rgls4326 confers efficacy and modulate aberrant...

RGLS4326 confers efficacy and modulate aberrant signaling and metabolic pathways

in PKD mouse modelsTania M. Valencia1,*, Sole Gatto1, Annelie E. Schairer1, Steven Lockton1, Michael Kim1, Kara Kersjes1,

Jian Li1, Andrea N. Flaten2, Vishal Patel2, Edmund C. Lee1

1Regulus Therapeutics Inc., San Diego, California. *Presenting author2Department of Internal Medicine and Division of Nephrology, University of Texas Southwestern Medical Center, Dallas, Texas.

Background:

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2

genes, where expansion of fluid-filled cysts and renal fibrosis often leads to end-stage renal disease.

MicroRNAs are short non-coding RNAs that modulate several biological processes. We have previously

shown that aberrant expression of miR-17 family of microRNAs is involved in human ADPKD

pathogenesis. RGLS4326 is a chemically-modified oligonucleotide designed to sterically inhibit miR-17

functions and has been shown to reduce cyst growth in vitro and in vivo. The goal of this study was to

determine the signaling pathways modulated by RGLS4326 treatment in PKD mouse models

Methods:

RGLS4326 is efficacious in the Pkd2KO and Pcy/CD-1 mouse models of PKD. We performed RNA

sequencing and metabolite profiling using kidney samples from both mouse models following RGLS4326

treatment. Ingenuity Pathway Analysis was used to provide novel insights into signaling pathways

modulated by RGLS4326 treatment

Results:

Through RNA sequencing, we identified >1000 differentially expressed genes in the PKD kidney samples

compared to their age- and strain-matched normal controls. Comparative pathway analysis identified

several dysregulated signaling pathways in the two PKD mouse models, including the Pparα, WNT/ β-

catenin and PCP signaling, that were in turn modulated following RGLS4326 treatment. Next, we

performed kidney global metabolite profiling comparing Pkd2KO and normal kidneys, and identified

several biochemical alterations in the Pkd2KO model, including substantial changes in lipid metabolism.

In particular, decrease in β-fatty acid oxidation pathway was observed in the Pkd2KO kidneys, which

corroborates with previously observed Pparα-dysregulation in PKD mouse models

Conclusion: Our results indicate that RGLS4326 confers efficacy and modulates aberrant PKD signaling

and metabolic pathways. These results support the clinical development of RGLS4326 for the treatment

of ADPKD

Abstract RNA Seq differential expression analysis in efficacy models Kidney global metabolite profiling

RGLS4326 is efficacious in Pkd2KO and Pcy/CD-1 mouse models

(A) Decrease in Kidney-Weight-to-Body-Weight (KW/BW) in the Pkd2KO efficacy model by

subcutaneous administration of RGLS4326 at 20 mg/kg on days P11, P12 and P13 and P19; Kidneys

were collected at P28. (B) Reduction in proliferation after RGLS4326 treatment as shown by phospho-

histone3 staining in the Pkd2KO model. (C) Decrease in KW/BW in the Pcy/CD-1 efficacy model

following Q4W dose of RGLS4326 at 25 mg/kg from 5 to 30 weeks of age. (D) Reduction of cyst index

after RGLS4326 treatment in the Pcy/CD-1 efficacy model

Global metabolic profile: Pkd2KO vs WT

Lipid Metabolism

Glucose Metabolism

Sphingolipid Metabolism

Purine Metabolism

NAD+ Metabolism

Nucleotide Changes

TCA Intermediates

BCAA Catabolism

Microbially-Associated

401 363

Lipid metabolism in the kidney: Pkd2KO vs WT

Dysregulated metabolism

Comparative pathway analysis –Pkd2KO and Pcy efficacy models

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T h y ro id H o rm o n e M e ta b o l ism I I (v ia C o n ju g a tio n a n d /o r D e g ra d a tio n )

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Retinoate Biosynthesis I

Pancreatic Adenocarcinoma Signaling

Wnt/b-catenin Signaling

Planar Cell Polarity (PCP) Pathway

LPS/IL-1 Mediated inhibition of RXR Function

Neuroprotective role of THOP1 in Alzheimer’s Disease

Leukocyte Extravasation Signaling

Basal Cell Carcinoma Signaling

Colorectal Cancer Metastasis Signaling P

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Ingenuity pathway analysis of the DE genes from Pkd2KO and Pcy/CD-1 efficacy models

(A) Comparative pathway analysis revealed that dysregulated pathways in the disease models (when

compared to age matched WT controls) are restored upon RGLS4326 treatment in both models

(B) Genes from selected dysregulated pathways identified in IPA comparative analysis were assessed by

qPCR in both models

References

Altered lipid metabolism noted in Pkd2KO kidney reflected the differential uptake, synthesis or utilization

of lipids. Several lipid classes were significantly decreased in Pkd2KO vs WT including monoacylglyerols

(MAG), long chain fatty acids (LCFA), polyunsaturated fatty acids (PUFA), endocannabinoids and

lysolipids. Preliminary data demonstrated that treatment with RGLS4326 modulated some dysregulated

metabolites.

