title: Enhancing potency of siRNA targeting fusion genes by optimization outside of target sequence.
abstract: Canonical siRNA design algorithms have become remarkably effective at predicting favorable binding regions within a target mRNA, but in some cases (e.g., a fusion junction site) region choice is restricted. In these instances, alternative approaches are necessary to obtain a highly potent silencing molecule. Here we focus on strategies for rational optimization of two siRNAs that target the junction sites of fusion oncogenes BCR-ABL and TMPRSS2-ERG. We demonstrate that modifying the termini of these siRNAs with a terminal G-U wobble pair or a carefully selected pair of terminal asymmetry-enhancing mismatches can result in an increase in potency at low doses. Importantly, we observed that improvements in silencing at the mRNA level do not necessarily translate to reductions in protein level and/or cell death. Decline in protein level is also heavily influenced by targeted protein half-life, and delivery vehicle toxicity can confound measures of cell death due to silencing. Therefore, for BCR-ABL, which has a long protein half-life that is difficult to overcome using siRNA, we also developed a nontoxic transfection vector: poly(lactic-coglycolic acid) nanoparticles that release siRNA over many days. We show that this system can achieve effective killing of leukemic cells. These findings provide insights into the implications of siRNA sequence for potency and suggest strategies for the design of more effective therapeutic siRNA molecules. Furthermore, this work points to the importance of integrating studies of siRNA design and delivery, while heeding and addressing potential limitations such as restricted targetable mRNA regions, long protein half-lives, and nonspecific toxicities.
authors.affiliation: Department of Cellular & Molecular Physiology, Yale University, New Haven, CT 06511; Department of Biomedical Engineering, Yale University, New Haven, CT 06511.
authors.forename: Kseniya
authors.initials: K
authors.lastname: Gavrilov
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, CT 06511.
authors.forename: Young-Eun
authors.initials: YE
authors.lastname: Seo
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, CT 06511.
authors.forename: Gregory T
authors.initials: GT
authors.lastname: Tietjen
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, CT 06511.
authors.forename: Jiajia
authors.initials: J
authors.lastname: Cui
authors.affiliation: Department of Biomedical Engineering, Yale University, New Haven, CT 06511.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Cheng
authors.affiliation: Department of Cellular & Molecular Physiology, Yale University, New Haven, CT 06511; Department of Biomedical Engineering, Yale University, New Haven, CT 06511 mark.saltzman@yale.edu.
authors.forename: W Mark
authors.initials: WM
authors.lastname: Saltzman
mesh_terms: D017209:Apoptosis; D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D002470:Cell Survival; D016503:Drug Delivery Systems; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D015973:Gene Expression Regulation, Leukemic; D018390:Gene Targeting / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D020014:K562 Cells; D019344:Lactic Acid / Q000737:chemistry; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000378:metabolism; D053758:Nanoparticles; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D011100:Polyglycolic Acid / Q000737:chemistry; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D014162:Transfection
chemical_list: C581634:BCR-ABL1 fusion protein, human; D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C515553:TMPRSS2-ERG fusion protein, human; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D016044:Fusion Proteins, bcr-abl
keywords: BCR-Abl; RNA interference; TMPRSS2-ERG; drug delivery; leukemia