2005 — 2012 |
Mahato, Ram I |
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. |
Growth Factor and Antiapoptotic Gene Delivery to Human Islets @ University of Tennessee Health Sci Ctr
DESCRIPTION (provided by applicant): Transplantation of human islets has great potential as an effective means of treating insulin dependent diabetes mellitus. Primary non-function is the main cause of islet graft failure and it results in the need for multi-donor transplants. We will test the hypothesis that islet engraftment can he enhanced simultaneously by expressing growth factor gene like human Vascular Endothelial Growth Factor (hVEGF) that promotes islet revascularization, and antiapoptotic gene like human interleukin-1 receptor antagonist (hIL-IRa) to prevent apoptosis of transplanted islets in the host. In preliminary studies, higher levels of hVEGF were secreted from human islets transfected with bicistronic adenoviral vector encoding hVEGF and Green Fluorescent Protein (Acv-GFP-hVEGF), while hVEGF secretion from Adv-GFP transfected and mock-transfected islets was very low. Insulin release from transfected islets was comparable to mock-transfected islets. Proapoptotic cytokines IFN-gamma and TNF-alpha did not induce apoptosis when islets were transfected with Adv-GFP-hVEGF. Our specific aims are to determine whether i) adenoviral vectors encoding hVEGF and hIL-IRa (AdvhVEGF- hlL-lRa) will efficiently transfect islets and improve their function; and ii) ex vivo transfection with Adv-hVEGF-hlL-IRa will prevent primary islet nonfunction and reduce the islet mass needed for restoring normoglycemia. The significance of this research is that the proposed ex vivo gene therapy will promote islet revascularization, prevent apoptosis and decrease the number of islets required for achieving normoglycemia. The data will be beneficial to successful human islet transplantation.
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0.988 |
2007 — 2010 |
Mahato, Ram I |
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. |
Targeted Delivery of Tfos For Treatment of Liver Fibrosis @ University of Tennessee Health Sci Ctr
DESCRIPTION (provided by applicant): Fibrosis leads to organ dysfunction and is characterized by an excessive production of extracellular matrix (ECM) components, namely type I and III collagens. Although activated hepatic stellate cell (HSC) remains the principal cell type responsible for liver fibrosis, other liver cell type of fibroblast lineage derived from portal and centrolobular veins also have fibrogenic potential. The overall hypothesis is that liver fibrosis can be treated by systemic administration of a1(I) collagen gene promoter specific triplex forming oligonucleotides (TFOs) conjugated with mannose 6-phosphate-bovine serum albumin (M6P-BSA) via a disulfide bond. In preliminary studies, antiparallel phosphorothioate polypurine TFOs specific for CI region formed triplexes, inhibited transcription of a1(I) collagen promoter and improved rat liver fibrosis. TFOs rapidly distributed throughout the body after systemic administration, with the highest accumulation in the liver. TFO accumulation in the liver was decreased when injected into liver fibrotic rats. Kupffer, sinusoidal endothelial and hepatic stellate cells accounted for approximately 70% of the liver uptake, and the remaining 30% in the hepatocytes. Bioconjugation with M6P-BSA significantly enhanced the cellular uptake of the TFOs by HSC-T6 cells in vitro, leading to enhanced inhibition of type a1(I) collagen transcription. TFO delivery to the liver and to the HSCs was significantly increased when M6P-BSA-TFO was injected intravenously into fibrotic rats. Our specific aims are to determine whether i) conjugation of targeting ligands to TFOs affect triplex formation;ii) TFOs inhibit fibrosis by inhibiting transcription of a1(I) collagen and/or blocking inflammation and activation of liver fibrogenic cells;and iii) M6P-BSA-TFO can be delivered efficiently to liver fibrogenic cells and prevent fibrosis. The significance of this research is that the proposed targeted TFO delivery to liver fibrogenic cells will inhibit disproportionate accumulation of a1(I) collagen, which is essential for the treatment of liver fibrosis. The data will also be beneficial to successful treatment of other organ fibrosis.
