Document text
Principal Investigator: Bruce Shapiro
Organization: DIVISION OF BASIC SCIENCES - NCI
Fiscal Year: 2020
Award: $1,074,314
Funding agency: National Cancer Institute
Recently we developed a new type of RNA nanostructure that forms a truncated tetrahedron in collaboration with Luc Jaeger, UC Santa Barbara. The structure was built from our hexameric ring where 4 sides of the tetrahedral structure each contain the hexmeric ring, but each ring contains 3 H-shaped crossover connectors to the other rings. This type of construct allows for the incorporation of up to 12 functional entities such as Dicer substrates, beacons and/or aptamers. We found that cells take up these constructs better than some of the other RNA nanoconstructs. The hypothesis that nanoparticle shape and size matter regarding functionality seems to be true. Due, at least in part, to the better uptake and we believe better processivity, we found that knockdown of targeted genes to induce cell death, using incorporated Dicer substrate PLK1 is more efficacious than some of our other particles. Several different methods were used to stunningly verify the assembly of this particle including an atomic force microscope (AFM) and Cryo-EM in collaborations with the Optical Microscopy and Image Analysis Lab and with the Electron Microscopy Lab respectively, of CCR. ----Typical methodologies that utilize RNAs as targeting agents to control diseased cells rely on pre-analysis of the state of the cells to be targeted followed by delivery of an agent e.g. antisense, antimiR or siRNA, thus separating the therapeutic step and the diagnonostic step. We developed a set of context-sensitive RNA-based logic switches based on AND and NOT functionalities that combines both steps into one logic system. This permits the the conditional activation or deactivation of the the release of single-stranded or double-stranded RNAs as a function of expressed RNAs. The switches are designed using RNA/DNA hybrids, significantly limiting issues related to nuclease degradation. ---To achieve control over deliverable functionality and stability of RNA-based nanoparticles, the properties of DNA and RNA were merged in the development of computationally designed nanoparticles that were constructed from RNA/DNA hybrids. These molecules allow higher stability in blood serum, attachment of fluorescent markers for tracking, and the ability to split the components of functional elements inactivating them, but allowing later activation under the control of complementary toeholds by which the kinetics of re-association can be tuned. Diceable substrate siRNA could be split into two components, each consisting of an RNA/DNA hybrid. Complementary RNA single-stranded toeholds rather than DNA can be used in the construction of the hybrids. The two hybrids, when transfected into cells recombine into two products due to the toeholds and the computationally determined thermodynamic difference between the hybrids and the products. From the perspective of thermodynamics, the use of RNA toeholds is advantageous as it reduces the length of the single stranded ends required to unzip the hybrids and generate the functional RNA element. In addition, less DNA is needed since the DNA portion of the hybrid is shortened. This has the added advantage of reducing innate cellular immune responses as a result of shorter double stranded DNA helical products. From a design perspective, the RNA toehold can be part of the functional DS RNA, or other potential RNA moiety, reducing the size and simplifying the design of the resulting hybrid duplexes. RNA-based hybrids containing 3 Dicer substrate siRNAs for synergistic simultaneous targeting of apoptosis-related genes in HT29 tumors have been tested in vivo after significant testing in cell cultures, in a comprehensive mouse study funded, in part, by the Invention Development Program. Results look encouraging showing retardation of tumor growth both intratumorally and more so by tail vein injection. Further studies are being performed using alternative delivery agents. ----Since we can control immune response with RNA-based nanoparticles, we have been collaborating with Joost Oppenheim, CCR, to take advantage of these properties to activate the immune system for anti-cancer treatment. Working with Joost Oppenheim's group we found significant in vivo results showing a "cure" in 3 out 10 immune competent mice. Most of the other mice showed signficant regression ot their tumors. Further experiments are planned to characterize more fully the pathways of action.