Martin Koser

Evaluation of Efficacy, Biodistribution, and Inflammation for a Potent siRNA Nanoparticle: Effect of Dexamethasone Co-treatment

Despite recent progress, systemic delivery remains the major hurdle for development of safe and effective small inhibitory RNA (siRNA)-based therapeutics. Encapsulation of siRNA into liposomes is a promising option to overcome obstacles such as low stability in serum and inefficient internalization by target cells. However, a major liability of liposomes is the potential to induce an acute inflammatory response, thereby increasing the risk of numerous adverse effects. In this study, we characterized a liposomal siRNA delivery vehicle, LNP201, which is capable of silencing an mRNA target in mouse liver by over 80%. The biodistribution profile, efficacy after single and multiple doses, mechanism of action, and inflammatory toxicity are characterized for LNP201. Furthermore, we demonstrate that the glucocorticoid receptor (GR) agonist dexamethasone (Dex) inhibits LNP201-induced cytokine release, inflammatory gene induction, and mitogen-activated protein kinase (MAPK) phosphorylation in multiple tissues. These data present a possible clinical strategy for increasing the safety profile of siRNA-based drugs while maintaining the potency of gene silencing.

Quantitative evaluation of siRNA delivery in vivo

Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem-loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design.

Inhibition of Glycolate Oxidase With Dicer-substrate siRNA Reduces Calcium Oxalate Deposition in a Mouse Model of Primary Hyperoxaluria Type 1

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive, metabolic disorder caused by mutations of alanine-glyoxylate aminotransferase (AGT), a key hepatic enzyme in the detoxification of glyoxylate arising from multiple normal metabolic pathways to glycine. Accumulation of glyoxylate, a precursor of oxalate, leads to the overproduction of oxalate in the liver, which accumulates to high levels in kidneys and urine. Crystalization of calcium oxalate (CaOx) in the kidney ultimately results in renal failure. Currently, the only treatment effective in reduction of oxalate production in patients who do not respond to high-dose vitamin B6 therapy is a combined liver/kidney transplant. We explored an alternative approach to prevent glyoxylate production using Dicer-substrate small interfering RNAs (DsiRNAs) targeting hydroxyacid oxidase 1 (HAO1) mRNA which encodes glycolate oxidase (GO), to reduce the hepatic conversion of glycolate to glyoxylate. This approach efficiently reduces GO mRNA and protein in the livers of mice and nonhuman primates. Reduction of hepatic GO leads to normalization of urine oxalate levels and reduces CaOx deposition in a preclinical mouse model of PH1. Our results support the use of DsiRNA to reduce liver GO levels as a potential therapeutic approach to treat PH1.

Effect of biological matrix and sample preparation on qPCR quantitation of siRNA drugs in animal tissues.

INTRODUCTION Quantitative pharmacokinetic measurement of short nucleotide sequences in animal tissues is critical to the successful development of siRNA-based drugs. Stem-loop qRT-PCR is a sensitive and precise methodology, but the effect of biological matrix and purity of the input sample has yet to be investigated. RESULTS The impact of lipid encapsulation, siRNA chemical modification and purity of the biological matrix on the stem-loop qRT-PCR assay was investigated. A comparison of siRNA standard curves in mouse liver homogenates before and after isolation of total RNA uncovered the potential for erroneous measurement due to significant loss of siRNA on purification columns. Recovery of chemically stabilized siRNA was improved by omission of the DNAse I digestion during RNA isolation. The stem-loop qRT-PCR method demonstrated excellent sensitivity and efficiency in mouse liver homogenates, plasma and whole blood. An optimized protocol based on these findings was used to quantitate siRNA in tissues after dosing mice with two different lipid nanoparticle formulations containing siRNA payloads. CONCLUSIONS Assay of crude homogenates, whole blood or plasma is more accurate, less resource intensive and more amenable to clinical translation than measurement of column-purified total RNA.

Direct Pharmacological Inhibition of β-Catenin by RNA Interference in Tumors of Diverse Origin.

