Christopher Daige

MicroRNAs (miRNAs) are emerging as potential cancer therapeutics, but effective delivery mechanisms to tumor sites are a roadblock to utility. Here we show that systemically delivered, synthetic miRNA mimics in complex with a novel neutral lipid emulsion are preferentially targeted to lung tumors and show therapeutic benefit in mouse models of lung cancer. Therapeutic delivery was demonstrated using mimics of the tumor suppressors, microRNA-34a (miR-34a) and let-7, both of which are often down regulated or lost in lung cancer. Systemic treatment of a Kras-activated autochthonous mouse model of non-small cell lung cancer (NSCLC) led to a significant decrease in tumor burden. Specifically, mice treated with miR-34a displayed a 60% reduction in tumor area compared to mice treated with a miRNA control. Similar results were obtained with the let-7 mimic. These findings provide direct evidence that synthetic miRNA mimics can be systemically delivered to the mammalian lung and support the promise of miRNAs as a future targeted therapy for lung cancer. MicroRNAs (miRNAs) are emerging as potential cancer therapeutics, but effective delivery mechanisms to tumor sites are a roadblock to utility. Here we show that systemically delivered, synthetic miRNA mimics in complex with a novel neutral lipid emulsion are preferentially targeted to lung tumors and show therapeutic benefit in mouse models of lung cancer. Therapeutic delivery was demonstrated using mimics of the tumor suppressors, microRNA-34a (miR-34a) and let-7, both of which are often down regulated or lost in lung cancer. Systemic treatment of a Kras-activated autochthonous mouse model of non-small cell lung cancer (NSCLC) led to a significant decrease in tumor burden. Specifically, mice treated with miR-34a displayed a 60% reduction in tumor area compared to mice treated with a miRNA control. Similar results were obtained with the let-7 mimic. These findings provide direct evidence that synthetic miRNA mimics can be systemically delivered to the mammalian lung and support the promise of miRNAs as a future targeted therapy for lung cancer. IntroductionLung cancer is a deadly disease with millions of victims worldwide each year. Non-small cell lung cancers (NSCLC) make up the majority of these deaths. Current therapies fail to treat this disease in the vast majority of cases, with <15%, 5 year survival rate.1Jemal A Siegel R Ward E Hao Y Xu J Murray T et al.Cancer statistics, 2008.CA Cancer J Clin. 2008; 58: 71-96Crossref PubMed Scopus (10173) Google Scholar Novel therapies based on a better understanding of the disease are desperately needed to save more lives.MicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression to affect a multitude of biological processes including cell proliferation, differentiation, survival, and motility.2Bartel DP MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29010) Google Scholar In addition, miRNAs are often found misexpressed or damaged in many cancers and have been implicated causally in promoting proliferation and metastasis of tumor cells.3Calin GA Sevignani C Dumitru CD Hyslop T Noch E Yendamuri S et al.Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers.Proc Natl Acad Sci USA. 2004; 101: 2999-3004Crossref PubMed Scopus (3539) Google Scholar,4Dykxhoorn DM MicroRNAs and metastasis: little RNAs go a long way.Cancer Res. 2010; 70: 6401-6406Crossref PubMed Scopus (183) Google Scholar,5Esquela-Kerscher A Slack FJ Oncomirs - microRNAs with a role in cancer.Nat Rev Cancer. 2006; 6: 259-269Crossref PubMed Scopus (6136) Google Scholar Two classes of oncogenesis-associated miRNAs (oncomiRs) have been described, those that are overexpressed in tumors and act as oncogenes and those that are underexpressed in tumors and act as tumor suppressors.5Esquela-Kerscher A Slack FJ Oncomirs - microRNAs with a role in cancer.Nat Rev Cancer. 2006; 6: 259-269Crossref PubMed Scopus (6136) Google Scholar Two well-characterized families of tumor suppressor miRNAs are let-7 and miR-34. let-7 is normally expressed in differentiated tissues but frequently lost in cancer, notably, lung cancers.3Calin GA Sevignani C Dumitru CD Hyslop T Noch E Yendamuri S et al.Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers.Proc Natl Acad Sci USA. 2004; 101: 2999-3004Crossref PubMed Scopus (3539) Google Scholar,6Johnson SM Grosshans H Shingara J Byrom M Jarvis R Cheng A et al.RAS is regulated by the let-7 microRNA family.Cell. 2005; 120: 635-647Abstract Full Text Full Text PDF PubMed Scopus (3089) Google Scholar let-7 negatively regulates multiple cell cycle oncogenes, such as RAS, MYC, and HMGA26Johnson SM Grosshans H Shingara J Byrom M Jarvis R Cheng A et al.RAS is regulated by the let-7 microRNA family.Cell. 2005; 120: 635-647Abstract Full Text Full Text PDF PubMed Scopus (3089) Google Scholar,7Lee YS Dutta A The tumor suppressor microRNA let-7 represses the HMGA2 oncogene.Genes Dev. 2007; 21: 1025-1030Crossref PubMed Scopus (1016) Google Scholar,8Sampson VB Rong NH Han J Yang Q Aris V Soteropoulos P et al.MicroRNA let-7a down-regulates MYC and reverts MYC-induced growth in Burkitt lymphoma cells.Cancer Res. 2007; 67: 9762-9770Crossref PubMed Scopus (666) Google Scholar and exogenous application of let-7 to human lung cancer cells reduces proliferation and radiosensitizes the cells.9Johnson CD Esquela-Kerscher A Stefani G Byrom M Kelnar K Ovcharenko D et al.The let-7 microRNA represses cell proliferation pathways in human cells.Cancer Res. 2007; 67: 7713-7722Crossref PubMed Scopus (1089) Google Scholar,10Weidhaas JB Babar I Nallur SM Trang P Roush S Boehm M et al.MicroRNAs as potential agents to alter resistance to cytotoxic anticancer therapy.Cancer Res. 2007; 67: 11111-11116Crossref PubMed Scopus (347) Google Scholar miR-34 is also lost in lung cancer and acts as a tumor suppressor by regulating multiple cell cycle and cell survival genes.11Bommer GT Gerin I Feng Y Kaczorowski AJ Kuick R Love RE et al.p53-mediated activation of miRNA34 candidate tumor-suppressor genes.Curr Biol. 2007; 17: 1298-1307Abstract Full Text Full Text PDF PubMed Scopus (957) Google Scholar,12Chang TC Wentzel EA Kent OA Ramachandran K Mullendore M Lee KH et al.Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis.Mol Cell. 2007; 26: 745-752Abstract Full Text Full Text PDF PubMed Scopus (1696) Google Scholar,13Lodygin D Tarasov V Epanchintsev A Berking C Knyazeva T Körner H et al.Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer.Cell Cycle. 2008; 7: 2591-2600Crossref PubMed Scopus (681) Google Scholar miR-34 is directly transcribed by the p53 tumor suppressor gene and is required for a radiation response in vitro and in vivo.14He L He X Lim LP de Stanchina E Xuan Z Liang Y et al.A microRNA component of the p53 tumour suppressor network.Nature. 2007; 447: 1130-1134Crossref PubMed Scopus (2282) Google Scholar,15Kato M Paranjape T Müller RU Ullrich R Nallur S Gillespie E et al.The mir-34 microRNA is required for the DNA damage response in vivo in C. elegans and in vitro in human breast cancer cells.Oncogene. 2009; 28: 2419-2424Crossref PubMed Scopus (194) Google ScholarDelivery of endogenous tumor suppressor miRNAs as synthetic miRNA mimics has emerged as a promising to treat D M The promise of microRNA therapy.Cancer Res. 2010; 70: PubMed Scopus Google Scholar miRNAs have been that tumor growth in mouse models of cancer. these are the tumor let-7, and J Wentzel EA et microRNA delivery in a cancer 2009; Full Text Full Text PDF PubMed Scopus Google L M RU Y et delivery of synthetic the growth of tumors of multiple 2010; Full Text Full Text PDF PubMed Scopus Google P L Kelnar K M et of lung tumors by the let-7 2010; PubMed Scopus Google L Kelnar K M L D et of a lung cancer therapeutic based on the tumor suppressor Res. 2010; 70: PubMed Scopus Google Scholar In of these cases, the miRNA was delivered directly by or was expressed a a for miRNA delivery to the tumor in the mouse delivery that are to in the are to a of and tumors that have expression is to show the in gene such as as as the for of a DNA and of the gene Cancer gene or Rev Cancer. PubMed Scopus Google p53 gene 2006; 6: PubMed