MicroRNAs (miRNAs) are a class of small ~22 nt noncoding (nc) RNAs that regulate gene expression post-transcriptionally by direct binding to target sites on mRNAs. They comprise more than 1,000 no...

Introduction: Targeted therapies for aggressive breast cancers like triple negative breast cancer (TNBC) are needed. The use of small interfering RNAs (siRNAs) to disable expression of survival genes provides a tool for killing these cancer cells. Cyclin dependent kinase 11 (CDK11) is a survival protein kinase that regulates RNA transcription, splicing and mitosis. Casein kinase 2 (CK2) is a survival protein kinase that suppresses cancer cell death. Eliminating the expression of these genes has potential therapeutic utility for breast cancer. Methods: Expression levels of CDK11 and CK2 mRNAs and associated proteins were examined in breast cancer cell lines and tissue arrays. RNA expression levels of CDC2L1, CDC2L2, CCNL1, CCNL2, CSNK2A1, CSNK2A2, and CSNK2B genes in breast cancer subtypes were analyzed. Effects following transfection of siRNAs against CDK11 and CK2 in cultured cells were examined by viability and clonal survival assays and by RNA and protein measures. Uptake of tenfibgen (TBG) nanocapsules by TNBC cells was analyzed by fluorescence-activated cell sorting. TBG nanocapsules delivered siRNAs targeting CDK11 or CK2 in mice carrying TNBC xenograft tumors. Transcript cleavage and response parameters were evaluated. Results: We found strong CDK11 and CK2 mRNA and protein expression in most human breast cancer cells. Immunohistochemical analysis of TNBC patient tissues showed 100% of tumors stained positive for CDK11 with high nuclear intensity compared to normal tissue. The Cancer Genome Atlas analysis comparing basal to other breast cancer subtypes and to normal breast revealed statistically significant differences. Down-regulation of CDK11 and/or CK2 in breast cancer cells caused significant loss of cell viability and clonal survival, reduced relevant mRNA and protein expression, and induced cell death changes. TBG nanocapsules were taken up by TNBC cells both in culture and in xenograft tumors. Treatment with TBG- siRNA to CDK11 or TBG- siRNA to CK2αα’ nanocapsules induced appropriate cleavage of CDK11 and CK2α transcripts in TNBC tumors, and caused MDA-MB-231 tumor reduction, loss of proliferation, and decreased expression of targeted genes. Conclusions: CDK11 and CK2 expression are individually essential for breast cancer cell survival, including TNBC. These genes serve as promising new targets for therapeutic development in breast cancer.

Liver sinusoidal endothelial cells are a major endogenous source of Factor VIII (FVIII), lack of which causes the human congenital bleeding disorder hemophilia A. Despite extensive efforts, gene therapy using viral vectors has shown little success in clinical hemophilia trials. Here we achieved cell type–specific gene targeting using hyaluronan- and asialoorosomucoid-coated nanocapsules, generated using dispersion atomization, to direct genes to liver sinusoidal endothelial cells and hepatocytes, respectively. To highlight the therapeutic potential of this approach, we encapsulated Sleeping Beauty transposon expressing the B domain–deleted canine FVIII in cis with Sleeping Beauty transposase in hyaluronan nanocapsules and injected them intravenously into hemophilia A mice. The treated mice exhibited activated partial thromboplastin times that were comparable to those of wild-type mice at 5 and 50 weeks and substantially shorter than those of untreated controls at the same time points. Further, plasma FVIII activity in the treated hemophilia A mice was nearly identical to that in wild-type mice through 50 weeks, while untreated hemophilia A mice exhibited no detectable FVIII activity. Thus, Sleeping Beauty transposon targeted to liver sinusoidal endothelial cells provided long-term expression of FVIII, without apparent antibody formation, and improved the phenotype of hemophilia A mice.

The main strategy of gene therapy has traditionally been focused on gene augmentation. This approach typically involves the introduction of an expression system designed to express a specific protein in the transfected cell. Both the basic and clinical sciences have generated enough information to suggest that gene therapy would eventually alter the fundamental practice of modern medicine. However, despite progress in the field, widespread clinical applications and success have not been achieved. The myriad deficiencies associated with gene augmentation have resulted in the development of alternative approaches to treat inherited and acquired genetic disorders. One, derived primarily from the pioneering work of homologous recombination, is gene repair. Simply stated, the process involves targeting the mutation in situ for gene correction and a return to normal gene function. Site-specific genetic repair has many advantages over augmentation although it too is associated with significant limitations. This review outlines the advantages and disadvantages of gene correction. In particular, we discuss technologies based on chimeric RNA/DNA oligonucleotides, single-stranded and triplex-forming oligonucleotides, and small fragment homologous replacement. While each of these approaches is different, they all share a number of common characteristics, including the need for efficient delivery of nucleic acids to the nucleus. In addition, we review the potential application of a novel and exciting nonviral gene augmentation strategy--the Sleeping Beauty transposon system.

Liver regeneration after 70% partial hepatectomy (PH) in rats induces >95% of hepatocytes to undergo two rounds of semisynchronous cell replication. Gene expression is controlled primarily by posttranscriptional processing, including changes in mRNA stability. However, the translational activity of a specific mRNA can also be modulated after PH, resulting in significant uncoupling of protein and transcript levels relative to quiescent liver for many genes including c-myc and p53. Although the precise mechanism by which this uncoupling occurs is unknown, the polysomal association of mRNA and microRNA (miRNA) can significantly modulate rate of decay as well as translational activity. Thus we characterized the association of c-myc and p53 mRNAs and miRNAs in free and cytoskeleton- and membrane-bound polysome populations 3, 6, and 24 h after PH. The transcripts for c-myc and p53 were differentially distributed in the three discrete polysome populations, and this was dramatically modulated during liver regeneration. Nascent polysome-associated p53 and c-myc proteins were also differentially expressed in the free and cytoskeleton- and membrane-bound polysomes and significantly uncoupled from transcript levels relative to nonresected liver. At least 85 miRNAs were associated with the three polysome populations, and their abundance and distribution changed significantly during liver regeneration. These data suggest that posttranscriptional control of c-myc and p53 protein expression is associated with the translocation of transcripts between the different polyribosomes. The alteration of expression for the same transcript in different polysome populations may, in part, be due to the action of miRNAs.

MicroRNAs (miRNAs) are a class of approximately 22 nucleotide long, widely expressed RNA molecules that play important regulatory roles in eukaryotes. To investigate miRNA function, it is essential that methods to quantify their expression levels be available.