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0.988 |
2011 — 2017 |
Marky, Luis [⬀] Mahato, Ram |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Sequence Specific Targeting of Nucleic Acids Using Intramolecular Complexes: Energetics, Kinetics and Hydration @ University of Nebraska Medical Center
Intellectual Merit This research focuses on the thermodynamics of nucleic acid intramolecular structures, especially, DNA structures that model the secondary structures of RNA molecules. The broad and long term objectives of this project are to understand the molecular forces controlling the overall stability of complex intramolecular DNA structures; to quantify the energetics, kinetics, and hydration contributions governing the association of these unusual intramolecular structures with their complementary strands, including the role of cations; and to determine the thermodynamics for their favorable interaction with polycations for cellular delivery purposes. The hypothesis is: The presence of unpaired base nucleotides in the loops of nucleic acid secondary structures provides favorable free energy contributions in their reaction with complementary strands and the slightly more hydrophobic surface of these constrained loops contribute favorably towards the interaction with delivery vectors, such as polycations. To test this hypothesis the following aims are proposed: Aim 1: To characterize the melting behavior of stem-loop motifs containing bulges or internal loops, pseudoknots and three-way junctions as a function of sequence, stability of their end loops, and solution conditions. Aim 2: To elucidate and quantify the molecular forces governing the reaction of intramolecular secondary DNA structures with their partially complementary strands. Aim 3: To determine the kinetics of the bimolecular association reactions of aim 2, includes reaction rates and associated activation energies and activation entropies, and to correlate them with their thermodynamics. Aim 4: To elucidate the molecular forces influencing the stability and structure of DNA-polycation complexes, and to quantify the thermodynamics governing their formation; including the role of polycation composition, DNA secondary structure, and solution conditions. The complete thermodynamic characterization of these DNA complexes and their association reactions will provide a fundamental understanding of the physical factors that determine their stability as a function of its sequence and solution conditions. These factors are basic to the rational design of gene-targeting reagents, and for their proper cellular delivery, that can be used in therapeutic, diagnostic and biotechnological applications. Another impact is the global role of water in the physical and chemical properties of biological macromolecules, and their interaction behavior towards one another. The correlation of energetics with hydration should improve our picture of how hydration controls the stability, conformation and melting behavior of unusual nucleic acid structures. In addition, the resulting hydration data can be used in molecular modeling studies and in theoretical calculations, providing an insight into global water that is not available by NMR or X-ray crystallography techniques.
Broader Impacts The educational significance of this project involves the mentoring of students at all levels underrepresented in the sciences by training them in a wide variety of biophysical techniques. This training will improve their understanding of the molecular forces, kinetics and hydration effects controlling the structure and conformation of macromolecules, and their interaction with other molecules. Furthermore, the research findings generated by this group are routinely incorporated into lectures in Biophysical Chemistry, Quantitative Pharmaceutical Analysis and Biochemistry.