Scopus Google Scholar cancer cells frequently show in the of miRNA expression a is a S C H GA et al.A in human cancer microRNA and 2009; PubMed Scopus Google Scholar delivery of miRNA mimics the to and TC D LP to the treatment of human the 2008; PubMed Scopus Google we delivery of miR-34a mimics using a neutral lipid emulsion that has the potential to be the L Kelnar K M L D et of a lung cancer therapeutic based on the tumor suppressor Res. 2010; 70: PubMed Scopus Google Scholar Systemic delivery of miR-34a mimics led to of miR-34a in tumor of direct miR-34a and of in L Kelnar K M L D et of a lung cancer therapeutic based on the tumor suppressor Res. 2010; 70: PubMed Scopus Google Scholar these lung tumors were the of this novel miRNA we the of the delivery in mouse models of therapeutic delivery of synthetic agents to both lung tissues as as lung and show of let-7 and miR-34 in autochthonous mouse model of lung delivered miRNA in the of delivery was we the of a miRNA in lung and tissues was is expressed by cells of the and the of delivered miRNA mimics the expressed miRNAs in were a of is to that a mouse on and lung were and to and in were in tissues the in with a that neutral lipid preferentially in the A C R G et gene in vivo using neutral Res. 2005; PubMed Scopus Google Scholar the miRNA was up by cells or was in the found in the a of were with to with by in and which that the majority of in these tissues in In the of in we that the miRNA is up by lung and that be a to therapeutic miRNAs and RNAs to lung and lung of to lung tumors that delivery of miRNA mimics to was the miRNA was by lung cells and the miRNA is In addition, lung tumors a lung and delivery to lung is of delivery to lung a for therapeutic delivery to lung we in vivo based on lung tumors were in mice by DM A of and for in Google Scholar delivery of the to lung and lung including as by delivery of and this of to the of tumor of the growth was by cells the directly with tumor mice was as the at mice of in a mice were of a of the was and expressed as to the of each mouse at in a of led to a reduction of to that were on the of In mice treated with the which is to tumor the that delivery led to the majority of lung tumor the complex and of delivery of to lung tumors in of mice lung tumors were and of or with neutral lipid of in The are as to the at of delivery of let-7 or miR-34 tumor growth in the autochthonous mouse have that let-7 and miR-34 with tumor growth in mouse models of P L Kelnar K M et of lung tumors by the let-7 2010; PubMed Scopus Google L Kelnar K M L D et of a lung cancer therapeutic based on the tumor suppressor Res. 2010; 70: PubMed Scopus Google A Trang P L Cheng A L et al.The let-7 microRNA reduces tumor growth in mouse models of lung cancer.Cell Cycle. 2008; 7: PubMed Scopus Google Scholar the therapeutic of delivery of miRNA mimics in tumor model has been this we the autochthonous mouse K D R et of lung tumor and using expression of Dev. PubMed Scopus Google Scholar model is based on that is expressed a of lung tumors treated with mice were 5 of to and for synthetic miR-34a or miRNAs with were of for a of at a of each the mice were and lung tissues were and lung cell proliferation, and were by treated with and of the mice that were with tumor those with that the of the that were treated with with P L Kelnar K M et of lung tumors by the let-7 2010; PubMed Scopus Google Scholar as as proliferation, that systemically delivered proliferation The of the obtained as a of cells was for treated mice compared to for treated mice the that to treatment with in the to show a decrease in proliferation as by lung tumors this to delivery of reduces lung tumor in a non-small cell lung cancer and tumor are treated with a significant reduction in lung in of treated compared to mice treated with and of in mice treated with and at a are of tumor in treated with and treated with and The of tumor area lung area are as a The P is expression in mouse 5 for each the and and the P is The and are and mice treated with we tumor compared to those treated with 60% reduction