The Wnt/β-catenin pathway is among the most frequently altered signaling networks in human cancers. Despite decades of preclinical and clinical research, efficient therapeutic targeting of Wnt/β-catenin has been elusive. RNA interference (RNAi) technology silences genes at the mRNA level and therefore can be applied to previously undruggable targets. Lipid nanoparticles (LNP) represent an elegant solution for the delivery of RNAi-triggering oligonucleotides to disease-relevant tissues, but have been mostly restricted to applications in the liver. In this study, we systematically tuned the composition of a prototype LNP to enable tumor-selective delivery of a Dicer-substrate siRNA (DsiRNA) targeting CTNNB1, the gene encoding β-catenin. This formulation, termed EnCore-R, demonstrated pharmacodynamic activity in subcutaneous human tumor xenografts, orthotopic patient-derived xenograft (PDX) tumors, disseminated hematopoietic tumors, genetically induced primary liver tumors, metastatic colorectal tumors, and murine metastatic melanoma. DsiRNA delivery was homogeneous in tumor sections, selective over normal liver and independent of apolipoprotein-E binding. Significant tumor growth inhibition was achieved in Wnt-dependent colorectal and hepatocellular carcinoma models, but not in Wnt-independent tumors. Finally, no evidence of accelerated blood clearance or sustained liver transaminase elevation was observed after repeated dosing in nonhuman primates. These data support further investigation to gain mechanistic insight, optimize dose regimens, and identify efficacious combinations with standard-of-care therapeutics. Mol Cancer Ther; 15(9); 2143–54. ©2016 AACR.

Dose-dependent effects of siRNA-mediated inhibition of SCAP on PCSK9, LDLR and plasma lipids in mouse and rhesus monkey

SREBP cleavage-activating protein (SCAP) is a key protein in the regulation of lipid metabolism and a potential target for treatment of dyslipidemia. SCAP is required for activation of the transcription factors SREBP-1 and -2. SREBPs regulate the expression of genes involved in fatty acid and cholesterol biosynthesis, and LDL-C clearance through the regulation of LDL receptor (LDLR) and PCSK9 expression. To further test the potential of SCAP as a novel target for treatment of dyslipidemia, we used siRNAs to inhibit hepatic SCAP expression and assess the effect on PCSK9, LDLR, and lipids in mice and rhesus monkeys. In mice, robust liver Scap mRNA knockdown (KD) was achieved, accompanied by dose-dependent reduction in SREBP-regulated gene expression, de novo lipogenesis, and plasma PCSK9 and lipids. In rhesus monkeys, over 90% SCAP mRNA KD was achieved resulting in approximately 75, 50, and 50% reduction of plasma PCSK9, TG, and LDL-C, respectively. Inhibition of SCAP function was demonstrated by reduced expression of SREBP-regulated genes and de novo lipogenesis. In conclusion, siRNA-mediated inhibition of SCAP resulted in a significant reduction in circulating PCSK9 and LDL-C in rodent and primate models supporting SCAP as a novel target for the treatment of dyslipidemia.

β-catenin mRNA silencing and MEK inhibition display synergistic efficacy in preclinical tumor models

Colorectal carcinomas harbor well-defined genetic abnormalities, including aberrant activation of Wnt/β-catenin and MAPK pathways, often simultaneously. Although the MAPK pathway can be targeted using potent small-molecule drugs, including BRAF and MEK inhibitors, β-catenin inhibition has been historically challenging. RNAi approaches have advanced to the stage of clinical viability and are especially well suited for transcriptional modulators, such as β-catenin. In this study, we report therapeutic effects of combined targeting of these pathways with pharmacologic agents. Using a recently described tumor-selective nanoparticle containing a β-catenin–targeting RNAi trigger, in combination with the FDA-approved MEK inhibitor (MEKi) trametinib, we demonstrate synergistic tumor growth inhibition in in vivo models of colorectal cancer, melanoma, and hepatocellular carcinoma. At dose levels that were insufficient to significantly impact tumor growth as monotherapies, combination regimens resulted in synergistic efficacy and complete tumor growth inhibition. Importantly, dual MEKi/RNAi therapy dramatically improved survival of mice bearing colorectal cancer liver metastases. In addition, pharmacologic silencing of β-catenin mRNA was effective against tumors that are inherently resistant or that acquire drug-induced resistance to trametinib. These results provide a strong rationale for clinical evaluation of this dual-targeting approach for cancers harboring Wnt/β-catenin and MAPK pathway mutations. Mol Cancer Ther; 17(2); 544–53. ©2017 AACR.

Abstract B20: EnCore-LNP mediated tumor delivery of MYC and CTNNB1 Dicer Substrate RNAs (DsiRNAs)

