2007 — 2008 |
Andreev, Oleg A |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
New Technology For Selective Delivery of Pnas in Cancer Cells in Vitro and in Viv @ University of Rhode Island
[unreadable] DESCRIPTION (provided by applicant): Many cancers arise from the gradual accumulation of genetic changes in cells. Gene therapy approaches as well as techniques for recognizing cancer cells with abnormal genes or elevated levels of certain mRNAs include the design and delivery into cancerous cells of antisense and antigene oligonucleotides or their synthetic mimics such as peptide nucleic acids (PNAs). PNAs are highly stable, resistant to nucleases and proteases, and bind RNA and DNA targets in a sequence-specific manner with high affinity. One of the main obstacles for gene therapy is a lack of technology for selective delivery of gene agents into cancer cells in vivo. Here we propose a new technology for selective delivery into cancer cells of PNAs targeting mRNAs involved in tumor growth and metastasis. It is well established that tumors develop a hypoxic and acidic extracellular environment, especially in the earlier stages. We designed a short peptide that is soluble in water and able to insert into the membrane as a transmembrane alpha-helix at low pH (<6.5) but not at normal pH (7.4). The peptide acts as a nanosyringe: it inserts in the membrane at low pH, translocates and releases in the cytoplasm various molecules, including dyes, toxins, and PNAs (Reshetnyak et al., PNAS, 2006, 103, 6460). The fluorescent PNAs are translocated into cells and stain the cytoplasm and nuclei. The mechanism of translocation of pH Low Insertion Peptides (pHLIPs) is based on a protonation of two Asp residues in the transmembrane domain, and this mechanism is fundamentally different from all reported peptide delivery agents. Whole-body imaging revealed that fluorescent pHLIPs accumulate in tumors in mice. The accumulation in tumors occurs because pHLIPs insert in the membrane at low pH while they interact only weakly with the surfaces of cells in tissues at normal pH. The replacement of two Asp residues by Lys or Asn residues eliminates the ability of pHLIPs to accumulate in tumors, which confirms the proposed mechanism of insertion of pHLIPs into cells. Our goal is to develop this nanosyringe technology for selective intracellular delivery of antisense and antigene PNAs into cancer cells in vitro and in vivo. We plan to conjugate various PNAs via disulfide linkages to the end of the peptide that inserts inside a cell. The efficiency of translocation of PNAs mediated by pHLIPs will be tested on different cell lines in vitro and in vivo in mice using fluorescence microscopy, flow cytometry, spectroscopy, whole-body imaging, and by measuring of level of expression of target proteins and rates of cell proliferation and tumor growth. The pHLIP nanosyringe could be a very effective tool for molecular analysis of cancer cells and diagnosis and treatment of cancer. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]
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2008 — 2012 |
Andreev, Oleg A Engelman, Donald M. (co-PI) [⬀] Reshetnyak, Yana K [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Phlip Nanotechnology Platform For Cancer Imaging and Therapy @ University of Rhode Island
DESCRIPTION (provided by applicant): Our project is based on the use of a water-soluble membrane peptide, pHLIP, which we have shown, by whole-body fluorescence and PET imaging, to selectively target acidic solid tumors in vivo and to translocate polar cargo molecules into the cytoplasms of cultured cancer cells. pHLIP inserts unidirectionally across the lipid bilayer of a cell membrane as a monomer under mildly acidic conditions, as are found in tumors and forms a transmembrane alpha helix, whereas there is practically no insertion across the membranes of cells with the normal extracellular pH of healthy tissue. To date, no toxic effects of pHLIP exposure have been observed either for cells in culture or for mice. Here we propose to develop a nanotechnology platform for selective delivery of imaging and therapeutic agents to tumors based on the use of the pHLIP-bionanosyringe. By attaching cargo molecules to the end of pHLIP that stays outside of the membrane, we can anchor imaging or therapeutic probes to the surfaces of cancer cells, facilitating diagnosis, treatment and therapeutic monitoring. By attaching cargo to its inserted end via cleavable links, pHLIP can be used for the selective translocation of polar, cell-impermeable molecules into cancer cells. By combining the efforts of three laboratories, a broad development of this promising technology will be possible. We will use pHLIP targeting to test cancer models and establish how tumor growth and development correlate with tumor acidity. To improve pHLIP technology, we will design, synthesize and test various dendrimeric-pHLIP constructs to enable delivery of multiple therapeutic and/or imaging probes to tumors. We will introduce a synthetic scheme of