in tumor to a significant of miR-34a in the lung as by tissues miR-34a treated mice expression of with of compared to for treated mice and in cells compared to treated mice These results show that delivery of miR-34a mimics can reduction of lung tumors in a mouse model of proliferation and of mimics lung tumor in autochthonous non-small cell lung cancer and tumor are mice tumors were to for synthetic miR-34a or with neutral lipid emulsion were delivered by treated with miR-34a a significant reduction in lung and compared to mice treated with and of in mice treated with miR-34a and at a are show at a of tumor in treated with and miR-34a treated with and The of tumor area lung area are as a The P is miR-34a expression in mouse 5 for each the and and the P is The and are and provide evidence that miRNA mimics delivered systemically is a therapeutic for the treatment of lung cancer. a miRNA can a of the of a miRNA in cancer can affect multiple pathways to a therapeutic for this is miR-34a is of a tumors the that is to be directly by the of cancer of RAS, is to the of miR-34a in this mouse let-7 and miR-34 a such as and VB Rong NH Han J Yang Q Aris V Soteropoulos P et al.MicroRNA let-7a down-regulates MYC and reverts MYC-induced growth in Burkitt lymphoma cells.Cancer Res. 2007; 67: 9762-9770Crossref PubMed Scopus (666) Google CD Esquela-Kerscher A Stefani G Byrom M Kelnar K Ovcharenko D et al.The let-7 microRNA represses cell proliferation pathways in human cells.Cancer Res. 2007; 67: 7713-7722Crossref PubMed Scopus (1089) Google Scholar,13Lodygin D Tarasov V Epanchintsev A Berking C Knyazeva T Körner H et al.Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer.Cell Cycle. 2008; 7: 2591-2600Crossref PubMed Scopus (681) Google R E M M et of miR-34a represses 2010; 17: PubMed Scopus Google Scholar many of that mechanisms of tumor are also be by that miR-34 treated tumors displayed proliferation and let-7 treated tumors proliferation that let-7 and miR-34a are frequently in human lung and that affect cancer a therapeutic of the miRNAs to cell cycle and of miR-34a has also been to in cell D Tarasov V Epanchintsev A Berking C Knyazeva T Körner H et al.Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer.Cell Cycle. 2008; 7: 2591-2600Crossref PubMed Scopus (681) Google L He X Lim LP de Stanchina E Xuan Z Liang Y et al.A microRNA component of the p53 tumour suppressor network.Nature. 2007; 447: 1130-1134Crossref PubMed Scopus (2282) Google Scholar and of miR-34a has been demonstrated to to the of K I K et and are endogenous of Biol. 2009; PubMed Scopus Google Scholar to and is a with p53 a miR-34a direct a miR-34a direct M M miR-34a of regulates Natl Acad Sci USA. 2008; PubMed Scopus Google M and the Cycle. 2009; PubMed Scopus Google Scholar is that regulates and M M miR-34a of regulates Natl Acad Sci USA. 2008; PubMed Scopus Google J Q Han L J H Y et with in human 2008; PubMed Scopus Google Scholar and of is H E et as p53 Full Text Full Text PDF PubMed Scopus Google Scholar miR-34a to have in cell types in in cell and of cell proliferation and in These be to that with miR-34a or of cell the of miR-34a is in of of miR-34a in elegans and mammalian cells to DNA agents and cell M Paranjape T Müller RU Ullrich R Nallur S Gillespie E et al.The mir-34 microRNA is required for the DNA damage response in vivo in C. elegans and in vitro in human breast cancer cells.Oncogene. 2009; 28: 2419-2424Crossref PubMed Scopus (194) Google R E M M et of miR-34a represses 2010; 17: PubMed Scopus Google Scholar of miR-34a is of the p53 R E M M et of miR-34a represses 2010; 17: PubMed Scopus Google Scholar The potential for the of pathways miR-34a the of this microRNA as a tumor delivery of miRNA mimics to lung tumors was with a novel delivery that in vivo delivery of miR-34a mimics to tumors in L Kelnar K M L D et of a lung cancer therapeutic based on the tumor suppressor Res. 2010; 70: PubMed Scopus Google Scholar delivery and of the that can be to based on neutral are to in be by the to the or be up by A C R G et gene in vivo using neutral Res. 