Antisense Oligonucleotide Gapmers Research Articles

Inhibition of Fli‐1 attenuates inflammation, pericyte loss and cognitive deficits in Alzheimer’s disease

AbstractBackgroundBrain pericytes maintain the integrity of the blood‐brain barrier (BBB) and facilitate the removal of amyloid β (Aβ) which is critical to healthy brain activity (Ethan et al., Brain Pathol., 24: 371‐386, 2014). Pericyte degeneration has been observed in brains from patients with Alzheimer’s disease (AD) and animal models (Jesse et al., Brain Pathol., 23: 303‐10, 2013; Wu et al., Aging (Albany NY), 11: 6120‐6133, 2019). Our previous data demonstrated that friend leukemia virus integration 1 (Fli‐1), an ETS transcription factor, regulates pericyte viability and vascular injury in murine sepsis (Li et al., J Infect Dis, 218: 1995‐2005, 2018); however, the role of Fli‐1 in AD remains unknown.MethodsFli‐1 levels and pericyte number were detected in postmortem brains from 21 patients with AD and 17 healthy controls, and in 5xFAD transgenic mice. Control or Fli‐1 antisense oligonucleotide Gapmers were injected into the hippocampus of 5xFAD mice at 3 and 4.5 months of age. Pericyte number, inflammatory mediators, Aβ deposition, BBB function and cognitive deficits were evaluated at 6 months of age. Human brain pericytes were transfected with control or Fli‐1 antisense Gapmers for 48 h and cultured with or without freshly aggregated Aβ40 for 7 consecutive days. Cell viability and apoptosis were detected.ResultsWe demonstrated that Fli‐1 expression was increased in postmortem brains from a cohort of human AD donors and in 5xFAD mice, which corresponded with a decreased pericyte number, elevated inflammatory mediators, and increased Aβ accumulation as compared to cognitively normal individuals and WT mice, respectively. Antisense oligonucleotide Fli‐1 Gapmer administrated via intrahippocampal injection decelerated pericyte loss, reduced inflammatory responses, ameliorated cognitive deficits, improved BBB dysfunction, and decreased Aβ deposition in 5xFAD mice. Fli‐1 Gapmer‐mediated inhibition of Fli‐1 protected against Aβ accumulation induced human brain pericyte apoptosis in vitro.ConclusionThese composite results indicate that Fli‐1 contributes to pericyte loss, inflammatory response, Aβ deposition, vascular dysfunction and cognitive decline, and suggest that suppression of Fli‐1 may represent a novel therapeutic strategy for AD.

LncRNA Neat1 regulates neuronal dysfunction post-sepsis via stabilization of hemoglobin subunit beta

Sepsis-associated encephalopathy (SAE) is characterized by acute and diffuse brain dysfunction and correlates with long-term cognitive impairments with no targeted therapy. We used a mouse model of sepsis-related cognitive impairment to examine the role of lncRNA nuclear enriched abundant transcript 1 (Neat1) in SAE. We observed that Neat1 expression was increased in neuronal cells from septic mice and that it directly interacts with hemoglobin subunit beta (Hbb), preventing its degradation. The Neat1/Hbb axis suppressed postsynaptic density protein 95 (PSD-95) levels and decreased dendritic spine density. Neat1 knockout mice exhibited decreased Hbb levels, which resulted in increased PSD-95 levels, increased neuronal dendritic spine density, and decreased anxiety and memory impairment. Neat1 silencing via the antisense oligonucleotide GapmeR ameliorated anxiety-like behavior and cognitive impairment post-sepsis. In conclusion, we uncovered a previously unknown mechanism of the Neat1/Hbb axis in regulating neuronal dysfunction, which may lead to a novel treatment strategy for SAE.

