Glycogen 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. We show that a single systemic injection of wild-type or native human G6PC mRNA results in significant improvements in fasting blood glucose levels for up to 7 days post-dose. These changes were associated with significant reductions in liver mass, hepatic G6P, glycogen, and triglycerides. In addition, an engineered protein variant of human G6Pase, designed for increased duration of expression, showed superior efficacy to the wild-type sequence by maintaining improved fasting blood glucose levels and reductions in liver mass for up to 12 days post-dose. Our results demonstrate for the first time the effectiveness of mRNA therapy as a potential treatment in reversing the hepatic abnormalities associated with GSD1a.
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Roseman et al. demonstrate that mRNA therapy could be a potential treatment to reverse hepatic abnormalities associated with GSD1a. The authors engineered G6PC protein to increase duration of protein expression, and mRNA-encoded G6PC enabled hepatic glucose secretion and reduction of liver glycogen, G6P, triglycerides, and liver weight in a GSD1a model.
mRNA 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. The current studies describe and validate screening methods using a diverse set of 5′UTR and 3′UTR combinations for improved expression of the Arginase 1 (ARG1) protein, a potential therapeutic mRNA target. Data revealed a number of critical aspects which need to be considered when developing a screening approach for engineering mRNA improvements. First, plasmid-based screening methods do not correlate with protein expression driven by exogenously expressed mRNA. Second, improved ARG1 protein production was driven by increased translation and not improved mRNA stability. Finally, the 5′ UTR appears to be the key driver in protein expression for exogenously delivered mRNA. From the testing of the combinatorial library, the 5′UTR for complement factor 3 (C3) and cytochrome p4502E1 (CYP2E1) showed the largest and most consistent increase in protein expression relative to a reference UTR. Collectively, these data provide important information for the development and optimization of therapeutic mRNAs.
Thrombotic 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. With technical breakthroughs in exploring mRNA as therapeutics, we hypothesized that restoration of ADAMTS13 activity for a prolonged duration of time can be achieved through systemic dosing of mRNA, wherein the dosed mRNA would utilize hepatic cells as bioreactors for continuous production of ADAMTS13. To test this hypothesis, mRNA encoding human ADAMTS13 WT or an ADAMTS13 variant, that had demonstrated resistance to predominant clinical TTP autoantibodies, was formulated in lipid nano-particles for liver-targeted delivery. In both ADAMTS13-sufficient and -deficient mice, a single dose of the formulated mRNAs at 1 mg/kg resulted in expression of hADAMTS13 at or above therapeutically relevant levels in mice for up to five days. This proof-of-concept study suggests that mRNA therapy could provide a novel approach for TTP treatment.
Small 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. We demonstrate that siRNA off-targets can be significantly reduced when cells are treated with a dose of siRNA that is relatively low (e.g. 1 nM), but sufficient to effectively silence the intended target. The reduction in off-targets was demonstrated for both modified and unmodified siRNAs that targeted either STAT3 or hexokinase II. Low concentrations reduced silencing of transcripts with complementarity to the seed region of the siRNA. Similarly, off-targets that were not complementary to the siRNA were reduced at lower doses, including up-regulated genes that are involved in immune response. Importantly, the unintended induction of caspase activity following treatment with a siRNA that targeted hexokinase II was also shown to be a concentration-dependent off-target effect. We conclude that off-targets and their related phenotypic effects can be reduced for certain siRNA that potently silence their intended target at low concentrations.
Arginase 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. 75% of patients whose symptoms can be managed through diet therapy continue to suffer neuro-cognitive deficits. In our research, we demonstrate in vitro and in vivo that administration of ARG1 mRNA increased ARG1 protein expression and specific activity in relevant cell types, including ARG1-deficient patient cell lines, as well as in wild type mice for up to 4 days. These studies demonstrate that ARG1 mRNA treatment led to increased functional protein expression of ARG1 and subsequently an increase in urea. Hence, ARG1 mRNA therapy could be a potential treatment option to develop for patients.
Alpha-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. AAT lung disease is often treated with one of several serum protein replacement products; however, long-term studies of the effectiveness of SerpinA1 replacement therapy are not available, and it does not reduce liver damage in AAT deficiency. mRNA therapy could potentially target both the liver and lungs of AAT deficient patients. AAT patient fibroblasts and AAT patient fibroblast-derived hepatocytes were transfected with SERPINA1-encoding mRNA and cell culture media were tested for SerpinA1 expression. Our data demonstrates increased SerpinA1 protein in culture media from treated AAT patient fibroblasts and AAT patient fibroblast-derived hepatocytes. In vivo studies in wild type mice demonstrate SERPINA1 mRNA biodistribution in liver and lungs, as well as SerpinA1 protein expression in these two target organs which are critically affected in AAT deficiency. Taken together, our data suggests that SerpinA1 mRNA therapy has the potential to benefit patients suffering from AAT deficiency.
The 14-3-3 proteins are a family of conserved regulatory molecules expressed in all eukaryotic cells. A striking feature of the 14-3-3 proteins is their ability to bind a multitude of functionally ...diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors. This plethora of interacting proteins allows 14-3-3 to play important roles in a wide range of vital regulatory processes, such as mitogenic signal transduction, apoptotic cell death, and cell cycle control. In this review, we examine the structural basis for 14-3-3-ligand interactions, proposed functions of 14-3-3 in various signaling pathways, and emerging views of mechanisms that regulate 14-3-3 actions.
Members 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. Previous studies emphasized the importance of Raf-1 and b-Raf in preventing apoptosis in addition to their roles in cell growth. In the present study, we examined whether inhibition of the Raf-1 or b-Raf kinase decreases cell growth and increases apoptosis in colon cancer cells. c-Raf and b-Raf were depleted in colon cancer cell lines, such as HCT116, HT29 and Colo205, containing Ras or b-Raf mutations by RNA interference (RNAi). The results showed that colon cancer cells with activating Ras mutations undergo apoptosis following Raf-1 inhibition, as determined by cell cycle analysis and the release of cytochrome c. Moreover, in b-Raf mutant colon cancers, the inhibition of b-Raf as compared to Raf-1 is crucial for cancer cell death. There is increasing evidence for both MEK-independent Raf signaling and Raf-independent MEK signaling. Thus, we investigated whether targeting multiple points of the mitogen-activated protein kinase (MAPK) pathway with a MEK inhibitor and Raf RNAi increases cancer cell death. The results showed that combination therapy, inhibiting Raf and MEK kinases simultaneously, increased apoptosis in colon cancer cells. Taken together, our data demonstrate that combination therapy targeting the MAPK pathway at two distinct points, Raf kinase and MEK, has greater efficacy in increasing cancer cell death and is likely to improve therapeutic outcomes for patients.
Recent 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. Moreover, using constitutively active versions of Akt1 and Rac1, we show that these downstream pathways of PI3K synergize to achieve anchorage-independent growth. At lower levels of c-
myc expression, activated PI3K also replaces st to complement H
-rasV12 and LT and confers both soft agar growth and tumorigenicity. However, elevated c-
myc expression cannot replace H
-rasV12 for tumorigenesis. These observations begin to define the pathways perturbed during the transformation of HMECs.
The 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. In vivo, MTOs synthesized in both RNase H-activating and steric-blocking oligonucleotide designs provide ≈4-5-fold increased potency and ≈2-fold increased efficacy, suggesting broad therapeutic applications.
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