Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCETM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide.
View Article and Find Full Text PDFPrimary Hyperoxaluria Type 1 (PH1) is an autosomal recessive disorder of glyoxylate metabolism. Loss of alanine glyoxylate aminotransferase (AGT) function to convert intermediate metabolite glyoxylate to glycine causes the accumulation and reduction of glyoxylate to glycolate, which eventually is oxidized to oxalate. Excess oxalate in PH1 patients leads to the formation and deposition of calcium oxalate crystals in the kidney and urinary tract.
View Article and Find Full Text PDFAlpha-1-antitrypsin (AAT) deficiency is a genetic disorder that produces inactive/defective AAT due to mutations in the SERPINA1 gene encoding AAT. This disease is associated with decreased activity of AAT in the lungs and deposition of excessive defective AAT protein in the liver. Currently there is no specific treatment for liver disease associated with AAT deficiency.
View Article and Find Full Text PDFArginase I (ARG1) deficiency is an autosomal recessive urea cycle disorder, caused by deficiency of the enzyme Arginase I, resulting in accumulation of arginine in blood. Current Standard of Care (SOC) for ARG1 deficiency in patients or those having detrimental mutations of ARG1 gene is diet control. Despite diet and drug therapy with nitrogen scavengers, ~25% of patients suffer from severe mental deficits and loss of ambulation.
View Article and Find Full Text PDFThrombotic thrombocytopenic purpura (TTP) is primarily caused by deficiency of ADAMTS13 within the blood stream due to either genetic defects or presence of inhibitory autoantibodies. Preclinical and clinical studies suggest that enzyme replacement therapy with recombinant human ADAMTS13 protein (rhADAMTS13) is effective and safe in treatment of TTP. However, frequent dosing would be required due to the relatively short half-life of rhADAMTS13 in circulation as well as the presence of inhibitory autoantibodies that collectively result in the poor pharmacological profile of rhADAMTS13.
View Article and Find Full Text PDFmRNA based therapies hold great promise for the treatment of genetic diseases. However, this therapeutic approach suffers from multiple challenges including the short half-life of exogenously administered mRNA and subsequent protein production. Modulation of untranslated regions (UTR) represents one approach to enhance both mRNA stability and translation efficiency.
View Article and Find Full Text PDFMessenger RNA (mRNA) is a promising new class of therapeutics that has potential for treatment of diseases in fields such as immunology, oncology, vaccines, and inborn errors of metabolism. mRNA therapy has several advantages over DNA-based gene therapy, including the lack of the need for nuclear import and transcription, as well as limited possibility of genomic integration. One drawback of mRNA therapy, especially in cases such as metabolic disorders where repeated dosing will be necessary, is the relatively short in vivo half-life of mRNA (∼6-12 h).
View Article and Find Full Text PDFGlycogen storage disease type Ia (GSD1a) is an inherited metabolic disorder caused by the deficiency of glucose-6-phosphatase (G6Pase). GSD1a is associated with life-threatening hypoglycemia and long-term liver and renal complications. We examined the efficacy of mRNA-encoding human G6Pase in a liver-specific G6Pase mouse model (L-G6PC) that exhibits the same hepatic biomarkers associated with GSD1a patients, such as fasting hypoglycemia, and elevated levels of hepatic glucose-6-phosphate (G6P), glycogen, and triglycerides.
View Article and Find Full Text PDFLeukocyte infiltration of adventitial and perivascular tissues is an early event in the development of vascular remodeling after injury. We investigated whether Slit/Robo-an axonal chemorepellent system in vertebrate and invertebrate development-is activated during the inflammatory phase that follows endothelial denudation. Using the rat carotid artery model of angioplasty, we conducted a time course analysis of mRNAs encoding Slit ligands (Slit2 and Slit3) and Robo receptors (Robo1, Robo2, and Robo4), as well as proinflammatory cell adhesion molecule (CAM) genes.
