These are exciting times in the scientific world. The public all over the world is fascinated by the spectacular advances in gene therapy, sequencing of the human genome, and stem cell research. These advances promise to prevent, correct or modulate genetic and acquired diseases, which use genes to produce therapeutic proteins or inhibit aberrant protein production.
The launching of the human proteome project has turned functional genomics and proteomics into powerful bullworks, which will give us an integrated scenario of turning nucleic acids into therapeutics. The development of effective nucleic acid therapeutics demands teamwork among scientists with expertise in molecular and cell biology, biochemistry, biophysics, polymer chemistry, colloid science, pharmaceutics, and medicine.
In the last decade, significant progress has been made in the use of nucleic acids, such as plasmid DNA, antisense oligonucleotides, siRNA, ribozymes, peptide nucleic acids (PNA) and aptamer nucleic acids for nucleic acid therapy.
ONGOING RESEARCH PROJECTS IN MAHATO'S LAB
Design and Synthesis of Novel Polymer and Lipid Carriers
We are working on the Site-specific Delivery of Oligonucleotides and microRNA to Hepatocytes or Hepatic Stellate Cells for treatment of hepatitis and liver fibrosis. We have previously shown that the conjugation of siRNA to mannose-6-phosphate- PEG (M6P-PEG) can significantly enhance its delivery to hepatic stellate cells and this M6P-PEG-siRNA has the potential to treat liver fibrosis by inhibiting collagen synthesis (Zhu et al, Bioconjug Chem, 2010, 21: 2119-2127).
We are developing several novel amphiphilic copolymers and lipopolymers for use as micellar delivery of small molecular weight hydrophobic anti-cancer drugs (Danquah et al, J. Polym. Sci.A Polym. Chem. 2013, 51: 347–362, Danquah et al, Biomaterials. 2010, 31:2358-70. and Li et al., Biomacromolecules. 2010, 11:2610-20).
Design and Construction of Plasmid and Adenovirus-based Gene Expression Systems
Despite tremendous progress in islet isolation, culture, and preservation, the clinical use of human islet transplantation for treating type I diabetes is limited due to post-transplantation challenges to the islets such as the failure to revascularize and immune destruction of the islet graft (Narang and Mahato, Pharmacol Rev.2006;58(2):194-243).
We are working on genetic modification of human islets prior to transplantation to prevent beta-cell death (Wu H et al., J. Gene Med. 2011, 13: 658–669). We are currently working on construction and characterization of adenoviral vectors with different therapeutic genes for ex vivo infection into human and rat islets (Wu H et al., Mol Pharm. 2010, 7:1655-66). In addition, we are also exploring siRNA delivery to human and rat islets for silencing antiapoptotic genes for improving the outcome of islet transplantation.
Combination of Stem Cells and Gene Delivery for Improving Islet Transplantation
Human bone marrow-derived mesenchymal stem cells (hBMSCs) have the potential to improve the outcome of human islet transplantation by repairing damaged islets and modulating immune responses. We have shown that using genetically modified hBMSCs promoted revascularization and inhibited apoptosis of transplanted islet (Wu et al. Pharm Res. 2011, 28: 2098-109). We tested immunosuppressive effect hBMSCs after co-transplantation with human islets in a humanized NSG mice model and shown that hBMSCs can improve the outcome of islet transplantation (Wu et al., Mol. Ther. 2013, doi: 10.1038/mt.2013.147). This will significantly reduce the number of islets required to achieve normoglycemia.
Our research in pancreatic cancer is based upon the hypothesis that simultaneous delivery of anti-cancer molecule gemcitabine and a suitable miRNA could render a more effective treatment strategy for advanced pancreatic cancer. miRNAs are the key regulators of cancer stem cells (CSCs) which play a central role in inducing chemoresistance and inferring metastatic potential to the cells of the pancreatic tumor tissue (Singh S., et al., Cancer Lett. 2013, 334:211-20). To meet this objective, we have synthesized gemcitabine conjugated copolymer which undergoes self-assembly into micelles. These micelles provided high drug payload, sustained drug release, prevented its plasma degradation and significantly inhibited tumor growth compared to free drug when injected intravenously into pancreatic tumor bearing NSG mice (Chitkara D., et al., Bioconjug. Chem. 2013, DOI: 10.1021/bc400032x). To identify miRNAs involved in chemoresistance, a series of dysregulated miRNAs were identified from the CSCs isolated from gemcitabine resistant MIA PaCa-2R cells and human pancreatic cancer tissues. Then, we synthesized gemcitabine conjugated copolymer for complex formation with miRNA of our choice and conducted preliminary experiments to establish the proof-of-concept and indeed observed an increase in chemosensitivity of pancreatic cancer cells and a significant reduction in their invasive ability. Studies are in progress to further optimize the combination formulation, study their spatio-temporal kinetics and determine efficacy in orthotopic pancreatic tumor model.
Our research in prostate cancer is mainly focused on the following objectives (a) designing an efficient drug delivery system which could target the tumor tissue specifically and prolong the residence time of the drugs in the tumor along with; (b) selection of an appropriate combination of therapeutic molecules. We have synthesized a lactic-acid and carbonate based polymer capable of forming core cross-linked micelles which offer higher thermodynamic (stability to dilution) and kinetic (stability to plasma proteins) stability over the conventional micellar formulations (Danquah et al. J. Polym. Sci. A Polym. Chem., 2013, 51, 347-362). Cross-linked (CM) micelles prepared using this copolymer were found to be stable to 1000- fold dilution and in BSA for 48 h unlike non cross-linked (NCM). We had also established the role of hedgehog signaling (Hh) and miRNA in chemo-resistant prostate cancer (Singh S., et al., PLoS One. 2012; 7(6), e40021). We further formulated dual-drug loaded CM of antimitotic drug paclitaxel (PTX) and Hh inhibitor, GDC-0449 with appreciable drug loading and carried out cytotoxicity assay in PTX resistant DU145 prostate cancer cells which showed significantly higher cell death in the presence of combination of drugs at even half-dose as compared to each of the individual drugs.
miRNA Delivery for Treatment of Liver Fibrosis and Diabetes
We are developing targeted system for miRNA site-specific delivery for treating liver fibrosis, and we are working on miRNA therapy for treating type I diabetes. We are also working on the design/construction of plasmid and adenovirus-based shRNA for treating these diseases.
Liver Ischemia Reperfusion Injury and Liver Transplantation
Hedgehog signaling is required for endodermal commitment and hepatogenesis after liver injury or ischemia reperfusion (I/R). We have determined the expression pattern of Hh signaling and its role in liver injury following I/R using Hh antagonists such as cyclopamine (CYA) and vismodegib (GDC 0449) (Pratap A., et al., Mol. Pharm. 8: 958–968, Pratap A., et al., J. Drug Target. 2012, 20:770-82). We plan to further study this approach by selectively targeting these drugs to the liver using mannose-6-phosphate ligand containing nanoparticles.