Genomes and Genes
SiRNA therapeutics: Gap junction delivery in vitro and in vivo
Principal Investigator: Richard T Mathias
Abstract: DESCRIPTION (provided by applicant): The cellular delivery of siRNA via gap junctions represents a unique and potentially clinically important delivery system. Our previous studies have shown that gap junctions composed of connexin43 (Cx43) are permeable to siRNAs and permeating siRNAs can subsequently reduce the mRNA levels of a specific gene. The aim of the studies proposed here is to characterize the transfer and permeation of siRNA from hMSCs and other communication competent cells into a target tissue. Previously we determined that gap junctions composed of Cx43 transfer siRNA, whereas those composed of Cx32 or Cx26 will not. Hence channel permeability for siRNA depends on the connexin. In aim 1 we will determine the permeability of gap junctions made of Cx40, Cx37 and Cx43, to morpholinos and siRNAs. Cx43, Cx40 and Cx37 are chosen because they are ubiquitously expressed in vivo in many organs. In aim 2 we will determine the synthesis and degradation rates of siRNA targeting HCN2 and GFP. We will also investigate the efficacy and time course of functional silencing of the membrane protein, the pacemaker channel HCN2. We will test the hypothesis that cellular delivery of siRNA via gap junction channels can silence HCN2 channel function in target cells by characterizing the functional silencing of HCN2 via gap junction mediated delivery of siRNA from hMSCs or other communication competent cells to a target cell expressing HCN2. We follow HCN2 mRNA concentration using RT-PCR to allow an estimate of the relative content over time in the presence of siRNA. We will also determine the concentrations of tagged morpholinos/siRNAs to establish the effective concentration necessary to silence a gene (HCN2 or GFP) and also provide parameters for our 2D/3D model to determine penetration within a tissue. In aim 3 we will experimentally assess how far siRNA can penetrate multiple cell layers of a syncytium. In aim 4 we will derive a model for transfer of siRNA along a simple linear chain of cells or geometries in 2 or 3 dimensions. It will be used to predict the number and position of siRNA containing cells (hMSCs) required to silence function in a tissue or an organ/tumor. In aim 5 we will assess siRNA effectiveness in silencing GFP in vivo. We use nude mice and inject a bolus of 10 million cells expressing GFP into the dermis or intramuscularly followed at various times with an injection of hMSCs loaded with siRNA targeting GFP. We will track the GFP fluorescence image over time using whole animal imaging. PUBLIC HEALTH RELEVANCE: Small interfering RNA (siRNA) targets a single protein reducing its expression. As such it has great potential as a highly selective drug. However systems for its in vivo delivery are not optimal. The present application investigates the ability of the immuno-privileged adult mesenchymal stem cell (MSC) as well as other cell types to deliver small interfering RNA (siRNA) to a target cell or tissue. The basis of this cell based delivery is the gap junction channel. These channels connect the intracellular compartments of coupled cells and allow transfer of small molecules without entry into the extracellular space. We have already established that cells that make connexins (the building block of gap junctions) can transfer siRNAs. This application asks whether cells can serve as a delivery system for siRNA. By a combination of experiment and mathematical modeling we seek to determine the ability of cellular delivery of siRNA to penetrate tissues in vitro and in vivo.
Funding Period: 2010-09-01 - 2014-08-31
more information: NIH RePORT
- Gap junction permeability: selectivity for anionic and cationic probesG Kanaporis
Dept of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, NY 11794 8661, USA
Am J Physiol Cell Physiol 300:C600-9. 2011..In conclusion, these results confirm that channels formed from individual connexins can discriminate for solutes based on size and charge, suggesting that channel selectivity may be a key factor in cell signaling...
- Lens intracellular hydrostatic pressure is generated by the circulation of sodium and modulated by gap junction couplingJunyuan Gao
Department of Physiology and Biophysics, SUNY at Stony Brook, NY 11794, USA
J Gen Physiol 137:507-20. 2011....
- The effect of size and species on lens intracellular hydrostatic pressureJunyuan Gao
Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, New York 11794 8661, USA
Invest Ophthalmol Vis Sci 54:183-92. 2013..Model calculations predicted that in larger lenses, all else equal, pressure should increase as the lens radius squared. To test this prediction, lenses of different radii from different species were studied...
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Department of Physiology and Biophysics and Institute for Molecular Cardiology, Health Sciences Center, Stony Brook University, Stony Brook, NY 11794 8661, USA
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Osteoarticular and Aging Research Group, Rheumatology Division, INIBIC Hospital Universitario A Coruña, A Coruna, Spain
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Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794, USA
Am J Physiol Cell Physiol 304:C1150-8. 2013..These results show that these two mutations exhibit a shared gain of functional activity and support the hypothesis that increased hemichannel activity is a common feature of human Cx26 mutations responsible for KID syndrome...
- Biochemical evidence for gap junctions and Cx43 expression in immortalized human chondrocyte cell line: a potential model in the study of cell communication in human chondrocytesR Gago-Fuentes
Cartilage Biology Research Group, Rheumatology Division, INIBIC Hospital Universitario A Coruña, Xubias de Arriba 84, 15006 A Coruna, Spain
Osteoarthritis Cartilage 22:586-90. 2014..This study was performed to investigate if T/C-28a2 cells contain Cx43 protein and form functional channels...
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