Antisense oligonucleotide gapmers containing phosphoryl guanidine groups reverse MDR1-mediated multiple drug resistance of tumor cells

Antisense gapmer oligonucleotides containing phosphoryl guanidine (PG) groups, e.g., 1,3-dimethylimidazolidin-2-imine, at three to five internucleotidic positions adjacent to the 3′ and 5′ ends were prepared via the Staudinger chemistry, which is compatible with conditions of standard automated solid-phase phosphoramidite synthesis for phosphodiester and, notably, phosphorothioate linkages, and allows one to design a variety of gapmeric structures with alternating linkages, and deoxyribose or 2′-O-methylribose backbone. PG modifications increased nuclease resistance in serum-containing medium for more than 21 days. Replacing two internucleotidic phosphates by PG groups in phosphorothioate-modified oligonucleotides did not decrease their cellular uptake in the absence of lipid carriers. Increasing the number of PG groups from two to seven per oligonucleotide reduced their ability to enter the cells in the carrier-free mode. Cationic liposomes provided similar delivery efficiency of both partially PG-modified and unmodified oligonucleotides. PG-gapmers were designed containing three to four PG groups at both wings and a central “window” of seven deoxynucleotides with either phosphodiester or phosphorothioate linkages targeted to MDR1 mRNA providing multiple drug resistance of tumor cells. Gapmers efficiently silenced MDR1 mRNA and restored the sensitivity of tumor cells to chemotherapeutics. Thus, PG-gapmers can be considered as novel, promising types of antisense oligonucleotides for targeting biologically relevant RNAs.

C-Casitas b-Lineage Lymphoma Downregulation Improves the Ability of Long-term Cultured Mesenchymal Stem Cells for Promoting Angiogenesis and Diabetic Wound Healing

The chronic wound induced by diabetes has poor efficacy and could lead to amputation. The repair function of mesenchymal stem cells (MSCs) impaired after long-term culture in vitro. Studies have shown that the proto-oncogene c-Casitas b-lineage lymphoma (c-Cbl) can regulate receptor- and non-receptor tyrosine kinase, which was also involved in the angiogenesis process. This study aimed to explore the regulative effect of c-Cbl on the proangiogenic functions of long-term cultured MSCs and evaluate its pro-healing effect on diabetic wounds. In this study, the c-Cbl level was downregulated by locked nucleic acid–modified antisense oligonucleotide gapmers (LNA Gapmers). We detected the effect of c-Cbl downregulation on long-term cultured MSCs in terms of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signal, cellular proliferation, senescence, migration, and angiogenic factors paracrine activity in vitro. In vivo, we observed the pro-healing effect of long-term cultured MSCs, with or without c-Cbl downregulation, on the diabetic wound. We found that the phosphorylation level of c-Cbl increased and that of Akt decreased in passage 10 (P10) MSCs compared with passage 3 (P3) MSCs (P < 0.05). Additionally, the proliferation, paracrine, and migration capacity of P10 MSCs decreased significantly, accompanied by the increase of cellular senescence (P < 0.05). However, these functions, including PI3K/Akt activity of P10 MSCs, have been improved by c-Cbl downregulation (P < 0.05). Compared with P10 MSCs treatment, treatment with c-Cbl downregulated P10 MSCs accelerated diabetic wound healing, as defined by a more rapid wound closure (P < 0.05), more neovascularization (P < 0.05), and higher scores of wound histological assessment (P < 0.05) in a diabetic rat model. Our findings suggested that c-Cbl downregulation could attenuate the impairment of proangiogenic functions in MSCs induced by long-term culture in vitro and improve the effect of long-term cultured MSCs in promoting diabetic wound healing.

Development of Antisense Oligonucleotide Gapmers for the Treatment of Dyslipidemia and Lipodystrophy.