Metabolic profiles between disease Pkd2KO and healthy WT kidney displayed significant differences in

metabolites (401 upregulated and 363 downregulated) across all biochemical nodes, indicating a broadly

altered metabolism in the disease kidney such as glucose, lipid, sphingolipids, Purine, NAD+ and amino

acids metabolism

RNAseq differential expression (DE) analysis of Pkd2KO and Pcy/CD-1 efficacy models. DE genes

upregulated by disease and downregulated by RGLS4326 treatment and vice-versa for the Pkd2KO (A)

and Pcy/CD-1 (B) efficacy models. Criteria FDR<0.05, Log2FC>0.5 in disease and <-0.5 in RGLS4326

vs PBS or Log2FC<-0.5 in disease and >0.5 in RGLS4326 vs PBS.

Kidney expression of selected genes following RLGS4326 treatment in

Pkd2KO and Pcy efficacy models

• Selected genes identified key PKD-disease related pathways in Pkd2KO (A) and Pcy/CD-1 (B) and

their modulation after RGLS4326 treatment

• Upregulation of Ppara upon RGLS4326 treatment in the Pkd2KO model corroborates with previous

findings where genetic deletion of miR17-92 cluster in the Pkd2KO kidney restores the de-repressed

Ppara pathway (Hajarnis et al. 2017)

• Overexpression of claudins in ADPKD patients compared to normal tissue has been reported

previously (Almeida et al. 2016). Consistently we detected elevated levels of claudins in disease

kidney and downregulation following RGLS4326 treatment in both efficacy models

• Aberrant regulation of PCP and Wnt signaling in Pkd1 Knockout cystic kidneys has been already

described (Luyten et al. 2010; Quin et al. 2012). In agreement with this, we also observed the

overexpression of Wnt7a and Wnt7b in Pkd2KO and Pcy mouse models, where their levels were

significantly downregulated after RGLS4326 treatment

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miRNA targets multiple signaling pathways

Repression of target mRNAs

by miRNAs

De-repression of target mRNAs

by Anti-miRNAsRegulation of multiple

Targets & Pathways

994 genes 658 genes

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Metabolyzer™

Eliminate Noise

Data Reduction

Library Search

RT, Mass, MS/MS

Biochemical

Extraction

UHPLC (HILIC)-MS/MS

UHPLC-MS/MS (+ESI) 2

UHPLC-MS/MS (+ESI) 1

UHPLC-MS/MS (-ESI)

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List of selected genes in

dysregulated pathways

51 209

A C

B D

A B

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Lipid Metabolites

Kidney Metabolites

Activated in disease

Repressed in disease

Upregulated in disease Upregulated in disease

Downregulated in disease Downregulated in disease

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• Almeida RMC., Clendenon SG., Richards WG., Boedigheimer M., Damore M., Rossetti S., Harris PC., Herbert B., Xu WM.,

Wandinger-Ness A., Wrd HH., Glazier JA., Bacallao RL., 2016. Transcriptome analysis reveals manifold mechanisms of cyst

development in ADPKD. Hum Genomics, 10: 37.

• Hajarnis S., Lakhia R., Yheskel M., Williams D., Sorourian M., Lui X., Aboudehen K., Zhang S., Kersjes K., Galasso R., Li J.,

Kimal V., Lockton S., Davis S., Flaten A., Johnson JA., Holland WL., Kusminski CM., Scherer PE., Harris PC., Trudel M., Wallace

DP., Igarashi P., Lee EC., Androsavich JR., Patel V., 2017. microRNA-17 family promotes polycystic kidney disease progression

through modulation of mitochondrial metabolism. Nature comm. 8: 14395.

• Luyten A., Su X., Gondela S., Chen Y., Rompani S., Takakura A., Zhou J., 2010. Aberrant regulation of Planar cell polarity in

polycystic kidney disease. J am Soc Nephrol, 21(9): 1521-1532.

• Quin S, Taglienti M., Cai L., Zhou J., Kreidberg JA., 2012. c-Met and NF-κB–Dependent overexpression of Wnt7a and -7b and

Pax2 promotes cystogenesis in polycystic kidney disease. J Am Soc Nephrol, 23(8): 1309-1318.

PBSPBS

PBS

PBS

CD-1

CD-1

WT

A B

Analysis

American Society of Nephrology - Kidney Week, San Diego, CA (Oct 23 - 28, 2018)Abstract Number: 3021851; Poster Board Number: TH-PO687

rgls4326 confers efficacy and modulate aberrant...

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