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0.915 |
2014 — 2017 |
Mahato, Ram I. |
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. |
Polymeric Nanomedicines of Small Molecules and Mirna For Treating Pancreatic Canc @ University of Nebraska Medical Center
DESCRIPTION (provided by applicant): Successful treatment of pancreatic cancer remains a challenge due to the presence of pronounced desmoplastic tumor microenvironment and emergence of chemoresistance resulting in recurrence and metastatic spread. Gemcitabine shows only limited efficacy due to its inefficient delivery to tumor because of its hydrophilicity and rapid metabolism and also due to the activity of cancer stem cells (CSCs), which are regulated by miRNAs. In our preliminary studies, gemcitabine was conjugated to poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate) (PEG- PCC) and formulated into micelles which significantly inhibited tumor growth compared to free drug when injected intravenously into pancreatic tumor bearing NSG mice. To achieve complete tumor regression, we identified miR-205 among a series of dysregulated miRNAs from the CSCs isolated from gemcitabine resistant MIA PaCa-2R cells and human pancreatic cancer tissues, to be significantly downregulated and playing a predominant role in regulating cell growth, epithelial to mesenchymal transition (EMT) and resistance. Transfection of MIA PaCa-2R cells with miR-205 mimic resulted in the restoration of chemosensitivity to gemcitabine. Then, we synthesized gemcitabine conjugated poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft- dodecanol-graft-tetraethylenepentamine) copolymer having cationic chains for polyplex formation with miR-205 mimic which showed improved stability in fetal bovine serum and efficiently transfected and reversed chemoresistance, invasion and metastasis in gemcitabine resistant cells. Therefore, we hypothesize that co-formulation of miR-205 mimic with gemcitabine may effectively treat pancreatic cancer by reversing the chemo-resistance of CSCs and simultaneously target bulk tumor cells as well. Major focus of the project is to: a) discover suitable miRNAs to target chemo-resistance and EMT (like miR205) in pancreatic cancer and b) co-deliver this miRNA with gemcitabine to the tumor using a actively targeted nanocarrier which can protect both these molecules from plasma degradation and ensure enhanced uptake. Our specific aims are to i) identify the aberrantly expressed miRNAs and validate their role in chemoresistance, invasion and metastasis; ii) co- formulate miRNA and gemcitabine in polymeric micelles and assess their synergistic effect on the inhibition of pancreatic cancer and, iii) assess the synergistic action of micelles carrying gemcitabine and miRNA on the regression of human pancreatic tumor bearing mice. Significance of the project is to develop nanomedicines of miRNA mimic and gemcitabine that effectively increase local drug concentrations within the fibrotic stroma of these tumors and bypass the chemo-resistant mechanisms that allow tumor growth and inhibit the efficacy of current standard chemotherapies.
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1 |
2015 — 2018 |
Batra, Surinder K. (co-PI) [⬀] Mahato, Ram I. |
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. |
Polymeric Nanomedicines of Hedgehog Inhibitor and Mirna For Treating Pancreatic Cancer @ University of Nebraska Medical Center
? DESCRIPTION: Successful treatment of pancreatic adenocarcinoma (PDAC) remains a challenge due to the desmoplastic microenvironment that promotes both tumor growth and metastasis and forms a barrier to chemotherapy. Hedgehog (Hh) signaling plays a crucial role in PDAC progression and contributes to desmoplasia. Hh inhibitor GDC-0449 can overcome desmoplastic reaction. While Hh levels are increased in pancreatic cancer stem cells (CSCs), tumor suppressor miR-let7b targets several genes involved in PDAC pathogenesis. Therefore, inhibition of Hh pathway and restoration of miR-let7b could effectively treat PDAC. In our preliminary studies, miR-let7b was downregulated in pancreatic cancer cell lines, human patient's cancer tissues and during progression of spontaneous pancreatic cancer in genetically engineered KPC mice. Our in-silico analysis and in vitro experiments indicate that miR-let7b targets several genes like MUC4, NCOA3, Kras, HMGA2 and TGFßR1, which are upregulated in PDAC. miR-let7b and GDC-0449 could inhibit the proliferation of human pancreatic cancer cells (Capan-1, HPAFII and T3M4) and there was synergistic effect when miR-let7b and GDC- 0449 were co-formulated into micelles using poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl- propylene carbonate-graft-dodecanol-graft-tetraethylenepentamine) (PEG-b-PCC-g-DC-g-TEPA) copolymer. This copolymer self-assembles into micelles and encapsulates hydrophobic GDC-0449 into its core and allows complex formation between miR-let7b and cationic pendant chains. This combination therapy effectively inhibited tumor growth when injected to ectopic tumor bearing mice compared to micelles carrying GDC-0449 or miR-let7b alone. Cetuximab, a monoclonal antibody for EGFR receptors, was conjugated to the nanomedicine to achieve active targeting to PDAC. Therefore, we hypothesize that combination therapy of miR-let7b with GDC-0449 using cetuximab conjugated nanomedicines can effectively chemosensitize PDAC cells by reducing desmoplasia and treat PDAC by reducing their tumorigenicity. Our overall objective is to develop novel combination therapy for PDAC using nanoformulations targeting multiple signaling pathways by miR-let7b and Hh pathway by GDC-0449. Our specific aims are to: i) determine the efficacy and molecular mechanisms of miR-let7b and GDC-0449 combination therapy in PC cells; ii) co-formulate miR-let7b and GDC- 0449 in cetuximab conjugated polymeric micelles and assess their synergistic effect on the inhibition of pancreatic cancer cells, iii) determine the therapeutic efficacy of micelles encapsulating miR-let7b and GDC-0449 in orthotopic NSG and genetically engineered KPC mouse models of PDAC. Long- term significance is to develop nanomedicines of miR-let7b and GDC-0449 that increase the local drug concentrations within the fibrotic stroma of these tumors and bypass the resistance mechanisms that allow tumor growth and inhibit the efficacy of current standard chemotherapies.
