Silence Therapeutics : SLN124, a GalNac-siRNA targeting transmembrane serine protease 6, in combination with deferiprone therapy reduces ineffective erythropoiesis and hepatic iron-overload in a mouse model of (β)-thalassaemia

research paper

SLN124, a GalNac-siRNA targeting transmembrane serine protease 6, in combination with deferiprone therapy reduces ineffective erythropoiesis and hepatic iron-overload in a mouse model of b-thalassaemia

Jim Vadolas,1,2 Garrett Z. Ng,3

Kai Kysenius,4 Peter J. Crouch,4

Sibylle Dames,5 Mona Eisermann,5

Tiwaporn Nualkaew,2 Shahla Vilcassim,6

Ute Schaeper5,* and

George Grigoriadis2,6,*

1Department of Molecular and Translational Sciences, Monash University, Clayton, 2Centre for Cancer Research, Hudson Institute of Medical Research, Clayton, VIC, 3Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, 4Department of Pharmacology and Therapeutics, Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Melbourne, VIC, Australia, 5Silence Therapeutics GmbH, Berlin, Germany, and 6School of Clinical Sciences at Monash Health, Monash University, Clayton, VIC, Australia

Received 3 February 2021; accepted for publication 2 March 2021 Correspondence: Jim Vadolas, Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, Australia.

E-mail: [e-mail]

Summary

Beta-thalassaemia is an inherited blood disorder characterised by ineffective erythropoiesis and anaemia. Consequently, hepcidin expression is reduced resulting in increased iron absorption and primary iron overload. Hepcidin is under the negative control of transmembrane serine protease 6 (TMPRSS6) via cleavage of haemojuvelin (HJV), a co-receptor for the bone morphogenetic protein (BMP)-mothers against decapentaplegic homologue (SMAD) signalling pathway. Considering the central role of the TMPRSS6/ HJV/hepcidin axis in iron homeostasis, the inhibition of TMPRSS6 expression represents a promising therapeutic strategy to increase hepcidin production and ameliorate anaemia and iron overload in b-thalassaemia. In the present study, we investigated a small interfering RNA (siRNA) conju-

gate optimised for hepatic targeting of Tmprss6 (SLN124) in b-thalassaemia mice (Hbbth3/+). Two subcutaneous injections of SLN124 (3 mg/kg) were

sufficient to normalise hepcidin expression and reduce anaemia. We also observed a significant improvement in erythroid maturation, which was associated with a significant reduction in splenomegaly. Treatment with the iron chelator, deferiprone (DFP), did not impact any of the erythroid parameters. However, the combination of SLN124 with DFP was more effective in reducing hepatic iron overload than either treatment alone. Collectively, we show that the combination therapy can ameliorate several disease symptoms associated with chronic anaemia and iron overload, and therefore represents a promising pharmacological modality for the treatment of b-thalassaemia and related disorders.

*U.S. and G.G. contributed equally.

Introduction

The b-thalassaemias are a group of inherited red blood cell (RBC) disorders caused by a reduction or absence of b-glo- bin synthesis.1 The clinical presentations of these disorders appear soon after birth when the fetal c-globin gene is progressively silenced and is replaced by the aberrant adult b- globin gene, contributing to atypical adult haemoglobin (HbA,a2b2) production.2 Affected individuals display extreme clinical heterogeneity, ranging from nearly asymptomatic to

displaying life-threatening severe anaemia. Dyserythropoiesis is a constant finding, which is characterised by extensive ery- throid expansion and maturation block at the polychro- matophilic erythroblast stage, as well as increased apoptosis of late erythroblasts.3-5 The failures of erythropoiesis and chronic anaemia lead to abnormal iron metabolism and systemic iron overload, which contributes to cardiac, hepatic and endocrine dysfunction.6 Treatment options are limited, consisting mainly of RBC transfusions, which exacerbates iron loading leading to end-organ damage. If left untreated,

ª 2021 Silence Therapeutics. British Journal of Haematology published by British Society for Haematology and	doi: 10.1111/bjh.17428
John Wiley & Sons Ltd.