2005; PubMed Scopus Google Scholar In to a of miRNA in but displayed delivery to lung and lung In addition, neutral delivery be those in with that miR-34a with to and in a response in L Kelnar K M L D et of a lung cancer therapeutic based on the tumor suppressor Res. 2010; 70: PubMed Scopus Google Scholar delivery to lung a therapeutic response in a mouse model and a support the of these let-7 and miR-34 as novel targeted therapies for lung cancer and in vivo was of and complex with synthetic in the were in with and a by the and at mice of were a of synthetic with to the by was the and were by delivery of the were with to the the and were the was with and was directly the of the and were and were in was tissues and the miRNA A was to in tissues and by cell of were a up of of synthetic lung tumor were by of human cells the gene a in et DM A of and for in Google Scholar that delivery the mice The were with a to and or were to and lung cancer mice The were by using a to cells directly to the in of growth with were by a of at of the mice lung tumors that are by and show in expression mice were with of a synthetic or a with in vivo to the was to of and and expressed as to the of each mouse at mouse tissues was using the the of and miR-34a was at for and transcribed using the and miR-34a miRNA with of miRNA was using the miRNA vivo and delivery of and miR-34a to mice were 5 of and for each mouse was with synthetic miR-34a or miRNA with by were were for a of at a of each the mice were and lung tissues were were for to tumor by the in in and with and was using a to that mice of the at the as K D R et of lung tumor and using expression of Dev. PubMed Scopus Google Scholar and tumor were using in as A Trang P L Cheng A L et al.The let-7 microRNA reduces tumor growth in mouse models of lung cancer.Cell Cycle. 2008; 7: PubMed Scopus Google Scholar The tumor was as a of tumor area to lung area by the of the and was by the and based on The is as of cells with mice with by lung with and mice with in of a mouse tumor of the mouse in on of tumor cells lung with of let-7 mimics with of tumor in treated with and treated with and The of tumor area lung area are as a The and are of The was using the with lung DNA to that of the mice treated with that the of the by DNA treated with and miR-34a is a control. The for the is at the is with mice treated with miR-34a a tumor IntroductionLung cancer is a deadly disease with millions of victims worldwide each year. Non-small cell lung cancers (NSCLC) make up the majority of these deaths. Current therapies fail to treat this disease in the vast majority of cases, with <15%, 5 year survival rate.1Jemal A Siegel R Ward E Hao Y Xu J Murray T et al.Cancer statistics, 2008.CA Cancer J Clin. 2008; 58: 71-96Crossref PubMed Scopus (10173) Google Scholar Novel therapies based on a better understanding of the disease are desperately needed to save more lives.MicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression to affect a multitude of biological processes including cell proliferation, differentiation, survival, and motility.2Bartel DP MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29010) Google Scholar In addition, miRNAs are often found misexpressed or damaged in many cancers and have been implicated causally in promoting proliferation and metastasis of tumor cells.3Calin GA Sevignani C Dumitru CD Hyslop T Noch E Yendamuri S et al.Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers.Proc Natl Acad Sci USA. 2004; 101: 2999-3004Crossref PubMed Scopus (3539) Google Scholar,4Dykxhoorn DM MicroRNAs and metastasis: little RNAs go a long way.Cancer Res. 2010; 70: 6401-6406Crossref PubMed Scopus (183) Google Scholar,5Esquela-Kerscher A Slack FJ Oncomirs - microRNAs with a role in cancer.Nat Rev Cancer. 2006; 6: 259-269Crossref PubMed Scopus (6136) Google Scholar Two classes of oncogenesis-associated miRNAs (oncomiRs) have been described, those that are overexpressed in tumors and act as oncogenes and those that are underexpressed in tumors and act as tumor suppressors.5Esquela-Kerscher A Slack FJ Oncomirs - microRNAs with a role in cancer.Nat Rev Cancer. 