Cell-Permeable Frataxin (CP-FXN) Ameliorate The Symptoms Of Friedreich’s Ataxia Through Regulation Of Cellular Iron Metabolism Joonno Lee, Sookeun Yeon, Wonheum Nah, Jeongheon Choi, Sujung Lee, Sunwoong Kim,Youngsil Choi and Daewoong Jo FXN Team, Cellivery R&D Institute, Cellivery Therapeutics, Inc., Seoul 03929, Korea BACKGROUND AIM Friedreich’s ataxia (FRDA) is monogenic autosomal recessive disease causing To ameliorate FRDA symptoms, cell-permeable FXN neurodegeneration and cardiomyopathy. FRDA is attributed to depletion of the (CP-FXN) has been developed by fusing sequence- mitochondrial
protein frataxin (FXN), which is caused by a GAA expansion in first optimized advanced macromolecule transduction intron of the frataxin gene. FXN is known to play a key role in iron metabolism in iron- domain (aMTD) to deliver FXN directly into cells and sulfur cluster (ISC) biogenesis and heme biosynthesis, both of which occur in tissues. mitochondria. METHODS A structurally stable backbone for CP-FXN has been established by examining structural stability and biological activity of various recombinant proteins. Next, to optimize the activity of FXN candidates, advanced macromolecule
transduction domains (aMTDs) were screened for enhanced solubility, biological activity, and cell permeability. RESULTS Figure 1. Development Of Structurally Stable Figure 2. Structure, Homogeneity & Figure 6. CP-FXN Interacts aMTD/SD-Fused FXM Recombinant Protein CD Analysis Of CP-FXN With Fe & Blocks Iron-Accumulation 2+ A A D A B C 100% 95% B Figure 3. CP-FXN Is Intracellularly Delivered A B B C 5770% C Figure 4. CP-FXN Is Co-Localized D With Mitochondria 84% A B D Figure 7. CP-FXN Restores Aconitase & Figure 5. CP-FXN Is Intracellularly SDH Activity In FXN-Knockdowned Cells Distributed In
Brain and Heart A A 110% 87% 700% 800% E B 70% 68% B C 100% 51% 55% CONCLUSION REFERENCES Contact Information CP-FXN is delivered into mitochondria, blocks iron Chung et al. (2020) Science Advances, 6: eaba 1193 Minyong Jung accumulation by binding to iron and restores Lim et al. (2013) Clinical Cancer Research, 19: 680-690 New Drug & Biusiness Development activity of ISC-dependent aconitase in FXN Lim et al. (2013) Biomaterials, 34: 6261-6271 deficient cells. Based on these data, CP-FXN has Lim et al. (2012) Molecular Therapy, 20: 1540-1549 Cellivery Therapeutics Inc. a therapeutic potential
for FRDA treatment Jo et al. (2005) Nature Medicine, 11: 892-898 jungmy@cellivery.com through mitochondrial delivery. +82-2-3151-8900 Jo et al. (2001) Nature Biotechnology, 19:929-933 [P. Molecular medicine and imaging-1] Mitochondrial Targeting Of Frataxin Rescues Abnormal Iron Accumulation By Activating Iron-Sulfur Cluster Biosynthesis Joonno Lee¹, Sookeun Yeon¹, Keumyoung Kang¹, Jeongheon Choi¹, Wonheum Na¹, Sujug Lee¹, Youngsil Choi¹, Daewoong Jo¹˙* ¹Cellivery R&D Institute, Cellivery Therapeutics, Seoul 03929, Korea Friedreich’s ataxia (FRDA) is monogenic autosomal recessive disease
causing neurodegeneration and cardiomyopathy. FRDA is attributed to partial depletion of the mitochondrial protein frataxin (FXN), which is caused by a GAA expansion in first intron of the frataxin gene. FXN is well known to play a key role in iron metabolism in iron-sulfur cluster (ISC) biogenesis and heme biosynthesis, both of which occur in mitochondria. Although treatment options for FRDA patients include antioxidant and iron chelation, these therapies have not shown substantial clinical improvement associated with neuronal loss. To overcome this problem, cell-permeable FXN (CP-FXN) has
been developed by fusing sequence-optimized advanced macromolecule transduction domain (aMTD) to deliver FXN directly into cells and tissues. CP-FXN is delivered into mitochondria, blocks iron accumulation by binding to iron and restores activity of ISC-dependent aconitase in FXN deficient cells. CP-FXN also shows cytoprotective effect under iron-mediated oxidative stress in FRDA patient-derived fibroblast, suggesting that CP-FXN is able to facilitate ISC biosynthesis, leading to maintaining cellular iron homeostasis. Based on these data, CP-FXN has a therapeutic potential for FRDA treatment