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J. Vadolas et al.

iron overload can rapidly worsen and become life-threaten- ing. Chelating agents allow for the active elimination of iron; however, chelation therapy is not sufficient to improve anaemia and to prevent iron overload-related complications.7,8

In response to excess iron, the liver peptide hepcidin inhibits gastrointestinal iron absorption. However, in b-thalas- saemia, hepcidin expression is suppressed contributing to uncontrolled absorption of dietary iron and insufficient iron retention by the reticuloendothelial system exacerbating ineffective erythropoiesis and anaemia.6 Thus, the manipulation of hepcidin and related pathways have become the target for development of novel therapies for iron overload disorders.9-

12 Hepcidin is under negative control of the transmembrane serine protease 6 (TMPRSS6) via the inhibition of haemoju- velin (HJV) activity, a co-receptor for the bone morpho- genetic protein (BMP)-mothers against decapentaplegic homologue (SMAD) signalling pathway, activated in a para-

crine manner by BMP2 and BMP6 produced by liver sinusoidal endothelial cells.6,10,13 Notably, mutations that disrupt TMPRSS6 expression in both humans and mice result in elevated hepcidin expression and iron deficiency, characterised as iron-refractory iron deficiency anaemia (IRIDA).14,15 Recent studies have shown that depletion of Tmprss6 expression using oligonucleotides such as anti-sense oligonu- cleotides (ASO) or small interfering RNA (siRNA) in b- thalassaemic mice not only decreased iron loading, but improved erythropoiesis and anaemia.16-20 Hence, the targeted reduction of TMPRSS6 expression represents a potential pharmacological modality to treat b-thalassaemia and other iron-loading anaemias associated with low hepcidin levels.16-21

The main challenge in oligonucleotide therapeutics is achieving efficient and targeted delivery to tissues and cells. The N-acetylgalactosamine (GalNAc) ligand is a well-definedliver-targeting moiety benefitting from its high affinity to the asialoglycoprotein receptor (ASGPR).22,23 GalNAc-conjugation of therapeutic oligonucleotides has led to development of a large number of drug candidates targeting liver hepato- cytes for a diverse range of conditions, including SLN124, which is composed of a trimeric GalNAc ligand conjugated to TMPRSS6-siRNA.22,24 We aimed to further investigate the therapeutic utility of SLN124, by treating b-thalassaemic mice (Hbbth3/+) to study the effect of Tmprss6 suppression.25

Our results indicate that two subcutaneous (s.c.) injections of SLN124 (3 mg/kg) in Hbbth3/+ mice, alone or in combination with the oral iron chelator deferiprone (DFP),26 not only suppressed Tmprss6 expression, but also normalised serum hepcidin levels, significantly improved erythropoiesis and reduced splenomegaly. Notably, the combination treatment achieved a significantly greater reduction in liver iron content when compared with each agent alone. Collectively, we show that the combination treatment can ameliorate several indicators of chronic anaemia and iron overload in Hbbth3/+ mice and therefore may provide a significant health benefit to patients with b-thalassaemia.

Materials and methods

Treatment of b-thalassaemia mice

The b-thalassaemic mice (Hbbth3/+) were bred on the C57BL/6 background, at the Monash Animal Research Plat- form, Monash University, under approved protocols. Mice were housed under a constant light-dark cycle and maintained on a standard mouse diet containing 200 parts per million (ppm) iron (Specialty Feeds, Glen Forrest, WA, Aus- tralia) with ad libitum access to food and water. The 6- month-old female mice were treated with either two doses of TMPRSS6 GalNAc-siRNA (SLN124) at 3 mg/kg, or the TMPRSS6 siRNA without GalNAc targeting ligand (CTRL) or the vehicle control [phosphate-buffered saline (PBS)], by s.c. injections on days 1 and 14. The oral iron chelator DFP was administered through the drinking water at 1 25 mg/ml, and maintained on standard commercial diet. The mice were humanely killed 21 days after the second dose for analysis.