---Besides using small molecules to target various RNA motifs for potential drugs as described under another project heading, we have been exploring, by the use of 3D modeling, molecular dynamics and molecular docking, the use of small molecule ligands that dock to specific RNA motifs that in turn alters the dynamics, shape and functionality of the nano constructs built from these motifs. We have shown that such affects are possible, i,e. changing of the biophysical characteristics of the targets. Further exploration of this type of nano design is being investigated.---The delivery of RNA-based nanoconstructs in cell culture and in vivo is essential for the development of therapeutic methodologies using these agents. Non-modified naked RNAs have short half-lives in blood serum due to nucleases and have difficulty crossing cell membranes due to their negative charge. Thus, we are developing lipid and polymer formulations. We have worked with Jonathan Lovell, U of Buffalo, on the development of sulfonated photoactivatable polymers for the delivery of our RNA-based nanoparticles. The concept being that the use of focused near infrared light on tumors that have been administered these photoactivatable polymers will specifically release drugs into the tumor with little of any release in normal tissues that have not be effected by the light. We have shown minimal leakage without laser treatment and significant functionality when laser treated. A second-generation chlorin-based photosensitizer, HPPH shows tremendous therapeutic potential in clinical trials in treatment of esophageal cancer. We, in collaboration with Sunil Dubey, Birla Institute of Technology & Science, have developed and validated by a bioanalytical method for estimation of HPPH (a compound used in photodynamic therapy) in rat plasma using High Performance Liquid Chromatography with PDA detector. Colon-26 mice using an HPPH LNP showed superior efficacy using PDT. ---We have also tested bolaamphiphile vesicles GLH-19 and GLH-20 formulations for delivery of siRNA to tumors and to the brain in collaboration with Eli Heldman, Ben Gurion University. We showed good delivery to both locations, including the brain which is difficult to target due to issues related to crossing the blood-brain barrier. The stability of the various formulations tested were also analyzed by molecular dynamics, which explained quite well the results we were seeing experimentally.---An exclusive license of two of our patents was established with a startup company, Sixfold that was established out of the NCI Nanochallenge. The company is using and further developing our RNA-based nanoparticles for targeting various tumor types.--- A relatively new lipid based on the use of oxime-ethers is being explored in collaboration with Michael Nantz, University of Louisville and Xiulang Lu, University of Connecticut. Preliminary results look quite promising for their use as a delivery agent in target lung cancer.--- A collaboration with Xandra Breakefield, Harvard Medical school is also being pursued to develop techniques to load exosomes with RNA for to be used as potential delivery agents. The technique utilizes highly positively charged proteins to enable this loading. Preliminary results look promising.