Although technological advances in molecular genetics over the last few decades have greatly expedited the identification of mutations in many genetic diseases, the translation of the genetic mechanisms into a clinical setting has been quite challenging, with a minimum number of effective treatments available. The advancements in antisense therapy have revolutionized the field of neuromuscular disorders as well as lipid-mediated diseases. With the approval of splice-switching antisense oligonucleotide (AO) therapy for nusinersen and eteplirsen for the treatment of spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD), several modified AOs are now being evaluated in clinical trials for the treatment of a number of disorders. In order to activate RNase H-mediated cleavage of the target mRNA, as well as to increase the binding affinity and specificity, gapmer AOs are designed that have a PS backbone flanked with the modified AOs on both sides. Mipomersen (trade name Kynamro), a 2'-O-methoxyethyl (MOE) gapmer, was approved by the Food and Drug Administration (FDA) for the treatment of homozygous familial hypercholesterolemia (HoFH) in 2013. Volanesorsen, another 20-mer MOE gapmer has shown to be successful in lowering the levels of triglycerides (TGs) in several lipid disorders and has received conditional approval in the European Union for the treatment of Familial chylomicronemia syndrome (FCS) in May 2019 following successful results from phase II/III clinical trials. This chapter focuses on the clinical applications of gapmer AOs for genetic dyslipidemia and lipodystrophy.

Development of Antisense Oligonucleotide Gapmers for the Treatment of Huntington's Disease.

The field of neuromuscular and neurodegenerative diseases has been revolutionized by the advent of genetics and molecular biology to evaluate the pathogenicity, thereby providing considerable insight to develop suitable therapies. With the successful translation of antisense oligonucleotides (AOs) from in vitro into animal models and clinical practice, modifications are being continuously made to the AOs to improve the pharmacokinetics and pharmacodynamics. In order to activate RNase H-mediated cleavage of the target mRNA, as well as to increase the binding affinity and specificity, gapmer AOs are designed to have a phosphorothioate (PS) backbone flanked with the modified AOs on both sides. Antisense-mediated knockdown of mutated huntingtin is a promising therapeutic approach for Huntington's disease (HD), a devastating disorder affecting the motor and cognitive abilities. This chapter focuses on the modified gapmer AOs for the treatment of HD.

Therapeutic Targeting of TGFβ Ligands in Glioblastoma Using Novel Antisense Oligonucleotides Reduces the Growth of Experimental Gliomas.

Transforming growth factor (TGF)-β is expressed at high levels by glioma cells and contributes to the malignant phenotype of glioblastoma. However, its therapeutic targeting remains challenging. Here, we examined an alternative therapeutic approach of TGFβ inhibition using two novel phosphorothioate-locked nucleic acid (LNA)-modified antisense oligonucleotide gapmers, ISTH1047 and ISTH0047, which specifically target TGFβ1 and TGFβ2. We characterized the effects of ISTH1047 and ISTH0047 on TGFβ1/2 expression, downstream signaling and growth of human LN-308, LN-229, and ZH-161 cells as well as murine SMA-560 glioma cells in vitro. Furthermore, we assessed their target inhibition and effects on survival in orthotopic xenogeneic and syngeneic rodent glioma models in vivo. Both antisense oligonucleotides specifically silenced their corresponding target and abrogated SMAD2 phosphorylation in several glioma cell lines. Moreover, inhibition of TGFβ1 or TGFβ2 expression by ISTH1047 or ISTH0047 reduced the migration and invasiveness of LN-308 and SMA-560 glioma cells. Systemic antisense oligonucleotide administration to glioma-bearing mice suppressed TGFβ1 or TGFβ2 mRNA expression as well as the expression of the downstream target PAI-1 in orthotopic gliomas. Glioma-bearing mice had significantly prolonged survival upon systemic treatment with ISTH1047 or ISTH0047, which was associated with a reduction of intratumoral SMAD2 phosphorylation and, in a fully immunocompetent model, with increased immune cell infiltration. Targeting TGFβ expression with the novel LNA antisense oligonucleotides ISTH1047 or ISTH0047 results in strong antiglioma activity in vitro and in vivo, which may represent a promising approach to be examined in human patients with glioma.

Biological Role and Therapeutic Targeting of TGF-β3 in Glioblastoma.