simultaneous conjugation of cargo molecules and fluorescent dyes to the C-terminus of pHLIP via a cleavable S-S bond and establish the properties (polarity, shape, charge and size) of cargo molecules that pHLIP can translocate through the lipid bilayer of a membrane, defining a new, polar class of therapeutic molecules that can be delivered for tumor treatment. We will test pHLIP for the intracellular delivery of two functional cell-impermeable molecules in vivo: a toxin (phalloidin) and a gene regulation agent (Peptide Nucleic Acid). Importantly, we will attempt the simultaneous detection and treatment of tumors by labeled pHLIP-phalloidin, which is our first lead for a potential antimetastatic drug. Further, we will develop a two-step delivery scheme for the specific tethering and assembly of nanoparticles at the surfaces of cancer cells in vivo: 1) targeting tumors using pHLIP with a binding domain, which will be tethered to the surface of cancer cells and 2) targeting the pHLIP with liposomes containing therapeutic and/or imaging payloads and having a surface-exposed complementary binding domain. Inspired by the properties of pHLIP in its current version, we will further evaluate the effect of pHLIP sequence variation on peptide insertion into a membrane, enabling the design of a second generation of the nanosyringe with a range of useful properties. pHLIP nanotechnology offers a new approach for the disease-specific imaging and treatment of cancers. Our ultimate goal is to improve the diagnosis and treatment of cancer, which is responsible for about 25% of all deaths in the USA and other developed countries. There are several aspects of the problem where our technology development could be useful, but the major concept is the selective delivery of therapeutic and imaging agents to cells in tumors. Another aspect of the technology is that it permits the use of a new class of therapeutic agents: cell-impermeable molecules that would be translocated into cells only in diseased tissue while not affecting healthy cells. A therapy based on these concepts would exhibit much higher efficacy and/or significantly reduced side effects. Such improvements are especially important for cancer treatment, since the majority of anti-cancer drugs are poisons that damage normal cells.
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2011 — 2021 |
Andreev, Oleg A Engelman, Donald M. [⬀] Reshetnyak, Yana K (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanism and Uses of Transmembrane Helix Insertion by Soluble Peptides
PROJECT SUMMARY/ABSTRACT The understanding we now have of the pHLIPs (pH-Low Insertion Peptides) enables the design of novel, pH- sensitive targeting agents that use the acidity of tumor cell surfaces as a biomarker. The work supported by this grant has already yielded a new diagnostic nuclear imaging agent (PET-pHLIP) and a new fluorescent imaging agent for image-guided surgical interventions (ICG-pHLIP), which are advancing to clinical trials at Memorial Sloan Kettering Cancer Center in 2019. The primary focus of this continuation is to enable targeted intracellular delivery of polar and moderately hydrophobic therapeutic molecules. We propose a systematic approach using representative cargoes from 3 classes of therapeutic molecules that possess different physical, chemical and functional properties: i) a moderately hydrophobic, sparingly cell-permeable, small drug molecule: mertansine;? ii) a moderately polar, cell-impermeable, cyclic, rigid, larger drug: amanitin;? and iii) a large, polar, cell-impermeable drug: calicheamicin. Each of these drugs is currently under development or in use as an antibody-drug conjugate warhead, but there are important limitations to the antibody approaches, including limited biomarker availability, resistance selection, a narrow therapeutic window and limited delivery, often with < 1% of a construct reaching the tumor. Our approach is based on targeting tumor acidity, especially cancer cell surface pH that is a general parameter within and among different tumors, and independent of tumor perfusion. We propose to explore variation of pHLIP sequences, introduce pHLIP-cycles and exploit pHLIP-bundles for delivery of these therapeutic cargoes. The pHLIP-cargo constructs will be tested for insertion stability and kinetics, using biophysical methods and computational modelling. Activity will be evaluated in cells, and promising constructs will be assessed in vivo. We will accumulate a parameter database of pHLIP-cargo properties and employ modern bioinformatics algorithms to analyze the entire data set to reveal major design principles. By studies of these therapeutic agents with pHLIPs, we hope to find principles for targeted delivery of a range of other compounds. Should we be successful, the limitations of antibody drug conjugates for therapy will be overcome, and greater understanding of the membrane barrier will also be in hand. Our expectation is to have a candidate for the treatment of bladder cancer as the practical outcome of this grant renewal.
