immunogenicity, production costs. Aptamers are an emerging class of molecules with unique characteristics that make them extremely appealing for islets imaging. Aptamers are oligonucleotide chains with a high affinity to their target, they are currently tested in clinical trial and commercialized. They can be developed with an unsupervised process called Cell-SELEX, that allows the parallel selection of hundreds of aptamers whose specificity for a given cell type (i.e. islet cells) is not based on a particular receptor but, rather, on all the surface markers that make the target unique. They are obtained by selection from a large random sequence pool, from which they are “evolved” to have a high binding affinity to their target. Given the small size and the ease of production in large scale, they represent extraordinary tools for imaging, proving superior to antibodies, and may prove useful for in vivo imaging and targeting strategies. We developed islet-specific aptamers with modified nuclease-resistant RNA structures useful for in vitro andin vivo applications. The selection approach was based on isolated living murine islets.A library of 1020 random aptamers was first depleted of non-specific aptamers using pancreatic exocrine tissue and positive selection was performed by incubation with islets. After removal of non-specific aptamers, islets-bound aptamers were isolated and amplified. The library obtained was used for the following cycle of selection, 12 cycles were performed. Aptamer libraries from the different cycles have been sequenced by the illumina high throughput sequencing and studied with bioinformatics tools for identification. We tested the resistance of aptamers to RNAse digestion and degradation. We demonstrated that our islet-specific aptamers are able to bind avidly to islet cells with a fluorescent staining approach in tissue sections.The aptamer staining colocalized in insulin-producing beta cells. We observed no specific binding to irrelevant tissues. We plan to scale up the production of the selected aptamers, combine the aptamers with contrast moieties and test them for in vivo imaging of islet cells. Additionally, these new class of reagents will be used for the delivery of therapeutic short hairpin silencing RNAs (shRNAs) with the purpose to limit cytokines induced cell death and promote the proliferation of the residual β cell mass at onset in T1D mice models.

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