Transforming growth factor (TGF)-β contributes to the malignant phenotype of glioblastoma by promoting invasiveness and angiogenesis and creating an immunosuppressive microenvironment. So far, TGF-β1 and TGF-β2 isoforms have been considered to act in a similar fashion without isoform-specific function in glioblastoma. A pathogenic role for TGF-β3 in glioblastoma has not been defined yet. Here, we studied the expression and functional role of endogenous and exogenous TGF-β3 in glioblastoma models. TGF-β3 mRNA is expressed in human and murine long-term glioma cell lines as well as in human glioma-initiating cell cultures with expression levels lower than TGF-β1 or TGF-β2 in most cell lines. Inhibition of TGF-β3 mRNA expression by ISTH2020 or ISTH2023, two different isoform-specific phosphorothioate locked nucleic acid (LNA)-modified antisense oligonucleotide gapmers, blocks downstream SMAD2 and SMAD1/5 phosphorylation in human LN-308 cells, without affecting TGF-β1 or TGF-β2 mRNA expression or protein levels. Moreover, inhibition of TGF-β3 expression reduces invasiveness in vitro Interestingly, depletion of TGF-β3 also attenuates signaling evoked by TGF-β1 or TGF-β2 In orthotopic syngeneic (SMA-560) and xenograft (LN-308) in vivo glioma models, expression of TGF-β3 as well as of the downstream target, plasminogen-activator-inhibitor (PAI)-1, was reduced, while TGF-β1 and TGF-β2 levels were unaffected following systemic treatment with TGF-β3 -specific antisense oligonucleotides. We conclude that TGF-β3 might function as a gatekeeper controlling downstream signaling despite high expression of TGF-β1 and TGF-β2 isoforms. Targeting TGF-β3in vivo may represent a promising strategy interfering with aberrant TGF-β signaling in glioblastoma. Mol Cancer Ther; 16(6); 1177-86. ©2017 AACR.

Potent and selective antisense oligonucleotides targeting the transforming growth factor beta (TGF-β) isoforms in advanced glaucoma: a preclinical evaluation

Purpose: ISARNA Therapeutics is developing highly potent and selective LNA-modified ASO gapmers targeting TGF-β1 and TGF-β2 mRNA. In the field of ophthalmology, glaucoma, the second leading cause for blindness in the US, has been linked to TGF-β activation as a key driver. ISTH0036 is a 14-mer phosphorothioate Locked Nucleic Acid- (LNA) modified antisense oligonucleotide gapmer, targeting the sequence of TGF-β2 mRNA and was developed for therapeutic intervention in ocular diseases. It was shown to effectively and potently downregulate target mRNA in a dose-dependent manner in relevant cell-based assays, as well as leading to target engagement in anterior eye segment tissues upon intravitreal administration (Isarna proprietary information). The aim of this study was to evaluate the therapeutic potential of ISTH0036 in murine models of glaucoma filtration surgery (GFS) following different intraocular administrations.Methods: A murine model of glaucoma filtration surgery has been used to evaluate the effect of intraocular ISTH0036 administration on post-operative wound healing. Bleb size and bleb survival were determined after different intraocular administrations of saline, control scrambled oligonucleotide or ISTH0036 (at day of surgery and after two weeks).Results: Intraocular administrations of ISTH0036 lead to a significant effect on bleb area and survival, as well as decreasing scarring (downregulation of collagen-1 and -3 fibers) in a murine glaucoma filtration surgery model.Conclusion: Consistent with the expected molecular mechanism of action and demonstrated pharmacokinetic (PK) and pharmacodynamic (PD) properties following intraocular administration, preclinical data with ISTH0036 in murine glaucoma filtration surgery model support the current exploration of the drug candidate in advanced glaucoma patients undergoing trabeculectomy.

Lipid Nanoparticles Loaded with an Antisense Oligonucleotide Gapmer Against Bcl-2 for Treatment of Lung Cancer.

