SiRNA therapeutics: Gap junction delivery in vitro and in vivo

Summary

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

Top Publications

  1. pmc Gap junction permeability: selectivity for anionic and cationic probes
    G 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
  2. pmc Lens intracellular hydrostatic pressure is generated by the circulation of sodium and modulated by gap junction coupling
    Junyuan Gao
    Department of Physiology and Biophysics, SUNY at Stony Brook, NY 11794, USA
    J Gen Physiol 137:507-20. 2011
  3. pmc The effect of size and species on lens intracellular hydrostatic pressure
    Junyuan 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
  4. pmc MATLAB implementation of a dynamic clamp with bandwidth of >125 kHz capable of generating I Na at 37 °C
    Chris Clausen
    Department of Physiology and Biophysics and Institute for Molecular Cardiology, Health Sciences Center, Stony Brook University, Stony Brook, NY 11794 8661, USA
    Pflugers Arch 465:497-507. 2013
  5. pmc Human articular chondrocytes express multiple gap junction proteins: differential expression of connexins in normal and osteoarthritic cartilage
    Maria D Mayan
    Osteoarticular and Aging Research Group, Rheumatology Division, INIBIC Hospital Universitario A Coruña, A Coruna, Spain
    Am J Pathol 182:1337-46. 2013
  6. pmc The human Cx26-D50A and Cx26-A88V mutations causing keratitis-ichthyosis-deafness syndrome display increased hemichannel activity
    Pallavi V Mhaske
    Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794, USA
    Am J Physiol Cell Physiol 304:C1150-8. 2013
  7. ncbi 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 chondrocytes
    R 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

Research Grants

  1. CENTER FOR GASTROINTESTINAL BIOLOGY AND DISEASE
    Robert S Sandler; Fiscal Year: 2013
  2. Mechanisms and Markers of Prostate Cancer Metastases
    ROBERT LOUIS VESSELLA; Fiscal Year: 2013
  3. The Virtual Physiological Rat Project
    Daniel A Beard; Fiscal Year: 2013
  4. CENTER FOR BIOMEDICAL RESEARCH
    Timothy Turner; Fiscal Year: 2013
  5. Connexin Distribution in Physiological Versus Pathological Cardiac Hypertrophy
    Michael R Zile; Fiscal Year: 2013
  6. TSH RECEPTOR MULTIMERIZATION
    TERRY FRANCIS DAVIES; Fiscal Year: 2013
  7. From human keratinocytes to biological pacemakers
    Ira S Cohen; Fiscal Year: 2013
  8. New Approaches To Cardiothoracic Tolerance Induction
    Joren C Madsen; Fiscal Year: 2013
  9. Mitochondrial Dysfunction in Neurodegeneration of Aging
    Gary E Gibson; Fiscal Year: 2013
  10. UNMC EPPLEY CANCER CENTER SUPPORT GRANT
    Kenneth H Cowan; Fiscal Year: 2013

Detail Information

Publications7

  1. pmc Gap junction permeability: selectivity for anionic and cationic probes
    G 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...
  2. pmc Lens intracellular hydrostatic pressure is generated by the circulation of sodium and modulated by gap junction coupling
    Junyuan Gao
    Department of Physiology and Biophysics, SUNY at Stony Brook, NY 11794, USA
    J Gen Physiol 137:507-20. 2011
    ....
  3. pmc The effect of size and species on lens intracellular hydrostatic pressure
    Junyuan 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...
  4. pmc MATLAB implementation of a dynamic clamp with bandwidth of >125 kHz capable of generating I Na at 37 °C
    Chris Clausen
    Department of Physiology and Biophysics and Institute for Molecular Cardiology, Health Sciences Center, Stony Brook University, Stony Brook, NY 11794 8661, USA
    Pflugers Arch 465:497-507. 2013
    ....
  5. pmc Human articular chondrocytes express multiple gap junction proteins: differential expression of connexins in normal and osteoarthritic cartilage
    Maria D Mayan
    Osteoarticular and Aging Research Group, Rheumatology Division, INIBIC Hospital Universitario A Coruña, A Coruna, Spain
    Am J Pathol 182:1337-46. 2013
    ....
  6. pmc The human Cx26-D50A and Cx26-A88V mutations causing keratitis-ichthyosis-deafness syndrome display increased hemichannel activity
    Pallavi V Mhaske
    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...
  7. ncbi 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 chondrocytes
    R 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...

Research Grants30

  1. CENTER FOR GASTROINTESTINAL BIOLOGY AND DISEASE
    Robert S Sandler; Fiscal Year: 2013
    ..Through all of its activities, the Center improves communication, promotes collaboration, develops careers and generally enriches the intellectual climate for digestive disease research. ..
  2. Mechanisms and Markers of Prostate Cancer Metastases
    ROBERT LOUIS VESSELLA; Fiscal Year: 2013
    ..It also conducts pre-clinical xenograft studies and provides statistical support to the overall P01 program. Core B is the Administrative Core which provides overall administrative support to the investigators. ..
  3. The Virtual Physiological Rat Project
    Daniel A Beard; Fiscal Year: 2013
    ..This proposal targets the grand challenge of understanding complex multi-faceted disease phenotypes through experiments and simulations that capture the complex genotype-environment-phenotype relationship. ..
  4. CENTER FOR BIOMEDICAL RESEARCH
    Timothy Turner; Fiscal Year: 2013
    ..abstract_text> ..
  5. Connexin Distribution in Physiological Versus Pathological Cardiac Hypertrophy
    Michael R Zile; Fiscal Year: 2013
    ..pathological hypertrophy, with ex- tensively characterized cytoskeletal properties in each setting. ..
  6. TSH RECEPTOR MULTIMERIZATION
    TERRY FRANCIS DAVIES; Fiscal Year: 2013
    ....
  7. From human keratinocytes to biological pacemakers
    Ira S Cohen; Fiscal Year: 2013
    ..If the HFKT-pacemaker functions well in the canine heart, a future goal would be to advance this novel autologous, cellular approach towards clinical deployment. ..
  8. New Approaches To Cardiothoracic Tolerance Induction
    Joren C Madsen; Fiscal Year: 2013
    ..We anticipate ongoing progress will continue to contribute to a reduction in the morbidity and mortality associated with solid organ transplantation. ..
  9. Mitochondrial Dysfunction in Neurodegeneration of Aging
    Gary E Gibson; Fiscal Year: 2013
    ..Successful completion of the goals of these projects can be expected to provide new insights into neurodegenerative processes and contribute to novel approaches to ameliorating age-related neurodegenerations. ..
  10. UNMC EPPLEY CANCER CENTER SUPPORT GRANT
    Kenneth H Cowan; Fiscal Year: 2013
    ....
  11. Discovery and Development of Therapeutic Genes for CHF
    H Kirk Hammond; Fiscal Year: 2013
    ..Four Cores will support the Program: Digital Imaging (Dr. Farquhar);Vector Production (Dr. Miyanohara);Translational Systems (Dr. Hammond) and Clinical &Administrative (Dr. Hammond). ..
  12. Baylor Intellectual and Developmental Disabilities Research Center
    Huda Y Zoghbi; Fiscal Year: 2013
    ..abstract_text> ..
  13. Neuro-Circulatory Function in Chronic Heart Failure
    Irving H Zucker; Fiscal Year: 2013
    ..The role of exercise training in modulating ROS generation and antioxidant enzymes in animals with CHF will also be investigated in this project. ..
  14. The Therapy of CML
    Richard E Champlin; Fiscal Year: 2013
    ..This is a productive, highly integrated program for development of novel therapies for CML. ..