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2012 — 2013 |
Andreev, Oleg A Reshetnyak, Yana K [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Mapping Ph At the Surface of Individual Cell @ University of Rhode Island
DESCRIPTION (provided by applicant): The acidity is associated with development of various pathological states such as solid tumors, ischemic stroke, neurotrauma, epileptic seizure, inflammation, infection, wounds, cystic fibrosis and others. Normal cell could be distinguished from highly glycolytic cell (for example, metastatic cancer cell) by transmembrane pH gradient and value of pH at surface of plasma membrane. We propose to develop novel tool to map pH at the extracellular and intracellular surfaces of individual cell in highly heterogeneous environment of cells in vivo. The tool would allow opening an opportunity to contribute in understanding of diseases progression and development of approaches of pH-based image-guided intervention. We will employ optical spectroscopic and imaging approaches, which allow achieving cellular resolution. Our strategy is based on use of peptides of pHLIP (pH Low Insertion Peptide) family. pHLIPs are water-soluble membrane peptides, which insert and fold in lipid bilayer of membrane only at slightly acidic conditions. Since the equilibrium is strongly shifted toward membrane inserted form at low pH, pHLIP injected into blood, circulates in body and accumulates in acidic tissue of tumors, site of inflammatory arthritis and ischemic regions. At 24 h after i.p. or i.v. administration of pHLIP, it is washed out completely from the blood and stays in plasma membrane of cells with low extracellular pH. pHLIP labeled with optical, PET or SPECT probes is considered to be first acidity markers, which are currently under development for clinical uses. We plan to conjugate pHLIP peptides of different pKa of insertion into membrane ranging from 4.5 to 6.5 with pH-sensitive fluorophore, SNARF-1. The main goal of using pHLIPs is to deliver and tether optical probe to the outer or inner leaflet of bilayer of plasma membrane. The SNARF-1 was selected, since it demonstrates shift of the emission spectra in response to pH, which solves the problem of calibration for the probe concentration. The probe will be attached to the N- or C- terminus of pHLIPs. In first case, SNARF-1 will stay in the extracellular space being tethered to the cell surface. On the other hand, when SNARF-1 would be conjugated with the peptide inserting end (C-terminus), pHLIP would flip SNARF-1 across the bilayer and expose it to the intracellular space, while keeping it close to the inner leaflet of membrane. Thus, we propose to measure pH from the outer and inner leaflets of plasma membrane and identify transmembrane pH gradient. Experiments in solution, 2D and 3D cell culture, as well as on mouse cancer models will be performed. Our goals are: - to map pH at the surface of cancer cells in a process of cell division and migration in 3D culture; - to map pH on the surface of individual cells in tumors implanted into mice; - to monitor kinetics of pH changes at the surface of cancer cells in real time induced by the glucose infusion; - to establish the minimal size of metastatic and non-metastatic tumors, which can acidify microenvironment below pH 7.0. PUBLIC HEALTH RELEVANCE: The acidity is associated with development of various pathological states such as solid tumors, ischemia, stroke, inflammation, infection, wounds, cystic fibrosis and others. We propose to develop a novel tool to map pH at the extracellular and intracellular surfaces of individual cell.
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