2006; 6: 259-269Crossref PubMed Scopus (6136) Google Scholar Two well-characterized families of tumor suppressor miRNAs are let-7 and miR-34. let-7 is normally expressed in differentiated tissues but frequently lost in cancer, notably, lung cancers.3Calin GA Sevignani C Dumitru CD Hyslop T Noch E Yendamuri S et al.Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers.Proc Natl Acad Sci USA. 2004; 101: 2999-3004Crossref PubMed Scopus (3539) Google Scholar,6Johnson SM Grosshans H Shingara J Byrom M Jarvis R Cheng A et al.RAS is regulated by the let-7 microRNA family.Cell. 2005; 120: 635-647Abstract Full Text Full Text PDF PubMed Scopus (3089) Google Scholar let-7 negatively regulates multiple cell cycle oncogenes, such as RAS, MYC, and HMGA26Johnson SM Grosshans H Shingara J Byrom M Jarvis R Cheng A et al.RAS is regulated by the let-7 microRNA family.Cell. 2005; 120: 635-647Abstract Full Text Full Text PDF PubMed Scopus (3089) Google Scholar,7Lee YS Dutta A The tumor suppressor microRNA let-7 represses the HMGA2 oncogene.Genes Dev. 2007; 21: 1025-1030Crossref PubMed Scopus (1016) Google Scholar,8Sampson VB Rong NH Han J Yang Q Aris V Soteropoulos P et al.MicroRNA let-7a down-regulates MYC and reverts MYC-induced growth in Burkitt lymphoma cells.Cancer Res. 2007; 67: 9762-9770Crossref PubMed Scopus (666) Google Scholar and exogenous application of let-7 to human lung cancer cells reduces proliferation and radiosensitizes the cells.9Johnson CD Esquela-Kerscher A Stefani G Byrom M Kelnar K Ovcharenko D et al.The let-7 microRNA represses cell proliferation pathways in human cells.Cancer Res. 2007; 67: 7713-7722Crossref PubMed Scopus (1089) Google Scholar,10Weidhaas JB Babar I Nallur SM Trang P Roush S Boehm M et al.MicroRNAs as potential agents to alter resistance to cytotoxic anticancer therapy.Cancer Res. 2007; 67: 11111-11116Crossref PubMed Scopus (347) Google Scholar miR-34 is also lost in lung cancer and acts as a tumor suppressor by regulating multiple cell cycle and cell survival genes.11Bommer GT Gerin I Feng Y Kaczorowski AJ Kuick R Love RE et al.p53-mediated activation of miRNA34 candidate tumor-suppressor genes.Curr Biol. 2007; 17: 1298-1307Abstract Full Text Full Text PDF PubMed Scopus (957) Google Scholar,12Chang TC Wentzel EA Kent OA Ramachandran K Mullendore M Lee KH et al.Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis.Mol Cell. 2007; 26: 745-752Abstract Full Text Full Text PDF PubMed Scopus (1696) Google Scholar,13Lodygin D Tarasov V Epanchintsev A Berking C Knyazeva T Körner H et al.Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer.Cell Cycle. 2008; 7: 2591-2600Crossref PubMed Scopus (681) Google Scholar miR-34 is directly transcribed by the p53 tumor suppressor gene and is required for a radiation response in vitro and in vivo.14He L He X Lim LP de Stanchina E Xuan Z Liang Y et al.A microRNA component of the p53 tumour suppressor network.Nature. 2007; 447: 1130-1134Crossref PubMed Scopus (2282) Google Scholar,15Kato M Paranjape T Müller RU Ullrich R Nallur S Gillespie E et al.The mir-34 microRNA is required for the DNA damage response in vivo in C. elegans and in vitro in human breast cancer cells.Oncogene. 2009; 28: 2419-2424Crossref PubMed Scopus (194) Google ScholarDelivery of endogenous tumor suppressor miRNAs as synthetic miRNA mimics has emerged as a promising to treat D M The promise of microRNA therapy.Cancer Res. 2010; 70: PubMed Scopus Google Scholar miRNAs have been that tumor growth in mouse models of cancer. these are the tumor let-7, and J Wentzel EA et microRNA delivery in a cancer 2009; Full Text Full Text PDF PubMed Scopus Google L M RU Y et delivery of synthetic the growth of tumors of multiple 2010; Full Text Full Text PDF PubMed Scopus Google P L Kelnar K M et of lung tumors by the let-7 2010; PubMed Scopus Google L Kelnar K M L D et of a lung cancer therapeutic based on the tumor suppressor Res. 2010; 70: PubMed Scopus Google Scholar In of these cases, the miRNA was delivered directly by or was expressed a a for miRNA