Oligonucleotides

SLN124 comprises a double-stranded 19mer RNA oligonu- cleotide with 20-O-methyl,20-fluoro-20-deoxy modifications and phosphorothioate bonds targeting TMPRSS6, which is linked to a tri-antennary GalNAc unit at the 50 end of the sense strand. The non-targeting control oligonucleotide (CTRL) contains the same siRNA and modifications but lacks the tri-antennary GalNAc unit. The SLN124 and the CTRL oligonucleotide used in this study were produced by Silence Therapeutics GmbH (Berlin, Germany), by chemical synthesis using standard solid phase technology for oligonu- cleotides.27 Briefly, the anti-sense strand and the sense strand were individually synthesised, purified by anion-exchange (AEX) chromatography and desalted. After hybridisation of the single strands, the final double-stranded molecules were obtained as lyophilised powder. The identity of the single strands was confirmed by denaturing ion-pairreversed-phasehigh-performance liquid chromatography mass spectrometry (IP-RP-HPLC-MS), whereby the recorded single strand masses complied with the calculated molecular weights. The purity of the singe strands was analysed by AEX-HPLC with ultraviolet detection (AEX-HPLC- UV) and resulted in purities ≥85%. Purity of the final double strands was analysed by native IP-RP-HPLC-UV and resulted in values of ≥90%.

Gene expression analysis

Approximately 10 mg of liver tissue was disrupted and homogenised in a Mixer Mill MM 400 (Retsch GmbH, Haan, Germany) using tungsten carbid beads (Qiagen, Hilden, Ger- many) and total RNA was prepared with InviTrap Spin Tissue RNA Mini Kit (Stratec, Berlin, Germany) according to the manufacturer's instruction (InviTrap Protocol). In all,

2 ª 2021 Silence Therapeutics. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.

100 ng total RNA was used for real-time quantitative poly- merase chain reaction (RT-qPCR) with the following ampli- con sets/sequences for Tmprss6, hepcidin anti-microbial peptide (Hamp) and Actin (mACTB) (Eurogentec, Seraing, Belgium) respectively Table SI. The RT-qPCR reactions were carried out with a QuantStudio 6 Flex (Applied Biosystems part of Thermo Fisher Scientific Inc., Waltham, MA, USA) using TakyonTM One-Step Low Rox Probe 5X MasterMix dTTP (Eurogentec). The data were calculated by using the comparative CT method also known as the 2 DDCt method.28,29

Hepcidin assay

Serum hepcidin was quantified using the 'Hepcidin Murine- Compete ELISA Kit' (Intrinsic Lifesciences, La Jolla, CA, USA) according to manufacturer's instructions.

Full blood examination

Blood was collected from the submandibular vein in hep- arinised tubes. Full blood examination was performed using an automated Roche Applied Science Cobas Helios haemato- logical analyser at the Walter and Eliza Hall Institute, Mel- bourne, Australia.

GalNac-siRNA conjugate targeting Tmprss6 in b-thalassaemia

and immunophenotyping was carried out on 7-AADve cells. A minimum of 10 000 events were recorded for erythrocytes in BM and spleen. For all the analyses, cells were acquired using the LSR Fortessa X-20 using FACSDiva software (BD Biosciences) and the results analysed with FlowJo software (Tree Star Inc.).

Tissue iron, magnesium and zinc measurements

Tissue sections were cut using the Microm HM330 at 10 µm and put onto Superfrost slides. Laser ablation-inductively coupled plasma-MS(LA-ICP-MS) was used to quantitate the distribution of iron in liver and spleen in formalin fixed tissue sections using external reference stan- dards.32 Iron (Fe), magnesium (Mg) and zinc (Zn) measurements were performed using the New Wave Research UP213 (Kennelec Scientific, Mitcham, Victoria, Australia) (neodymium-doped yttrium aluminum garnet (Nd:YAG) laser system with a two-volume large format cell connected to an Agilent 7500 cs ICP-MS (Agilent Technologies Inc., Santa Clara, CA, USA) for isotopes 56Fe, 24Mg and 66Zn. This technique involves vaporising material with a UV laser that is transferred into an ICP-MS via argon gas.33,34 Consecutive sections at 4 µm were also stained for Perls' Prus- sian blue iron stain for iron distribution and structural identification.