Terms: <2-(1-Hexyloxyethyl)-2-Devinyl Pyropheophorbide-a><3-D modeling><3D modeling><Affect><Alkanesulfonates><Alkyl Sulfonates><Alkylsulfonate Compound><Apoptosis><Apoptosis Pathway><Atomic Force Microscopes><BBB crossing><Binding Proteins><Biocompatible Materials><Biologic Development><Biomaterials><Blood Plasma><Blood Serum><Brain><Brain Nervous System><Buffaloes><CCR><Cancers><Cell Body><Cell Culture Techniques><Cell Death><Cell membrane><Cells><Charge><Clinical Trials><Collaborations><Colon><Common Rat Strains><Complementary RNA><Complex><Connecticut><Cryo-electron Microscopy><Cryoelectron Microscopy><Cytoplasmic Membrane><DNA><Deoxyribonucleic Acid><Detection><Development><Dimensions><Disease><Disorder><Docking><Double-Stranded DNA><Double-Stranded RNA><Drugs><Electric Field Microscopes><Electron Cryomicroscopy><Electron Microscopy><Elements><Encephalon><Environment><Esophageal Cancer><Esophagus Cancer><Ethers><Extravasation><Formulation><Functional RNA><Funding><Generations><Genes><HPLC><HPPH><High Performance Liquid Chromatography><High Pressure Liquid Chromatography><High Speed Liquid Chromatography><Hybrids><Hydroxyimino Compounds><Image Analyses><Image Analysis><Immune response><Immune system><Immunocompetent><Immunological response><Injections><Institutes><Kinetics><Laser Electromagnetic><Laser Radiation><Lasers><Lateral Force Microscopes><Leakage><Legal patent><Length><Licensing><Ligand Binding Protein><Ligand Binding Protein Gene><Ligands><Light><Lipids><Location><Logic><Magnetic Force Microscopes><Malignant Esophageal Neoplasm><Malignant Esophageal Tumor><Malignant Neoplasms><Malignant Tumor><Malignant Tumor of the Esophagus><Malignant Tumor of the Lung><Malignant neoplasm of esophagus><Malignant neoplasm of lung><Medication><Methodology><Methods><Mice><Mice Mammals><Molecular><Molecular Analysis><Molecular Configuration><Molecular Conformation><Molecular Dynamics Simulation><Molecular Stereochemistry><Murine><Mus><Nanostructures><Non-Coding><Non-Coding RNA><Non-Polyadenylated RNA><Non-translated RNA><Noncoding RNA><Nontranslated RNA><Normal Tissue><Normal tissue morphology><Optics><Oximes><PLK Gene><PLK1><PLK1 gene><PUVA><PUVA Photochemotherapy><Patents><Pathway interactions><Pharmaceutic Preparations><Pharmaceutical Preparations><Photochemotherapy><Photodynamic Therapy><Photoradiation><Photosensitization><Photosensitizers><Photosensitizing Agents><Plasma><Plasma Membrane><Plasma Serum><Polo-Like Kinase><Polymers><Program Development><Programmed Cell Death><Property><Protein Binding><Proteins><Pulmonary Cancer><Pulmonary malignant Neoplasm><RNA><RNA Gene Products><RNA Stability><RNA delivery><Rat><Rats Mammals><Rattus><Receptor Cell><Reticuloendothelial System, Serum, Plasma><Ribonucleic Acid><STPK13><Scanning Probe Microscopes><Scanning Thermal Microscopes><Scanning Tunneling Microscopes><Science><Serine/Threonine Protein Kinase 13><Serum><Shapes><Short interfering RNA><Side><Small Interfering RNA><Spillage><Structure><System><Tail><Techniques><Technology><Testing><Therapeutic><Thermodynamic><Thermodynamics><Two Hybrid><Universities><Untranslated RNA><Veins><Vesicle><Work><Yeast One Hybrid System><Yeast One/Two-Hybrid System><allergic/immunologic body system><allergic/immunologic organ system><anti-cancer treatment><anticancer treatment><aptamer><base><biological material><biophysical characteristics><biophysical characterization><biophysical measurement><biophysical parameters><biophysical properties><blood-brain barrier crossing><bloodbrain barrier crossing><bound protein><catalyst><cell culture><chlorin><conformation><conformational state><cryo-EM><cryoEM><design><designing><detector><developmental><disease control><disorder control><drug/agent><ds-DNA><dsDNA><dsRNA><exosome><experiment><experimental research><experimental study><genetic information><host response><image evaluation><image interpretation><immune competent><immunoresponse><in vivo><in vivo evaluation><in vivo testing><invention><knock-down><knockdown><light effects><lung cancer><malignancy><medical college><medical schools><microscope imaging><microscopic imaging><microscopy imaging><molecular dynamics><nano><nano biology><nano device><nano particle><nano-sized particle><nano-structures><nanobiology><nanodevice><nanoparticle><nanosized particle><necrocytosis><neoplasm/cancer><neoplasm/cancer photoradiation therapy><noncoding><nuclease><oesophageal cancer><optical><particle><pathway><photosensitizer><plasmalemma><polo-like kinase 1><school of medicine><siRNA><siRNA delivery><small molecule><sulfonate><synthetic biology><targeted agent><therapeutic agent development><therapeutic development><three-dimensional modeling><tumor><tumor growth><uptake><yeast two hybrid system>