delivery to the tumor in the mouse delivery that are to in the are to a of and tumors that have expression is to show the in gene such as as as the for of a DNA and of the gene Cancer gene or Rev Cancer. PubMed Scopus Google p53 gene 2006; 6: PubMed Scopus Google Scholar cancer cells frequently show in the of miRNA expression a is a S C H GA et al.A in human cancer microRNA and 2009; PubMed Scopus Google Scholar delivery of miRNA mimics the to and TC D LP to the treatment of human the 2008; PubMed Scopus Google we delivery of miR-34a mimics using a neutral lipid emulsion that has the potential to be the L Kelnar K M L D et of a lung cancer therapeutic based on the tumor suppressor Res. 2010; 70: PubMed Scopus Google Scholar Systemic delivery of miR-34a mimics led to of miR-34a in tumor of direct miR-34a and of in L Kelnar K M L D et of a lung cancer therapeutic based on the tumor suppressor Res. 2010; 70: PubMed Scopus Google Scholar these lung tumors were the of this novel miRNA we the of the delivery in mouse models of therapeutic delivery of synthetic agents to both lung tissues as as lung and show of let-7 and miR-34 in autochthonous mouse model of lung cancer.

miR34a is a tumor-suppressor miRNA that functions within the p53 pathway to regulate cell-cycle progression and apoptosis. With apparent roles in metastasis and cancer stem cell development, miR34a provides an interesting opportunity for therapeutic development. A mimic of miR34a was complexed with an amphoteric liposomal formulation and tested in two different orthotopic models of liver cancer. Systemic dosing of the formulated miR34a mimic increased the levels of miR34a in tumors by approximately 1,000-fold and caused statistically significant decreases in the mRNA levels of several miR34a targets. The administration of the formulated miR34a mimic caused significant tumor growth inhibition in both models of liver cancer, and tumor regression was observed in more than one third of the animals. The antitumor activity was observed in the absence of any immunostimulatory effects or dose-limiting toxicities. Accumulation of the formulated miR34a mimic was also noted in the spleen, lung, and kidney, suggesting the potential for therapeutic use in other cancers.

Abstract Metabolic dysregulation is a hallmark of cancer. Many tumors exhibit auxotrophy for various amino acids, such as arginine, because they are unable to meet the demand for these amino acids through endogenous production. This vulnerability can be exploited by employing therapeutic strategies that deplete systemic arginine in order to limit the growth and survival of arginine auxotrophic tumors. Pegzilarginase, a human arginase-1 enzyme engineered to have superior stability and enzymatic activity relative to the native human arginase-1 enzyme, depletes systemic arginine by converting it to ornithine and urea. Therapeutic administration of pegzilarginase in the setting of arginine auxotrophic tumors exerts direct antitumor activity by starving the tumor of exogenous arginine. We hypothesized that in addition to this direct effect, pegzilarginase treatment indirectly augments antitumor immunity through increased antigen presentation, thus making pegzilarginase a prime candidate for combination therapy with immuno-oncology (I-O) agents. Tumor-bearing mice (CT26, MC38, and MCA-205) receiving pegzilarginase in combination with anti–PD-L1 or agonist anti-OX40 experienced significantly increased survival relative to animals receiving I-O monotherapy. Combination pegzilarginase/immunotherapy induced robust antitumor immunity characterized by increased intratumoral effector CD8+ T cells and M1 polarization of tumor-associated macrophages. Our data suggest potential mechanisms of synergy between pegzilarginase and I-O agents that include increased intratumoral MHC expression on both antigen-presenting cells and tumor cells, and increased presence of M1-like antitumor macrophages. These data support the clinical evaluation of I-O agents in conjunction with pegzilarginase for the treatment of patients with cancer.