MYC and CTNNB1 are well-characterized drivers of numerous tumor types. Human and preclinical genetic evidence suggest that pharmacological intervention to reduce transactivation of MYC and CTNNB1-regulated genes would yield therapeutic benefit to many cancer patients. Since the proteins encoded by these genes are challenging to target via conventional modalities, progress in new therapeutic agents has been slow despite decades of research. RNA interference technology has enabled the inhibition of previously-undruggable genetic targets at the mRNA level, and has advanced to clinical development for several indications. DCR-MYC is a Phase I-stage lipid nanoparticle (LNP)-formulated Dicer substrate siRNA (DsiRNA), representing a potent class of RNAi triggers being developed by Dicerna Pharmaceuticals. Here we describe new preclinical data that increase our understanding of the parameters that impact tumor delivery and activity of DsiRNA. We demonstrate that the cationic lipid and PEG-lipid components of Dicerna9s unique EnCore LNP platform can be modulated to improve delivery of DsiRNA to both orthotopic and spontaneous liver tumors, as well as xenograft tumors of diverse non-hepatic tissue origin. Characterization of LNP formulations with respect to plasma PK, tissue exposure and target mRNA knockdown was employed towards understanding the pharmacology of LNP-mediated tumor delivery. Citation Format: Marc Abrams, Shanthi Ganesh, Bo Ying, Girish Chopda, Utsav Saxena, Anee Shah, Martin Koser, Rokhand Arvan, Dongyu Chen, Serena Shui, Rohan Diwanji, Wei Zhou, Benjamin Holmes, Boyoung Kim, Hailin Yang, Mihir Patel, Wendy Cyr, Wendy Cyr, Natalie Pursell, Nicole Avitahl-Curtis, Hank Dudek, Cheng Lai, Weimin Wang, Bob D. Brown. EnCore-LNP mediated tumor delivery of MYC and CTNNB1 Dicer Substrate RNAs (DsiRNAs). [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B20.

Specific Inhibition of Hepatic Lactate Dehydrogenase Reduces Oxalate Production in Mouse Models of Primary Hyperoxaluria

Primary hyperoxalurias (PHs) are autosomal recessive disorders caused by the overproduction of oxalate leading to calcium oxalate precipitation in the kidney and eventually to end-stage renal disease. One promising strategy to treat PHs is to reduce the hepatic production of oxalate through substrate reduction therapy by inhibiting liver-specific glycolate oxidase (GO), which controls the conversion of glycolate to glyoxylate, the proposed main precursor to oxalate. Alternatively, diminishing the amount of hepatic lactate dehydrogenase (LDH) expression, the proposed key enzyme responsible for converting glyoxylate to oxalate, should directly prevent the accumulation of oxalate in PH patients. Using RNAi, we provide the first in vivo evidence in mammals to support LDH as the key enzyme responsible for converting glyoxylate to oxalate. In addition, we demonstrate that reduction of hepatic LDH achieves efficient oxalate reduction and prevents calcium oxalate crystal deposition in genetically engineered mouse models of PH types 1 (PH1) and 2 (PH2), as well as in chemically induced PH mouse models. Repression of hepatic LDH in mice did not cause any acute elevation of circulating liver enzymes, lactate acidosis, or exertional myopathy, suggesting further evaluation of liver-specific inhibition of LDH as a potential approach for treating PH1 and PH2 is warranted.

Abstract 3827: Preclinical characterization of DCR-BCAT as a component of combination therapy

Dicer-substrate siRNAs (DsiRNAs) are a potent class of RNA interference (RNAi) triggers capable of silencing any expressed mRNA with high specificity. DCR-MYC, a first-in-class DsiRNA targeting the MYC oncogene is currently in Phase 1b/II clinical trials [ASCO 2015, abstract 11006]. DCR-BCAT is an advanced preclinical development candidate that targets CTNNB1, the gene which encodes β-catenin. The β-catenin/Wnt pathway is consistently activated in human tumors, including >50% of hepatocellular carcinomas (HCC) and >80% of colorectal cancers (CRC). Robust preclinical and genetic evidence strongly suggests that inhibiting β-catenin function would yield broad therapeutic benefit in oncology, but efforts to target it using conventional drug modalities have been unsuccessful to-date. We have previously reported extensive preclinical pharmacology for DCR-BCAT in mouse tumor models of diverse origin. Here, we explore DCR-BCAT as a monotherapy and in combination with both standard-of-care and experimental therapeutics. Interestingly, when mice bearing HCC tumors were treated with a combination of CTNNB1 and MYC DsiRNAs, the antitumor efficacy was additive or synergistic relative to either single agent alone. Additionally, since up to 50% of colorectal tumors have both activated Wnt signaling and mutant KRAS, we also explored combination therapy of DCR-BCAT and FDA-approved MEK inhibitors. A major advantage of DCR-BCAT is that the improved nanoparticle formulation is more selective for siRNA delivery to tumors over liver and other normal tissues. These data support continued development of DCR-BCAT as a first-in-class RNAi therapeutic, and highlight the potential for use in combination with other promising agents. Citation Format: Shanthi Ganesh, Wendy Cyr, Martin Koser, Girish Chopda, Edmond Chipumuro, Zakir Siddiquee, Kevin Craig, Serena Shui, Dongyu Chen, Cheng Lai, Hank Dudek, Weimin Wang, Bob Brown, Marc Abrams. Preclinical characterization of DCR-BCAT as a component of combination therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3827.