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1 |
2015 |
Mahato, Ram I. |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Research and Development of Novel Drug Delivery Systems Symposium @ University of Nebraska Medical Center
? DESCRIPTION (provided by applicant): High throughput screening and emergence of new therapeutic targets led to discovery of highly potent molecules with nanomolar efficacy. Clinical translation of the new therapeutic agents is limited by poor physicochemical and biopharmaceutical properties. To resolve this potential challenge, the need of the hour is the concerted effort by academia, pharmaceutical industry and regulatory body towards the development of novel delivery systems. Several new drug therapies fail due to non- selective drug disposition, poor accessibility to intracellular targets, dose limiting off target effects and resistance upon prolonged administration. These limitations can be potentially overcome through the development and scale up of novel drug delivery technologies. To expedite the translation of new molecules, the purpose of this conference is to share knowledge on drug product development principles among key players of pharmaceutical drug development, including representatives from academia, industry, and regulatory agencies. Participation from a diverse group of individuals including those representing minorities and traditionally underrepresented communities in science careers is expected. The two day symposium will include oral as well as poster presentations. Financial support provided for this conference will create opportunity to students to interact with top scientist in the field of drug delivery. In addition, registration fee waivers and travel awards will be provided to encourage participation by minorities and underrepresented communities. Based on the presentations and discussions at the conference, a manuscript will be prepared within 3 months after the conference and submitted for publication in a timely manner.
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1 |
2016 |
Mahato, Ram I. |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Research and Development of Nucleic Acid Based Nanomedicines @ University of Nebraska Medical Center
PROJECT SUMMARY Human genome project evolved with the promise of offering genetic modification as a tool to treat severe and debilitating diseases. Gene therapy has emerged as an important tool to modulate gene expression for treating various diseases. Development of gene medicines requires the rational nucleic acid drug design with unparalleled specificity and rapidity of development. Despite of many advantages, only two nucleic acid-based therapies have been approved: fomivirsen (Vitravene, an antisense phosphorothioate oligonucleotides) and pegaptanib (Macugen, an aptamer) for local intraocular therapy highlighting the challenges in systemic therapy of nucleic acids. Inefficient cellular uptake, poor plasma stability, intracellular trafficking and immunogenicity are some of the key hurdles in the translation of nucleic acids into therapeutics. These limitations can be potentially overcome through the development and scale up of nanoparticulate based nucleic acid delivery technologies. Although innovative and exciting work is being led by the biotech industry and academics, there is still a lack of platforms where scientists from pharma, biotech, academia and regulatory agencies can meet to share their expertise and discuss strategies to overcome challenges related to systemic delivery of nucleic acids and product development. Nucleic acid-based therapeutics requires diverse disciplines including biochemistry and molecular biology, material science, drug delivery, pharmacokinetics, genomics, bioengineering and informatics. This two day symposium will provide opportunity to the key players working on different aspects of gene medicines to share their knowledge and thoughts on further improvement. The symposium will have interdisciplinary focus with participation of scientists from diverse institutions and scientific disciplines to share/discuss/debate topics related to challenges of gene therapy and potential approaches for its rapid clinical development. Financial support provided for this symposium will allow the participation of trainees/young investigators as well as a diverse group of individuals including those representing minorities and traditionally underrepresented communities in science careers. In addition, registration fee waivers and travel awards will be provided to encourage the participation by minorities and underrepresented communities. There will also be oral and poster presentations from selected graduate students and young investigators.