Abstract MicroRNA (miRNA) mimics have emerged as a novel class of therapeutics with promising anti-oncogenic activity. These mimics are modeled after naturally occurring tumor suppressor miRNAs that are ubiquitously expressed in normal cells but frequently show a loss-of-function in human malignancies. The premise for the strong inhibitory activity is based on the observation these endogenous miRNAs control multiple oncogenic pathways commonly deregulated in cancer. Therefore, “miRNA replacement therapy” acts in accordance with our current understanding of cancer as a pathway disease that can only be successfully treated when intervening with multiple cancer pathways. We have identified a series of key tumor suppressor miRNAs, including miR-34, and validated the therapeutic potential in cultured cancer cells and mouse models of cancer. The translation of this potential into future medicines, however, was hampered by the lack of a robust clinically relevant delivery system. To facilitate a rapid route to the clinic, we have screened a panel of external delivery systems that are in pre-clinical development or have already reached the clinic featuring another oligonucleotide. Here, we present the pharmacologic and pharmacodynamic parameters of miRNA mimics complexed in ionizable NOV340 liposomes (SMARTICLEs, Marina Biotech, Bothell, WA) in an orthotopic tumor model of hepatocellular carcinoma. Treatment of mice carrying existing tumors mimics led to significant tumor regression, prolonged survival and lacked notable drug-related side effects. Some of the mice appeared to be tumor-free. The data demonstrate the therapeutic utility of the NOV340/miRNA formulation and support the initiation of IND-enabling studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5636. doi:1538-7445.AM2012-5636

Abstract Tumors unable to synthesize L-Arginine (arginine) due to decreased expression of enzymes of the arginine biosynthetic pathway show increased sensitivity to arginine depletion. Pegzilarginase (AEB1102) is a bioengineered human PEGylated arginase 1 with enhanced pharmacological properties that enables marked depletion of arginine in plasma and slows tumor growth in pre-clinical in vivo models. Extracellular depletion of arginine directly affects tumor cells, increasing protein turnover, inhibiting proliferation, inducing apoptosis, and increasing autophagy (PMID:27109103); however, the impact of arginine availability on tumor immunogenicity has not been clearly established. Depletion of extracellular arginine induces autophagy, and given the known relationship between autophagy and antigen processing for MHC presentation (PMID:29058602) we hypothesized that pegzilarginase could trigger an enhanced recruitment of immune cells to the tumor microenvironment. Pegzilarginase, both as monotherapy or in combination with anti-PD-L1 mAb (10F.9G2), was administered to Balb/c mice bearing palpable, syngeneic, subcutaneous CT26 tumors. At pre-determined time points, tumor measurements were taken and tumor cell viability and immunophenotyping were assessed via flow cytometry. Treatment with both pegzilarginase or anti-PD-L1 mAb alone slowed tumor growth compared to control. Combination treatment of pegzilarginase and anti-PD-L1 mAb resulted in an enhancement of anti-tumor activity with 12.5% complete response (CR) observed in the anti-PD-L1 mAb monotherapy group and 25% CR observed in the combination therapy group. All mice with CR were re-challenged with fresh CT26 cells and failed to develop new tumors, consistent with induction of an immune memory response. We observed a decrease in tumor cell viability in all treatment groups on days 7 and 17, with the combination treatment group showing the greatest reduction in viable cells and tumor volume at day 17. The observed anti-tumor activity in monotherapy and combination therapy groups was accompanied by an increase in CD45+ tumor-infiltrating cells, with the combination therapy group showing the highest proportion of CD45+ tumor-infiltrating cells, including total T cells, macrophages and dendritic cells, and an increase in serum IFN-γ. Combination of pegzilarginase and anti-PD-L1 mAb results in synergistically greater anti-tumor activity than either monotherapy, and is accompanied by an increase in tumor-infiltrating immune cells. The enhanced infiltration of immune cells into tumor following depletion of arginine with pegzilarginase monotherapy and combination therapy challenges some of the prevailing theories on the role of arginine in immune cell signaling and cancer biology. Citation Format: Giulia Agnello, Mark D. Badeaux, Danlee Enzler, Leslie Priddy, Jason F. Wiggins, Christopher L. Daige, Scott W. Rowlinson. Depletion of blood arginine with pegzilarginase (AEB1102) in combination with anti-PD-L1 increases tumor infiltration by immune cells and enhances antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 869.