Narayan Shivapurkar

Concentration of Gapmer measured in tumors and tissues

Viable cell numbers of AsPC-1 cells transfected with Gapmer-90

Serum chemistries (mean ± SEM) obtained at necropsy

Precision medicine and genomic profiling with target-specific therapy directed to cancer cell receptors have improved the outcome of many recalcitrant cancers. Strategies to deliver gene therapy to downregulate cancer driver genes have been challenging in vivo. Pancreatic cancer has the poorest survival of all solid tumors due to the lack of target-specific therapies and its characteristic tumor microenvironment with dense fibrosis and abundant immunosuppressive M2-polarized macrophages. In this study, we designed a panel of locked nucleic acid gapmer antisense oligonucleotides directed to human gastrin mRNA. We tested their efficacy by downregulation of mRNA and growth inhibition in vitro. The most effective gapmer, gapmer-90, was modified for in vivo therapeutics by thiol–maleimide click chemistry to render it target-specific to the cholecystokinin-B receptor. This G-protein–coupled receptor is overexpressed in pancreatic cancers. Mice bearing orthotopic human pancreatic tumors were treated with PBS (control), an untargeted gapmer, or receptor-targeted gapmers at low (60 nmol/L) and high (120 nmol/L) concentrations. Uptake of the gapmer was measured in tissues using a complementary probe. We found that the receptor-targeted gapmer significantly enhanced uptake in vivo and decreased growth and metastases of human pancreatic tumors in a dose-related fashion without off-target toxicity. The target-specific gapmer also altered the tumor microenvironment by decreasing fibrosis and reducing M2-polarized macrophages. Collectively, our results provide evidence that locked nucleic acid gapmers are a unique tool to deliver antisense oligonucleotides for therapy to recalcitrant cancers. Rendering the gapmers target-specific allows for selective uptake by receptor internalization, improving efficacy and decreasing off-target toxicity.

Chemical structure of the targeted LNA Gapmer-90.

Cytotoxicity assay was performed by flow cytometry

Precision medicine and genomic profiling with target-specific therapy directed to cancer cell receptors have improved the outcome of many recalcitrant cancers. Strategies to deliver gene therapy to downregulate cancer driver genes have been challenging in vivo. Pancreatic cancer has the poorest survival of all solid tumors due to the lack of target-specific therapies and its characteristic tumor microenvironment with dense fibrosis and abundant immunosuppressive M2-polarized macrophages. In this study, we designed a panel of locked nucleic acid gapmer antisense oligonucleotides directed to human gastrin mRNA. We tested their efficacy by downregulation of mRNA and growth inhibition in vitro. The most effective gapmer, gapmer-90, was modified for in vivo therapeutics by thiol–maleimide click chemistry to render it target-specific to the cholecystokinin-B receptor. This G-protein–coupled receptor is overexpressed in pancreatic cancers. Mice bearing orthotopic human pancreatic tumors were treated with PBS (control), an untargeted gapmer, or receptor-targeted gapmers at low (60 nmol/L) and high (120 nmol/L) concentrations. Uptake of the gapmer was measured in tissues using a complementary probe. We found that the receptor-targeted gapmer significantly enhanced uptake in vivo and decreased growth and metastases of human pancreatic tumors in a dose-related fashion without off-target toxicity. The target-specific gapmer also altered the tumor microenvironment by decreasing fibrosis and reducing M2-polarized macrophages. Collectively, our results provide evidence that locked nucleic acid gapmers are a unique tool to deliver antisense oligonucleotides for therapy to recalcitrant cancers. Rendering the gapmers target-specific allows for selective uptake by receptor internalization, improving efficacy and decreasing off-target toxicity.

This investigation identified a novel pathway involving the activation of hepatic stem cells and liver oncogenesis. Receptor blockade or genetic disruption of the cholecystokinin-B receptor (CCK-BR) signaling pathway decreased the activation and proliferation of hepatic stem cells after liver injury without eliminating the regenerative capacity of healthy hepatocytes.