through mitochondrial delivery. Frataxin Replenishment Ameliorates Heart Dysfunction Through Recovery Of Iron Metabolism In Frataxin Deficient Mice Wonheum Na, Joonno Lee, Hyunpyo Kim, Sookeun Yeon, Jeongheon Choi, Sujung Lee, Youngsil Choi, and Daewoong Jo Drug Development Division for Neurodegenerative Disease, Cellivery R&D Institute, Cellivery Therapeutics, Inc., Seoul 03929, Korea. BACKGROUND AIM Friedreich’s ataxia (FRDA) is a progressive cardiac and neurodegenerative disease To determine whether CP-FXN can rescue the iron with early life mortality resulted from cardiomyopathy. FRDA is
caused by a metabolism pathway in heart-specific FXN deficient deficiency of frataxin (FXN) which plays a role in regulating iron metabolism. Since mice, FXN conditional knockout mouse model which is there is no treatment for FRDA, cell-permeable FXN (CP-FXN) has been developed specifically deleting exon 2 of FXN gene in heart is by fusing sequence-optimized advanced macromolecule transduction domain currently being exploited. (aMTD) to replenish insufficient FXN in cells and animals. METHODS To determine in vivo efficacy [administration route (IP & IV), dose (30 & 50 mg/kg) & frequency],
CP-FXN was treated in FXN KO animal model. In addition, CP-FXN was treated for determining in vivo efficacy in Doxorubicin (DOX) mediated cardiomyopathy animal model. RESULTS Figure 1. Intravenous (IV) Administration of Figure 4. High Dose Of CP-FXN Prevents Figure 2. High Dose Of CP-FXN Is More Effective CP-FXN Is More Effective Route To Expand To Prevent Body Weight Loss In FXN KO Animals Body Weight And Heart Rate Reduction Life Span In FXN KO Animals In DOX-Mediated Cardiomyopathy Animals Protocol Optimization: Route Protocol Optimization: Dose Protocol Optimization: Dose (IP & IV, 50
mg/kg, 3 times/week) (IV, 30 & 100 mg/kg, 3 times/week) ((IV, 30, 50 & 100 mg/kg, 3/W) ) Histological Analysis Aconitase Activity Iron Accumulation Aconitase Activity Heart Rate & ECG (electrocardiogram) Histological Analysis Figure 3. Low & High Frequency Of CP-FXN Have Similar Effect On Body Weight Change In FXN KO Animals Protocol Optimization: Frequency Figure 5. High Frequency Of CP-FXN Prevents (IV, 100 mg/kg, 3/W & 7/W) Body Weight And Heart Rate Reduction In DOX-Mediated Cardiomyopathy Animals Protocol Optimization: Frequency (IV, 100 mg/kg, 2/W & 3/W) Apoptotic Cell Death Analysis
Histological Analysis Heart Rate & ECG (electrocardiogram) CONCLUSION REFERENCES Contact information In FXN KO and DOX mouse models, CP- Chung et al. (2020) Science Advances, 6: eaba 1193 Minyong Jung FXN recovers life span, iron metabolism and Lim et al. (2013) Clinical Cancer Research, 19: 680-690 New Drug & Business Development heart function. These results suggest that Lim et al. (2013) Biomaterials, 34: 6261-6271 Cellivery Therapiutics, Inc. CP-FXN has a therapeutic potential for Lim et al. (2012) Molecular Therapy, 20: 1540-1549 FRDA treatment by improving cardiac iron Jo et al. (2005)
Nature Medicine, 11: 892-898 jungmy@cellivery.com homeostasis. Jo et al. (2001) Nature Biotechnology, 19: 929-933 +82-2-3151-8900 [P. Molecular medicine and imaging-2] Frataxin Replenishment Ameliorates Heart Dysfunction Through Recovery Of Iron Metabolism In Frataxin Deficient Mice Wonheum Nah¹, Joonno Lee¹, Keumyoung Kang¹, Jeongheon Choi¹, SooJung Lee¹, Youngsil Choi¹, Daewoong Jo¹ ¹Cellivery R&D Institute, Cellivery Therapeutics, Seoul 03929, Korea Friedreich’s ataxia (FRDA) is a progressive cardiac and neurodegenerative disease with early life mortality resulted from cardiomyopathy. FRDA