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1 |
2021 |
Coulter, Donald Mahato, Ram I. |
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. |
Overcoming Resistance Mechanisms in Hedgehog and Myc-Amplified Medulloblastoma @ University of Nebraska Medical Center
PROJECT SUMMARY Medulloblastoma (MB) is a pediatric brain tumor arising from the cerebellum. MB treatment is challenging due to diverse genetic make-up, resistance to chemotherapy, inefficient drug transport across the blood brain barrier (BBB) and drug induced neurotoxicity. Hedgehog (Hh) and IGF/PI3K signaling pathways regulate cell growth, cancer stem cell (CSC) proliferation, and tumorigenicity in MB patients. Hh inhibitors are effective initially to treat SHH-MB, but their repeated use develops chemoresistance due to mutations in SMOothened (SMO) but can be overcome by modulating GLI, which is downstream of SMO using SF2523, which is a BRD4/PI3K dual inhibitor and inhibits MYCN expression. In our preliminary studies, we synthesize SMO inhibitor 2-chloro-N1-[4-chloro-3-(2-pyridinyl) phenyl]-N4, N4-bis (2-pyridinyl methyl)-1, 4-benzene- dicarboxamide (MDB5). MDB5 and SF2523 effectively inhibited the proliferation of ONS-76 and HD-MB03 cells in a dose dependent manner, with significantly higher cell killing when these drugs were used in combination. Treatment of HD-MB03 cells with the combination of these two drugs showed significantly higher decrease in colony formation and cyclin D1 expression but higher increase in Bax expression, compared to individual drugs. We synthesized mPEG-b-PCC-g-DC copolymer, with 5.1±0.21 and 6.5±0.1% loading for MDB5 and SF2523 when formulated into nanoparticles (NPs). There was sustained drug release from NPs, wherein 100% of MDB5 was released in 50 h, but only 60% of SF2523 was released in 80 h. Targeted NPs were prepared by mixing COG-133-PEG-b-PCC-g-DC and mPEG-b-PCC-g-DC at 10/90, 20/80 and 30/70 ratios, with the highest cellular uptake at 30/70 ratio. Systemic administration of COG-133-NPs loaded SF2523 into orthotopic SHH-MB tumor bearing NSG mice resulted in significantly higher drug concentration in the brain at 6 and 24h post administration compared to non-targeted NPs loaded with this drug while systemic injection of free drug showed negligible drug concentration in the brain. Moreover, systemic administration of COG-133-NPs loaded with MDB5 and SF2523 resulted in decreased tumor burden compared to non-targeted NPs of MDB5 and SF2523 as determined by IVIS imaging, with no hepatic toxicity. Our hypothesis is that BRD4/PI3K and Hh signaling pathways exert control over CSC proliferation in SHH-MB and hence represents a target for therapeutic exploitation with small molecules which can inhibit these signaling pathways. Our specific aims are to i) evaluate the resensitization effects of MDB5 and SF2523 on SHH and MYC driven MB and patient derived xenograft (PDX) cells; ii) formulate MDB5 and SF2523 into NPs with COG-133 conjugation and determine biodistribution and systemic/organ toxicity; and iii) determine effects of targeted NPs loaded with SF2523 and MDB5 in SHH and MYC driven orthotopic, patient derived xenograft (PDX) and transgenic SmoA1 MB mouse models. Long- term significance. Successful completion of this project will provide a platform technology for treating SHH- MB and other brain tumors using this innovative NP-based combination therapy of Hh and BRD4/PI3K inhibitors.
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1 |