Reactive oxygen species measurements

The reactive oxygen species (ROS) level was assessed as described before.30 Briefly, RBCs were washed and re-suspended in PBS supplemented with 2% fetal bovine serum and incubated with 5-(and-6)-chloromethyl-2',7'-dichlorodi- hydrofluorescein diacetate (CM-H2DCFDA) stain (5 lmol/l) in the dark for 5 min. The oxidative conversion of CM- H2DCFDA to its fluorescent product by ROS was measured immediately by flow cytometry. The ROS fluorescence signals (median fluorescence intensity) were recorded in these gated RBC populations. For all the analyses, cells were acquired using the LSR Fortessa X-20 using fluorescence-activated cell sorting (FACSDiva) software (BD Biosciences San Jose, CA, USA) and the results analysed with FlowJo software (Tree Star Inc., Ashland, OR, USA).

Erythroid differentiation in bone marrow and spleen

Erythroid cells were analysed from both the spleen and bone marrow (BM) by flow cytometry using fluorescein isothio- cyanate (FITC)-conjugated anti mouse-CD71, allophyco- cyanin (APC)-conjugatedanti-mouse Ter119 and phycoerythrin (PE)-conjugatedanti-mouse CD44 antibodies (BD Biosciences). This assay allows the separation of ery- throid cells into distinct populations corresponding to (i) pro-erythroblasts, (ii) basophilic, (iii) polychromatic, (iv) reticulocytes and (v) RBCs.31 For all the cytometric analyses 7-amino-actinomycin D (7-AAD) was used as viability dye

Statistical analysis

Experimental data are presented as box plots or stacked bar charts. One-way analysis of variance (ANOVA) with Brown-Forsythe test (F*-test) was performed followed by Dunnett's T3 post hoc tests against Vehicle group using GraphPad Prism Version 8.4.3 (GraphPad Software Inc., San Diego, CA, USA), unless indicated otherwise in the fig- ure legend. If the assumption of normal distribution by Q- Q plot investigation was violated, the measured values were log2-(RT-qPCR assay) or log10-transformed before the anal- ysis. Data were considered statistically significant, if adjusted P ≤ 0 05.

Results

SLN124 induces hepcidin expression and reduces serum iron levels in Hbbth3/+ mice

The development of novel therapies for the management of anaemia and iron overload in haemoglobinopathies represents a major field of focus. It has been shown that Tmprss6 siRNA can be used to increase hepcidin synthesis and reduce ineffective erythropoiesis in b-thalassaemic mice.19,20 Based on GalNAc-conjugate delivery technology, SLN124 was developed as an siRNA therapeutic to restore hepcidin expression and normalise iron homeostasis in b-thalassaemia. To evaluate the in vivo activity of SLN124, in silencing Tmprss6 and

ª 2021 Silence Therapeutics. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.

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J. Vadolas et al.

thereby reducing disease activity in b-thalassaemic mice, 6- month-old wild type (WT) and Hbbth3/+ mice, were treated with SLN124 (3 mg/kg on days 1 and 14) alone or in combination with the oral iron chelator DFP (1 25 mg/ml) in the drinking water and followed for 35 days. In this study, we used the same SLN124 dose, previously demonstrated to be effective in a murine model for hereditary haemochromatosis

1. type 1.24 The SLN124-treated mice had significantly reduced liver Tmprss6 mRNA levels, dramatically increased
   liver Hamp mRNA (which encodes hepcidin) and increased serum hepcidin levels in Hbbth3/+ mice compared to controls (Fig 1A-C). SLN124 was also effective in lowering serum
   iron levels (Fig 1D), while treatment with the oral iron chelator DFP alone did not affect Tmprss6, Hamp or serum iron levels (Fig 1). Importantly, these results provide evi- dence that SLN124 represents a potent in vivo modulator of Hamp expression and serum iron availability by the transient inhibition of Tmprss6 expression.