is caused by a deficiency of frataxin (FXN) which plays a role in regulating iron metabolism. Since there is no treatment for FRDA, cell-permeable FXN (CP-FXN) has been developed by fusing sequence-optimized advanced macromolecule transduction domain (aMTD) to replenish insufficient FXN in cells and animals. To determine whether CP-FXN can rescue the iron metabolism pathway in heart-specific FXN deficient mice, FXN conditional knockout mouse model which is specifically deleting exon 2 of FXN gene in heart and skeletal muscle is currently being exploited. Animals were administered CP-FXN at a
dose of 50 mg/kg for 6 weeks. In heart, CP-FXN restores expression of ISC containing enzyme molecules (aconitase, SDH B, NDUFS3) and iron storage molecule (H-ferritin). Also, aconitase activity (70%) and SDH activity (37%) were induced by CP-FXN. Histological analyses demonstrate that CP-FXN suppresses cardiac iron accumulation, myocardial post-necrotic fibrosis and cardiomyocyte apoptosis in FXN KO mice. These results suggest that CP-FXN has a therapeutic potential for FRDA treatment by improving cardiac iron homeostasis. Paeoniflorin, a Major Compound of P. lactoflora, Increases Endometrial
Receptivity by Upregulating the Expression of LIF Hye-Rin Park 1,2,† , Hee-Jung Choi 1,† , Mi-Ju Park , Dong-Ryeol Ryu , Ki-Tae Ha 1,2,* 1 3 Korean Medical Research Center for Healthy Aging, Pusan National University and 1 Department of Korean Medical science, School of Korean Medicine, Pusan National University, Yangsan, 2 Gyeongsangnam-do, Republic of Korea Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea 3 These authors contributed equally in this study. † ABSTRACT MATERIALS & METHODS Although recent progress of assisted reproduction
technologies, many patients have been suffering from recurrent Cell viability assay: Cytotoxic effect caused by paeoniflorin implantation failure (RIF) to achieve pregnancy after embryo transfer. Among some types of RIF, endometrial RIF treatment of cultured medium of Ishikawa cells was estimated by defined as low endometrial receptivity and thin endometrium can be good therapeutic target of traditional herbal MTT assay. medicine. To understand the endometrial RIF in genetic levels, we compared transcriptomic data from human Adhesion assay: The number of fluorescence-labeled JAr cells
endometrium obtained from patients undergoing RIF to normal samples. First, we performed gene set enrichment bound to Ishikawa cell monolayer were counted. RT-PCR: The mRNA levels of LIF, several adhesion molecules analysis using the NCBI GEO database, including GES71835, GES92324, GES26787, and GES4888. Next, we (Integrin αV, β1, β3 and β5), and β-actin were measured. categorized functionally related gene sets with the enrichment map visualization method and the result showed the Western blot analysis: The protein levels of LIF, adhesion cell adhesion-related gene sets were enriched in normal
human endometrium. Twelve genes of them had a molecules (Integrin αV, β1, β3 and β5), and GAPDH were positive correlation with leukemia inhibitory factor (LIF), a well-known regulator of endometrial receptivity. examined. In our study, paeoniflorin, a major compound of P. lactoflora, enhanced embryo implantation in vitro and in vivo via Knock-down of LIF expression: pLKO.1 vectors harboring induction of the leukemia inhibitory factor (LIF) and several integrin molecules. Therefore, our results suggest that shRNA for LIF were introduced by viral infection. paeoniflorin might be a potent
candidate for ameliorating the endometrial RIF by enhancing endometrial receptivity. In vivo study: A implantation failure model mice using RU-486, an antagonist of progesterone receptor, were treated with paeoniflorin(0.16mg/mouse/day). RESULTS Figure 1. Enriched Cell Adhesion-Related Figure 4. The Effect of Paeoniflorin on LIF Figure 5. Paeoniflorin Enhances Gene Set in Normal Human Endometrium. Expression in mRNA and Protein Levels. Endometrial Receptivity in an LIF- Dependent Manner. NES = 2.034 A) Paeoniflorin(μM) 3.0 NOM p-value = 0 * * FDR q-value = 0 0 10 30 50 2.5 LIF 2.0 β-actin
Adhesion cell count (fold of control) 1.5 ### B)