View Article and Find Full Text PDFThe in vivo potency of antisense oligonucleotides (ASO) has been significantly increased by reducing their length to 8-15 nucleotides and by the incorporation of high affinity RNA binders such as 2', 4'-bridged nucleic acids (also known as locked nucleic acid or LNA, and 2',4'-constrained ethyl [cET]). We now report the development of a novel ASO design in which such short ASO monomers to one or more targets are co-synthesized as homo- or heterodimers or multimers via phosphodiester linkers that are stable in plasma, but cleaved inside cells, releasing the active ASO monomers. Compared to current ASOs, these multimers and multi-targeting oligonucleotides (MTOs) provide increased plasma protein binding and biodistribution to liver, and increased in vivo efficacy against single or multiple targets with a single construct.
View Article and Find Full Text PDFMembers of the Ras protooncogene family are mutated in approximately 75% of colon cancers. The Raf kinases (Raf-1, b-Raf and a-Raf) directly interact with Ras and serve as mediators of mitogenic signals. Expression of the constitutively active alleles of Raf or Ras gene families results in oncogenesis in a number of model systems.
View Article and Find Full Text PDFInvestigation of therapy naïve human tumor and adjacent normal tissue biopsies demonstrated that expression levels of miRNAs are altered at and between stages of CRC. Targets of these altered miRNAs are members of the Insulin signaling pathways. Phosphorylation states of several molecules in the Insulin signaling pathways were altered between stages of CRC, and significantly the change in molecular phosphorylation state correlated with decreases in specific miRNAs that target them.
View Article and Find Full Text PDFAntisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues.
View Article and Find Full Text PDFSmall interfering RNAs (siRNAs) are routinely used to reduce mRNA levels for a specific gene with the goal of studying its function. Several studies have demonstrated that siRNAs are not always specific and can have many off-target effects. The 3' UTRs of off-target mRNAs are often enriched in sequences that are complementary to the seed-region of the siRNA.
View Article and Find Full Text PDFApolipoprotein CIII (apoCIII), a major constituent of triglyceride-rich lipoprotein, has been proposed as a key contributor to hypertriglyceridemia on the basis of its inhibitory effects on lipoprotein lipase. Many immunochemical methods have been developed for human apoCIII quantification, including ELISA. However, a sensitive and quantitative assay for nonhuman primates is not commercially available.
View Article and Find Full Text PDFPURPOSE: Androgen receptor abundance and androgen receptor-regulated gene expression in castration-recurrent prostate cancer are indicative of androgen receptor activation in the absence of testicular androgen. Androgen receptor transactivation of target genes in castration-recurrent prostate cancer occurs in part through mitogen signaling that amplifies the actions of androgen receptor and its coregulators. Herein we report on the role of 14-3-3eta in androgen receptor action.
View Article and Find Full Text PDFApoptosis signal-regulating kinase 1 (ASK1) is a critical mediator of apoptotic signaling pathways initiated by a variety of death stimuli. Its activity is tightly controlled by various mechanisms such as covalent modification and protein-protein interaction. One of the proteins that control ASK1 function is 14-3-3zeta, a member of the 14-3-3 protein family.
View Article and Find Full Text PDFRecent studies have demonstrated that introduction of hTERT in combination with SV40 large T antigen (LT), small t antigen (st), and H-rasV12 suffices to transform many primary human cells. In human mammary epithelial cells (HMECs) expressing elevated c-Myc, activated H-Ras is dispensable for anchorage-independent growth. Using this system, we show that st activates the PI3K pathway and that constitutive PI3K signaling substitutes for st in transformation.
View Article and Find Full Text PDFDEF-1/ASAP1 is an ADP-ribosylation factor GTPase-activating protein (ARF GAP) that localizes to focal adhesions and is involved in cytoskeletal regulation. In this paper, we use a cell-based ARF GAP assay to demonstrate that DEF-1 functions as a GAP for ARF1 and not ARF6 in vivo. This degree of substrate preference was unique to DEF-1, as other ARF GAP proteins, ACAP1, ACAP2, and ARFGAP1, were able to function on both ARF1 and ARF6.
View Article and Find Full Text PDF