SLN124 treatment improves RBC parameters in Hbbth3/+ mice

Treatment with SLN124 significantly reduced anaemia in Hbbth3/+ mice as measured by an increase in haemoglobin (Hb) levels, RBC counts and decreased red cell distribution width (RDW) when compared to Hbbth3/+ controls and DFP-treated mice (Fig 2A-C). In addition, SLN124 partially corrected retic- ulocytosis in Hbbth3/+ mice either alone or in combination with DFP (Fig 2D). Mice treated with SLN124 combined with DFP showed similar benefit as mice treated with SLN124 alone (mean Hb increase of 25 g/dl versus control) compared to vehicle control, whereas mice treated with DFP alone showed no improvement in RBC parameters. Moreover, the improvement in RBC parameters in SLN124 treated Hbbth3/+ mice was associated with a significant reduction in splenomegaly (Fig 2E).

In b-thalassaemia, the unpaired a-globin chains precipi- tate, forming large, insoluble aggregates in RBCs, which

Fig 1. SLN124 treatment induces hepcidin expression and reduces serum iron levels in b-thalassaemia intermedia (Hbbth3+/ ) mice. (A) Liver transmembrane serine protease 6 (Tmprss6) mRNA expression levels. (B) Liver hepcidin anti-microbial peptide (Hamp) mRNA expression levels.

1. Serum hepcidin levels and (D) serum iron levels following treatment. Hbbth3+/ and wild type (WT) (~6-month-old female) mice were injected twice with 3 mg/kg SLN124 alone or in combination with deferiprone (DFP) over a 35-day period. Control groups received either the vehicle (phosphate-buffered saline) or Tmprss6 small interfering RNA (siRNA) without targeting ligand (CTRL). Total liver mRNA was harvested
   21 days after the final injection from either vehicle control or treated groups. Tmprss6 or Hamp mRNA was assessed by for real-time quantitative polymerase chain reaction (RT-qPCR), normalised to b-actin and expressed relative to the Hbbth3+/- vehicle control group, which was defined as 1 0. Serum hepcidin and serum iron levels was assessed on day 35. Results are presented by box plots with n = 4-6 mice/group. Statistical signifi- cance was determined in a Brown-Forsythe test (F*-test) followed by Dunnett's T3 post hoc tests against associated vehicle control group and as indicated by the brackets [not statistically significant (ns), P > 0 05; *P ≤ 0 05; ***P ≤ 0 001].

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GalNac-siRNA conjugate targeting Tmprss6 in b-thalassaemia

Fig 2. Analysis of red blood cell (RBC) parameters following SLN124 administration alone, or in combination with deferiprone (DFP). The RBC parameters (A) Haemoglobin concentration (Hb), (B) RBC numbers, (C) RBC distribution width (RDW), (D) Reticulocyte counts (E) Spleen weight and (F) reactive oxygen species (ROS) levels in RBCs measured using dichlorofluorescein diacetate (DCFDA) in either vehicle control or treated groups. Hbbth3+/ and wild type (WT) (~6-month-old female) mice were injected twice with 3 mg/kg SLN124 alone or in combination with DFP over a 35-day period and RBC parameters were measured 21 days after the final injection. Results are represented by box plots, n = 4- 9 mice/group. Statistical significance was determined in a Brown-Forsythe test (F*-test) followed by Dunnett's T3 post hoc tests against associated vehicle control group and as indicated by the brackets [not statistically significant (ns), P > 0 05, *P ≤ 0 05; **P ≤ 0 01; ***P ≤ 0 001].

increases ROS production resulting in haemolysis and greatly exacerbating the anaemic phenotype in b-thalassaemia.2,35 We next assessed the ROS levels in RBCs obtained from SLN124-treated mice. FACS analysis of RBCs was used to measure the oxidative conversion of DCFH-DA in RBCs. Remarkably, mice treated with SLN124 alone or in

combination with DFP significantly reduced ROS to near normal levels, whereas DFP-treated mice showed no change in ROS levels (Fig 2F). Overall, these results demonstrate that SLN124 alone or in combination with DFP can improve RBCs and reticulocyte cellular indices as well as disease activ- ity.

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