Bcl-2 is an anti-apoptotic gene that is frequently overexpressed in human cancers. G3139 is an antisense oligonucleotide against bcl-2 that has shown limited efficacy in clinical trials. Here, we report the synthesis of a new antisense oligonucleotide containing additional chemical modifications and its delivery using nanoparticles. An oligonucleotide G3139-GAP was synthesized, which has 2'-O-methyl nucleotides at the 5' and 3' ends based on a "gapmer" design. Furthermore, G3139-GAP was incorporated into lipid nanoparticles (LNPs) composed of DOTAP/egg PC/cholesterol/Tween 80. The LNP-loaded G3139-GAP was evaluated in A549 lung cancer cells both in vitro and in a murine xenograft model for biological activity and therapeutic efficacy. The LNPs showed excellent colloidal and serum stability, and high encapsulation efficiency for G3139-GAP. They have a mean particle diameter and zeta potential of 134nm and 9.59mV, respectively. G3139-GAP-LNPs efficiently downregulated bcl-2 expression in A549 cells, as shown by 40% and 83% reduction in mRNA and protein levels, respectively. Furthermore, G3139-GAP-LNPs were shown to inhibit tumor growth, prolong survival, and downregulate tumor bcl-2 expression in an A549 murine xenograft tumor model. These data indicate that G3139-GAP-LNPs have excellent anti-tumor efficacy and warrant further evaluation.

Thrombin and Factor XII Drive Prostate Tumor Growth In Vivo

Several hemostatic factors are known to strongly influence the metastatic process by promoting the survival of early metastases. Despite this consistent phenomenon observed across multiple tumor cell types and multiple procoagulant components of the hemostatic system, the role of hemostasis in the establishment and growth of primary tumors is highly context dependent. One clinically relevant context where coagulation appears to be important for tumor development/outgrowth is prostate cancer. Men on long-term anticoagulation appear to be at significantly lower risk for developing prostate cancer compared to men on short-term anticoagulation or those not on anticoagulation. Here, we report that multiple hemostatic proteases drive prostate tumor growth in vivo. To test the hypothesis that the central hemostatic protease, thrombin, promotes prostate tumor growth, mice with a life-long genetically-imposed diminution in circulating prothrombin levels (fIILox/-), or mice administered a prothrombin-specific antisense oligonucleotide (ASO) gapmer that limits prothrombin expression, were inoculated subcutaneously with the murine-derived prostatic adenocarcinoma cell line TRAMP-C2Re3 in parallel with appropriate controls. Genetically or pharmacologically lowering prothrombin expression significantly impeded prostate tumor growth. In complementary experiments, treatment with the prothrombin-specific ASO gapmer significantly impeded the growth of human-derived prostate cancer cells (PC3) in immunodeficient mice. Detailed histological analysis revealed that tumors harvested from mice with diminished prothrombin expression had significantly lower mitotic indices, suggesting that thrombin promotes tumor proliferation in vivo. Lowering prothrombin levels did not affect apoptosis, vascular density, or the presence of tumor-associated macrophages. Previous studies have indicated that tumor cell-associated tissue factor expression is one important mechanism by which cancer cells can locally generate thrombin. However, the relative importance of the contact pathway of coagulation system activation in cancer progression is less defined. In order to determine whether fXII plays a role in prostate cancer growth, mice challenged with prostate cancer were treated with a fXII-specific ASO gapmer or a control oligonucleotide with no homology in the murine genome in parallel. Remarkably, tumors from anti-fXII ASO gapmer treated mice were ~3-fold smaller than those harvested from control animals at the end of the ~3 week experiment. To our knowledge, this finding is the first demonstration that the contact system protease fXII plays a significant role in tumor growth. In sum, these data show that thrombin promotes the growth of prostate cancer, and suggest that contact activation of the coagulation cascade may be a critical factor in prostate cancer growth. DisclosuresMonia:Isis Pharmaceuticals: Employment, Other: Shareholder. Revenko:SIS Pharmaceuticals Inc.: Employment.

MRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

Diminished Multi-Organ Pathologies and Inflammation Associated With Sickle Cell Disease In Mice With Genetically Limited Prothrombin Levels

Sickle cell disease (SCD) is characterized by chronic hemolytic anemia, vaso-occlusions, chronic inflammation and an oxidative state, with progressive and widespread organ dysfunction and reduced life expectancy. Systemic coagulation system activation has been well documented in both SCD patients and animal models of SCD. However, the precise causal relationship, if any, between hemostatic factors and SCD pathogenesis has remained uncertain. Here, we test the hypothesis that thrombin-mediated proteolysis is a key mediator of the end-organ pathologies in SCD by evaluating the phenotypic consequences of imposed deficits in prothrombin in the well-characterized humanized murine model of SCD, Berkeley sickle mice (HbS mice). Prothrombin (fII) specific anti-sense oligonucleotide (ASO) gapmers (ISIS Pharmaceutics) were used to pharmacologically suppress circulating plasma fII levels to less than 10% of normal in cohorts of HbS mice starting at 3 weeks of age. Kaplan-Meier analysis revealed a major survival benefit in HbS mice when hepatic fII expression and circulating fII levels were selectively lowered, with ∼90% of fIILow mice surviving to 15 weeks of age as compared to just 60% of HbS mice treated with control ASO gapmer (P<0.04). As a complementary approach to better define the importance of fII in SCD pathobiology, we extended these studies to explore SCD in previously described fIIlox/- mice with a life-long, genetic deficit in circulating prothrombin. These animals carry ∼10% the normal level of fII with a resulting INR of 1.7, yet exhibit normal developmental and reproductive success, and never develop spontaneous bleeding events. Here, we utilized a hematopoietic stem cell transplantation (HSCT) approach using HbS stem cell donors to generate HbS/fIIlox/- and HbS/fIIWT cohorts. These transplanted mice were followed for a year after HSCT to examine the inflammatory and hemostasis parameters and organ pathologies and function. There was no significant difference in the RBC indices between HbS/fIIWT and HbS/fIIlox/- mice. However, HbS/fIIlox/- mice exhibited significantly reduced neutrophil counts (4.9 ± 0.3 vs.8.7 ± 1.2, P<0.01) and platelet counts (984 ± 66 vs. 1208 ± 55, P<0.01) compared to HbS/fIIWT mice. Furthermore, circulating IL-6, sVCAM, and D-dimer levels were significantly lower in HbS/fIIlox/- mice. Taken together, these data indicate that baseline inflammation was decreased as a consequence of reduced fII. Lower fII levels in HbS/fIIlox/- mice also resulted in significantly improved organ pathologies, including reduced infarctions in the liver, reduced inflammation in the lungs, remarkably improved kidney glomerular and tubular pathology, and decreased right ventricular (RV) hypertrophy. This was accompanied with a much improvement in organ function. Here, there was significantly improved renal function in HbS/fIIlox/- mice relative to HbS/fIIWT animals was reflected by a markedly reduced albumin (477 ± 96 vs. 1694 ± 118, P<0.0001) and improved urine concentrating ability (urine osmolality 1731 ± 185 vs. 1208 ± 169; P<0.01). Similarly, diminished hepatic damage was reflected in reduced transaminitis. Pulmonary hypertension also appeared to be significantly ameliorated in the HbS/fIIlox/- animals relative to HbS/fIIWT mice as reflected in decreased pulmonary artery smooth muscle hyperplasia in the lungs, and reduced RV hypertrophy by cardiac MR. Left ventricular diastolic dysfunction was also significantly ameliorated as reflected in reduced end diastolic volumes by cardiac MR. Notably, the median INR values in HbS/fIIlox/- and HbS/fIIWTmice, were 2.0 and 1.2, respectively, and not associated with any spontaneous hemorrhage in either group. Therefore, these data suggest that long-term therapeutic intervention with direct thrombin inhibitors, fXa inhibitors, or warfarin, even at doses that would only modestly increase INRs, could significantly reduce organ dysfunction and favorably affect the long term outcome in patients with SCD. In summary, our data supports the hypothesis that thrombin significantly contributes to inflammation and end organ pathologies in SCD, and may represent a novel therapeutic target to improve renal, cardiac and pulmonary functions, thereby reducing the morbidity and mortality in this patient population.Arumugam P and Mullins E contributed equally. Malik P and Degen J are Co-corresponding authors. Disclosures:Mullins:Baxter : Consultancy.